STABLE ANTIBODY FORMULATION

专利摘要:
the present invention provides stable pharmaceutical formulations that comprise a human antibody that specifically binds to human programmed death protein-1 (pd-1). in certain embodiments, the formulations contain, in addition to an anti-pd-1 antibody, a buffer, an amino acid, a non-ionic surfactant and a sugar. the pharmaceutical formulations of the present invention exhibit a substantial degree of antibody stability after stress and storage.
公开号:BR112019020246A2
申请号:R112019020246-3
申请日:2018-03-23
公开日:2020-05-12
发明作者:Hu Qingyan；Liu Dingjiang
申请人:Regeneron Pharmaceuticals, Inc.；
IPC主号:

专利说明:

Invention Patent Descriptive Report for STABLE ANTIBODY FORMULATION.
[0001] This application is being filed on March 23, 2018 as a PCT International Patent Application and claims priority benefit to United States Provisional Patent Application No. 62 / 482,270, filed on April 6, 2017, whose description is hereby incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to the field of therapeutic antibody formulations. More specifically, the present invention relates to the field of pharmaceutical formulations that comprise a human antibody that specifically binds to human programmed death protein-1 (PD-1).
BACKGROUND OF THE INVENTION
[0003] Therapeutic macromolecules (eg, antibodies) should be formulated in a way that not only takes the appropriate molecules for administration to patients, but also maintains their stability during storage and subsequent use. For example, therapeutic antibodies in liquid solution are prone to unwanted degradation, aggregation or chemical modifications, unless the solution is formulated properly. The stability of an antibody in the liquid formulation depends not only on the types of excipients used in the formulation, but also on the quantities and proportions of the excipients in relation to each other. In addition, considerations other than stability must be taken into account when preparing a liquid antibody formulation. Examples of such additional considerations include the viscosity of the solution and the concentration of antibody that can be accommodated by a given formulation, and the visual quality or appeal of the formulation. Thus, when formulating an
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2/130 therapeutic antibodies, great care must be taken to arrive at a formulation that remains stable, contains an appropriate concentration of antibodies and has an adequate viscosity, in addition to other properties which allow the formulation to be conveniently administered to patients.
[0004] Antibodies to human programmed death protein-1 (PD-1) are an example of a therapeutically relevant macromolecuia that requires adequate formulation. Anti-PD-1 antibodies are clinically useful for the treatment of cancer (for example, lung cancer, melanoma and brain cancer) and viral infections and autoimmune diseases. Exemplary anti-PD-1 antibodies are described, inter alia, in United States Patents / Publications 7101550, 7595048, 7488802, 7563869, 8008449, 8168757, 8216996, 20110008369, 20130017199, 20130022595 and in documents W0200612119, W0200911114, W0200910114, W0200910114, W0200910114, W0200910114 and EP2504028. US20140234296 describes lyophilized formulations of an anti-PD-1 antibody.
[0005] Although anti-PD-1 antibodies are known, there is still a need in the art for innovative pharmaceutical formulations comprising anti-PD-1 antibodies that are sufficiently stable and suitable for administration to patients.
BRIEF SUMMARY OF THE INVENTION
[0006] The present invention satisfies the aforementioned need by providing stable pharmaceutical formulations that comprise a human antibody that specifically binds to human programmed death protein-1 (PD-1).
[0007] In one aspect, a stable, low viscosity liquid pharmaceutical formulation is provided which comprises: (i) a human antibody that specifically binds to human programmed death protein-1 (PD-1); (ii) a buffer; (iii) an organic cosolvent; (iv) one
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3/130 stabilizer: and (v) a viscosity modifier.
[0008] In several embodiments, the antibody is supplied in a concentration from about 5 ± 0.75 mg / mL to about 250 ± 37.5 mg / mL In one embodiment, the antibody is provided in a concentration of 12.5 mg / ml ± 1.85 mg / ml or about 12.5 mg / ml. In one embodiment, the antibody is supplied at a concentration of 25 mg / ml ± 3.75 mg / ml or about 25 mg / ml. In another embodiment, the antibody is supplied at a concentration of 50 mg / ml ± 7.5 mg / ml or about 50 mg / ml. In another embodiment, the antibody is supplied at a concentration of 100 mg / ml ± 15 mg / ml or about 100 mg / ml. In one embodiment, the antibody is supplied at a concentration of 150 mg / ml ± 22.5 mg / ml or about 150 mg / ml. In another embodiment, the antibody is supplied at a concentration of 175 mg / ml ± 26.25 mg / ml or about 175 mg / ml. In another embodiment, the antibody is supplied at a concentration of 200 mg / ml ± 30 mg / ml or about 200 mg / ml.
[0009] In certain embodiments, the formulation comprises any of the anti-PD-1 antibodies described in United States Patent Application Publication No. 20150203579, incorporated herein in full. In certain embodiments, the anti-PD-1 antibody comprises (a) a heavy chain variable region (HCVR) comprising the heavy chain complementarity determining regions 1, 2 and 3 (HCDR1-HCDR2-HCDR3), each comprising a sequence of SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, respectively; and (b) a light chain variable region (LCVR) comprising light chain complementarity determining regions 1, 2 and 3 (LCDR1-LCDR2-LCDR3), each comprising a sequence of SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively. In one embodiment, the antibody comprises an HCVR comprising the amino acid sequence of SEQ ID NO: 1 and an
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LCVR comprising the amino acid sequence of SEQ ID NO: 2. In one embodiment, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 9 and a light chain comprising the amino acid sequence of SEQ ID NO: 9 : 10. In one embodiment, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 11 and a light chain comprising the amino acid sequence of SEQ ID NO: 10. In one embodiment, the antibody comprises a heavy chain comprising the amino acid sequence selected from the group consisting of SEQ ID NOs: 9 and 11; and a light chain comprising the amino acid sequence of SEQ ID NO: 10. In one embodiment, the antibody comprises an HCVR that has 90% sequence identity with SEQ ID NO: 1. In one embodiment, the antibody comprises an LCVR which has 90% sequence identity with SEQ ID NO: 2. In one embodiment, the antibody comprises an HCVR that has 90% sequence identity with SEQ ID NO: 1 and an LCVR that has 90% sequence identity with SEQ ID NO: 2. [0010] In one embodiment, the pH of the liquid formulation is pH 6.0 ± 0.5, pH 6.0 ± 0.4, pH 6.0 ± 0.3, pH 6.0 ± 0.2, pH 6.0 ± 0.1, pH 6.0 ± 0.05, pH 6.0 ± 0.01 or pH 6.0. In one embodiment, the pH of the liquid formulation is a pH of about 6.0 ± 0.3.
[0011] In one embodiment, the buffer comprises histidine. In certain embodiments, the histidine buffer is in a concentration from 5 mM ± 1 mM to 50 mM ± 10 mM, preferably from 5 mM ± 1 mM to 25 mM ± 5 mM. In one embodiment, the histidine buffer is at a concentration of 10 mM ± 2 mM or about 10 mM. In one embodiment, the histidine buffer is at a concentration of 20 mM ± 4 mM or about 20 mM. In one embodiment, the histidine buffer is at a concentration of 40 nM ± 8 mM or about 40 nM. In certain embodiments, histidine buffer
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5/130 comprises L-histidine and L-histidine monohydrate monohydrate. In one embodiment, L-histidine is in a concentration from 2 mM ± 0.4 mM to 25 mM ± 5 mM, preferably from 4 mM ± 0.8 mM to 20 mM ± 4 mM. In one embodiment, the Lhistidine monohydrate monohydrate is in a concentration of 2 mM ± 0.4 mM to 25 mM ± 5 mM, preferably 4 mM ± 0.8 mM to 20 mM ± 4 mM. In one embodiment, the buffer comprises L-histidine at a concentration of 4.8 mM ± 0.96 mM and L-histidine monohydrate monohydrate at a concentration of 5.2 mM ± 1.04 mM. In one embodiment, the buffer comprises histidine at a concentration of 10 mM ± 2 mM, where histidine comprises L-histidine at a concentration of 4.8 mM ± 0.96 mM and L-histidine monohydrate monohydrate in a concentration of 5.2 mM ± 1.04 mM.
[0012] In certain embodiments, the organic cosolvent is a non-tonic polymer that contains a fraction of polyoxyethylene. In one embodiment, the organic solvent is a surfactant. In some embodiments, the organic cosolvent is any one or more of polysorbate, poloxamer 188 and polyethylene glycol 3350. In one embodiment, the organic cosolvent is polysorbate 80. In one embodiment, the organic cosolvent is polysorbate 20.
[0013] In one embodiment, the organic cosolvent is in a concentration from about 0.01% ± 0.005% to about 1% ± 0.5% by weight in volume or w / v where, for example, 0.1 g / ml ··· 10% and 0.01 g / ml = 1%. In certain embodiments, the organic solvent is polysorbate in a concentration from 0.05% ± 0.025% to 0.5% ± 0.25% (w / v). In one embodiment, the organic cosolvent is polysorbate 80, which is at a concentration of 0.2% ± 0.1% w / v or about 0.2%. In another embodiment, the organic co-solvent is polysorbate 80, which is in a concentration of 0.1% ± 0.05% w / v or about 0.1% w / v. In one embodiment, the organic co-solvent
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6/130 is polysorbate 20, which is at a concentration of 0.2% ± 0.1% w / v or about 0.2%. In another embodiment, the organic co-solvent is polysorbate 20, which is in a concentration of 0.1% ± 0.05% w / v or about 0.1% w / v.
[0014] In certain modalities, the stabilizer is a sugar. In one embodiment, sugar is sucrose. In several modalities, the stabilizer is in a concentration from 1% ± 0.2% w / v to 20% ± 4% w / v, from 5% ± 1% w / v to 15% ± 3% w / v v or from 1% ± 0.2% to 10% ± 2% w / v. In one embodiment, the stabilizer is sucrose at a concentration of 5% ± 1% w / v or about 5% w / v. In another embodiment, the stabilizer is sucrose at a concentration of 9% ± 1.8% w / v or about 9% w / v. In another embodiment, the stabilizer is sucrose at a concentration of 10% ± 2% w / v or about 10% w / v.
[0015] In one embodiment, the viscosity modifier is an amino acid. In one embodiment, the viscosity modifier is L-proline. In certain embodiments, the viscosity modifier is in a concentration from 1% ± 0.2% to 5% ± 1% w / v. In one embodiment, the viscosity modifier is proline at a concentration of 1.5% ± 0.3% or about 1.5%. In one embodiment, the viscosity modifier is proline at a concentration of 3% ± 0.6% or about 3%.
[0016] In certain embodiments, the viscosity of the liquid pharmaceutical formulation at 25 ° C is less than or equal to about 15 cPoise ± 10%. In certain embodiments, the viscosity at 25 ° C is between 1.0 cPoise ± 10% and 20 cPoise ± 10%. In certain embodiments, the viscosity of the liquid pharmaceutical formulation is <15 cPoise. In certain embodiments, the viscosity of the liquid pharmaceutical formulation is <20 cPoise. In certain modalities, the
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7/130 viscosity of the liquid pharmaceutical formulation is <10 cPoise. In certain embodiments, the viscosity at 25 ° C is 5 cPoise ± 10%, 6.0 cPoise ± 10%, 7.0 cPoise ± 10%, 7.1 cPoise ± 10%, 7.2 cPoise ± 10%, 7 , 9 cPoise ± 10%, 8.3 cPoise ± 10%, 9.0 cPoise ± 10%, 9.6 cPoise ± 10%, 10.0 cPoise ± 10%, 10.6 cPoise ± 10%, 11.4 cPoise ± 10%,
11.6 cPoise ± 10%, 11.8 cPoise ± 10%, 12.0 cPoise ± 10%, 13.0 cPoise ± 10%, 14.0 cPoise ± 10%, 15.0 cPoise ± 10% or 16 cPoise ± 10%. [0017] In one aspect, a stable low viscosity liquid pharmaceutical formulation is provided comprising: (i) from 5 ± 0.75 mg / ml to 250 ± 37.5 mg / ml of a human antibody that binds specifically to human PD-1; (ii) histidine buffer at from 0 mM to 40 ± 8 mM; (iii) polysorbate 80 in from 0% to 0.5% ± 0.25% (w / v); (iv) sucrose in from 0% to 15% ± 3% (w / v); and (v) proline from 0 to 5% ± 1% at a pH of about 5.3 to about 6.7; wherein the anti-PD-1 antibody comprises a heavy chain variable region (HCVR) and a light chain variable region (LCVR), so that the HCVR / LCVR combination comprises heavy and light chain complementarity determining regions (HCDR1HCDR2 -HCDR3 / LCDR1-LCDR2-LCDR3) comprising the amino acid sequences of SEQ ID NOs: 3-4-5 / SEQ ID NOs: 6-7-8, respectively. In one embodiment, the anti-PD-1 antibody comprises a heavy chain variable region (HCVR) and a light chain variable region (LCVR) comprising an amino acid sequence of SEQ ID NO: 1 and SEQ ID NO: 2, respectively. In certain embodiments, the anti-PD1 antibody comprises an Fc region chosen from the group consisting of isotypes of human IgG 1, IgG2, IgG3 and IgG4. In one embodiment, the antibody comprises an isotype of human IgG4. In one embodiment, the antibody comprises a heavy chain comprising the amino acid sequence selected from the group consisting of SEQ ID NOs: 9 and 11; is
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8/130 chain had to comprise the amino acid sequence of SEQ ID NO: 10. In one embodiment, the antibody comprises a heavy chain which comprises the amino acid sequence of SEQ ID NO: 9 and a chain had to comprise the amino acid sequence of SEQ ID NO: 10. In one embodiment, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 11 and a light chain comprising the amino acid sequence of SEQ ID NO: 10. In one embodiment, the antibody has a molecular weight of 143 kDa ± 5 kDa.
[0018] In certain embodiments, a stable, low-viscosity liquid pharmaceutical formulation is provided that comprises: (i) from 5 ± 0.75 mg / mL to 250 ± 37.5 mg / mL of a human antibody that specifically binds to human PD-1; (ii) histidine buffer at from 0 mM to 40 ± 8 mM; (iii) polysorbate 80 in from 0% to 0.5% ± 0.25% (w / v); (iv) sucrose in from 0% to 15% ± 3% (w / v); and (v) proline from 0 to 5% ± 1% at a pH of about 5.3 to about 6.7; wherein the anti-PD ~ 1 antibody comprises an HCVR and an LCVR, where the HCVR has 90% sequence identity with SEQ ID NO: 1 and / or the LCVR has 90% sequence identity with SEQ ID NO: 2. In one embodiment, the anti-PD-1 antibody comprises an HCVR which comprises the amino acid sequence of SEQ ID NO: 1 and an LCVR which comprises the amino acid sequence of SEQ ID NO: 2. In one embodiment, the antibody anti-PD-1 comprises a heavy chain comprising the amino acid sequence selected from the group consisting of SEQ ID NOs: 9 and 11; and a light chain comprising the amino acid sequence of SEQ ID NO: 10.
[0019] In certain embodiments, a stable, low-viscosity liquid pharmaceutical formulation is provided comprising: (i) from 5 ± 0.75 mg / mL to 250 ± 37.5 mg / mL of a human antibody
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9/130 which specifically binds to human PD-1; (ii) histidine buffer at from 0 mM to 40 ± 8 mM; (ii) polysorbate 80 in from 0% to 0.5% ± 0.25% (w / v); (iv) sucrose in from 0% to 15% ± 3% (w / v); and (v) proline from 0 to 5% ± 1% at a pH of about 5.3 to about 6.7; where the anti-PD-1 antibody comprises an HCVR and an LCVR, where the HCVR comprises an amino acid sequence of SEQ ID NO: 1 which has no more than five amino acid substitutions and where the LCVR comprises an amino acid sequence of SEQ ID NO: 2 which has no more than two amino acid substitutions. In one embodiment, the anti-PD-1 antibody comprises an HCVR comprising the amino acid sequence of SEQ iD NO: 1 and an LCVR comprising the amino acid sequence of SEQ ID NO: 2. In one embodiment, the anti- PD-1 comprises a heavy chain comprising the amino acid sequence selected from the group consisting of SEQ ID NOs: 9 and 11; and a light chain comprising the amino acid sequence of SEQ ID NO: 10.
[0020] In certain modalities, the formulation of any of the previous aspects has an attribute selected from the group consisting of: (i) the formulation is stable to long-term storage at 25 ° C, 5 ° C, -20 ° C -30 ° C and -80 ° C, as described here; (ii) the formulation is stable to agitation stress as described here; (ii) the formulation is of low viscosity (viscosity less than 20cPoise, preferably less than 15 ePoise); (iii) the formulation is stable, even with variation of up to ± 50% in the formulation's excipient concentrations, as described here; (iv) the formulation is iso-osmolar for physiological conditions; (v) the formulation is stable and compatible with the devices and procedures for intravenous administration; and (vi) the formulation is stable to long-term storage in a glass bottle or filled syringe.
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[0021] In certain embodiments of this aspect, a stable liquid formulation is provided which comprises: (i) from 5 ± 0.75 mg / ml to 250 ± 37.5 mg / ml of a human antibody that specifically binds to Human PD-1; (ii) from 5 mM ± 1 mM to 20 ± 4 mM histidine buffer; (iii) polysorbate 80 in from 0.05% ± 0.025% to 0.3% ± 0.15% (w / v); (iv) sucrose in from 1% ± 0.2% to 10% ± 2% (w / v); and (v) proline from 1% ± 0.2% to 5% ± 1% at a pH of about 6.0, wherein the antibody comprises an HCVR / LCVR comprising a pair of SEQ amino acid sequences ID Nos: 1/2. In one embodiment, the stable liquid formulation of this aspect has a viscosity of less than 15 cP. In one embodiment,> 90% of the antibodies have a molecular weight of 143 kDa ± 1 kDa. In one embodiment, the pharmaceutical formulation has a viscosity of less than 20 cP, less than 15 cP or less than 10 cP. In one embodiment, more than 96% of antibodies have a native conformation when stored for 12 months at 5 ° C. In one embodiment, at least 97% or more of the antibodies have a native conformation when stored at -80 ° C, - 30 ° C and / or -20 ° C for 6 months.
[0022] In one such embodiment, the stable liquid formulation comprises (i) 25 ± 3.75 mg / ml of an anti-PD-1 antibody; (ii) 10 ± 2 mM histidine buffer; (iii) polysorbate 80 at 0.2% ± 0.1% (w / v) 80; (iv) proline at 1.5% ± 0.3% (w / v); and (v) 5% ± 1% (w / v) sucrose at a pH 6.0 ± 0.3, where the antibody comprises an HCVR / LCVR comprising a pair of amino acid sequences of SEQ ID NOs: 1 /2.
[0023] In one embodiment of this aspect, the stable liquid formulation comprises (i) 25 ± 3.75 mg / ml of an anti-PD-1 antibody; (ii) 4.8 mM ± 0.96 mM Lhistidine; (iii) L-histidine monohydrate monohydrate at 5.2 mM ± 1.04 mM; (iv) polysorbate 80 at 0.2% ± 0.1% (w / v);
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11/130 (v) 1.5% ± 0.3% proline (w / v); and (vi) 5% ± 1% (w / v) sucrose at a pH 6.0 ± 0.3, wherein the antibody comprises an HCVR / LCVR comprising a pair of amino acid sequences of SEQ ID NOs: 1 /2.
[0024] In one embodiment of this aspect, the stable liquid formulation comprises (i) 50 ± 7.5 mg / ml of an anti-DP-1 antibody; (ii) 10 ± 2 mM histidine buffer; (iii) polysorbate 80 at 0.2% ± 0.1% (w / v) 80; (iv) proline at 1.5% ± 0.3% (w / v); and (v) 5% ± 1% (w / v) sucrose at a pH 6.0 ± 0.3, where the antibody comprises an HCVR / LCVR comprising a pair of amino acid sequences of SEQ ID NOs: 1 /2. In one embodiment of this particular formulation, the viscosity is less than 10 cPoise.
[0025] In one embodiment of this aspect, the stable liquid formulation comprises (i) 50 ± 7.5 mg / ml of an anti-PD-1 antibody; (ii) 4.8 mM ± 0.96 mM Lhistidine; (iii) L-histidine monohydrate monohydrate at 5.2 mM ± 1.04 mM; (iv) polysorbate 80 at 0.2% ± 0.1% (w / v) 80; (v) 1.5% ± 0.3% proline (w / v); and (vi) 5% ± 1% (w / v) sucrose at a pH 6.0 ± 0.3, where the antibody comprises an HCVR / LCVR comprising a pair of amino acid sequences of SEQ ID NOs: 1 /2.
[0026] In one embodiment, the stable liquid formulation comprises (i) 100 ± 15 mg / ml of an anti-PD-1 antibody; (ii) 10 ± 2 mM histidine buffer; (iii) polysorbate 80 at 0.2% ± 0.1% (w / v) (w / v) 80; (iv) proline at 1.5% ± 0.3% (w / v); and (v) 5% ± 1% (w / v) sucrose at a pH 6.0 ± 0.3, where the antibody comprises an HCVR / LCVR comprising a pair of amino acid sequences of SEQ ID NOs: 1 /2. In one embodiment of this particular formulation, the viscosity is less than 10 cPoise.
[0027] In such an embodiment, the stable liquid formulation comprises (i) 100 ± 15 mg / ml of an anti-PD-1 antibody; (ii) L
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12/130 histidine at 4.8 mM ± 0.96 mM; (iii) L-histidine monohydrate monohydrate at 5.2 mM ± 1.04 mM; (iv) polysorbate 80 at 0.2% ± 0.1% (w / v) 80; (v) 1.5% ± 0.3% proline (w / v); and (vi) 5% ± 1% (w / v) sucrose at a pH 6.0 ± 0.3, wherein the antibody comprises an HCVR / LCVR comprising a pair of amino acid sequences of SEQ ID NOs: 1 /2.
[0028] In one embodiment, the stable liquid formulation comprises (i) 150 ± 22.5 mg / ml of an anti-PD-1 antibody; (ii) 10 ± 2 mM histidine buffer; (iii) polysorbate 80 at 0.2% ± 0.1% (w / v) 80; (iv) 10% ± 2% (w / v) sucrose; and (v) 1.5% ± 0.3% (w / v) proline at a pH 6.0 ± 0.3, where the antibody comprises an HCVR / LCVR comprising a pair of SEQ amino acid sequences ID NOs: 1/2. In one embodiment of this particular formulation, the viscosity is less than 20 cPoise, preferably less than 15 cPoise.
[0029] In one embodiment of this aspect, the stable liquid formulation comprises (i) 150 ± 22.5 mg / ml of an anti-PD-1 antibody; (ii) 4.8 mM L-histidine ± 0.96 mM; (iii) L-hlstidine monohydrate monohydrate at 5.2 mM ± 1.04 mM; (iv) polysorbate 80 at 0.2% ± 0.1% (w / v) 80; (v) 1.5% ± 0.3% proline (w / v); and (vi) 5% ± 1% (w / v) sucrose at a pH 6.0 ± 0.3, where the antibody comprises an HCVR / LCVR comprising a pair of amino acid sequences of SEQ ID NOs: 1 /2.
[0030] In such an embodiment, the stable liquid formulation comprises (i) 175 ± 26.25 mg / ml of an anti-PD-1 antibody; (ii) 10 ± 2 mM histidine buffer; (iii) polysorbate 80 at 0.2% ± 0.1% (w / v) 80; (iv) sucrose at 5% ± 1% (w / v); and (v) 1.5% ± 0.3% (w / v) proline at a pH 6.0 ± 0.3, where the antibody comprises an HCVR / LCVR comprising a pair of SEQ amino acid sequences ID NOs: 1/2. In one embodiment of this particular formulation, the viscosity is less than 20 cPoise, preferably less than 15
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13/130 ePoise.
[0031] In one embodiment of this aspect, the stable liquid formulation comprises (i) 175 ± 26.25 mg / ml of an anti-PD-1 antibody; (li) 4.8 mM L-histidine ± 0.96 mM; (iii) L-histidine monohydrate monohydrate at 5.2 mM ± 1.04 mM; (Iv) polysorbate 80 at 0.2% ± 0.1% (w / v) 80; (v) 1.5% ± 0.3% proline (w / v); and (vi) 5% ± 1% (w / v) sucrose at a pH 6.0 ± 0.3, where the antibody comprises an HCVR / LCVR comprising a pair of amino acid sequences of SEQ ID NOs: 1 /2.
[0032] In one embodiment of this aspect, the stable liquid formulation comprises (i) 200 ± 30.00 mg / ml of an anti-PD-1 antibody; (ii) 10 ± 2 mM histidine buffer; (iii) polysorbate 80 at 0.2% ± 0.1% (w / v) 80; (iv) sucrose at 5% ± 1% (w / v); and (v) 1.5% ± 0.3% (w / v) proline at a pH 6.0 ± 0.3, where the antibody comprises an HCVR / LCVR comprising a pair of SEQ amino acid sequences ID NOs: 1/2. In one embodiment of this particular formulation, the viscosity is less than 20 cPoise.
[0033] In one embodiment of this aspect, the stable liquid formulation comprises (i) 200 ± 30.00 mg / ml of an anti-DP-1 antibody; (ii) 4.8 mM L-histidine ± 0.96 mM; (iii) L-histidine monohydrate monohydrate at 5.2 mM ± 1.04 mM; (iv) polysorbate 80 at 0.2% ± 0.1% (w / v) 80; (v) 1.5% ± 0.3% proline (w / v); and (vi) 5% ± 1% (w / v) sucrose at a pH 6.0 ± 0.3, where the antibody comprises an HCVR / LCVR comprising a pair of amino acid sequences of SEQ ID NOs: 1 /2.
[0034] In one embodiment, after storing the formulation at 45 ° C for 28 days,> 90% of the antibody is native and> 35% of the antibody is in the main charge form. In one embodiment, after storing the formulation at 25 ° C for three months,> 94% of the antibody is native and> 44% of the antibody is in charge form
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Main 14/130. In one embodiment, after storing the formulation at 5 ° C for 12 months,> 96% of the antibody is native and> 50% of the antibody is in the main charge form. In one embodiment, after storing the formulation at 20 ° C for 12 months,> 96% of the antibody is native and> 40% of the antibody is in the main charge form. In one embodiment, after storing the formulation at 30 ° C for 12 months,> 96% of the antibody is native and> 40% of the antibody is in the main charge form. In one embodiment, after storing the formulation at -80 ° C for 12 months,> 96% of the antibody is native and> 40% of the antibody is in the main charge form. In one embodiment, more than 96% of antibodies have a native conformation when stored for 12 months at 5 ° C. In one embodiment, at least 97% or more of the antibodies have a native conformation when stored at -80 ° C, -30 ° C and / or -20 ° C for 6 months.
[0035] In one aspect, the present invention provides a stable liquid formulation comprising: (i) up to 100 mg / ml of an anti-DP-1 antibody: (ii) histidine buffer in from 2 mM ± 0, 4 mM to 20 mM ± 4 mM; (iii) sucrose up to 20% ± 4% (w / v); and (iv) polysorbate up to 0.2% ± 0.1% w / v at a pH 6.0 ± 0.3. In one embodiment, the stable liquid formulation comprises 25 mg / mL of anti-PD-
1. In one embodiment, the stable liquid formulation comprises 50 mg / ml of anti-PD-1 antibody. In one embodiment, the stable liquid formulation comprises 75 mg / ml of anti-PD-1 antibody. In one embodiment, the stable liquid formulation comprises 10 mM ± 2 mM histidine buffer. In one embodiment, the stable liquid formulation comprises 5% sucrose. In one embodiment, the stable liquid formulation comprises 6% sucrose. In one embodiment, the stable liquid formulation comprises 9% sucrose. In one mode, the strength
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15/130 stable liquid mulation comprises 10% sucrose. In one embodiment, the stable liquid formulation comprises 0.1% polysorbate. In one embodiment, the polysorbate is polysorbate 80 or polysorbate
20. In one embodiment, the anti-PD-1 antibody comprises an HCVR / LCVR of SEQ ID NOs: 1/2.
[0036] In one aspect, a stable liquid pharmaceutical formulation of any of the above is provided in a container. In one embodiment, the container is a polycarbonate bottle. In one embodiment, the container is a glass bottle. In one embodiment, the glass bottle is a type 1 borosilicate glass bottle with a fluorocarbon-coated butyl rubber stopper. In one embodiment, the container is a micro-infuser. In one embodiment, the container is a syringe. In one embodiment, the container is a filled syringe. In one embodiment, the syringe comprises a fluorocarbon-coated plunger. In certain embodiments, the syringe is a 1 ml or 2.25 ml glass syringe containing less than about 500 parts per billion tungsten, equipped with a 27-G needle, a fluorocarbon-coated butyl rubber stopper and a latex-free rubber needle protector, non-cytotoxic. In one embodiment, the syringe is a 1 mL glass syringe fitted with a 27 G thin-walled needle, a FLUROTEC-coated 4023/50 rubber stopper and an FM 27 rubber needle protector. In one embodiment, the syringe is a 1 ml, 2 ml, 3 ml, 5 ml or 10 ml plastic syringe with a needle.
[0037] In one aspect, a kit is provided which comprises a stable pharmaceutical composition of any of the above, a container and instructions. In one embodiment, the container is a glass bottle. In one embodiment, the container is a filled syringe. In one embodiment, the syringe is a 1 ml or 2.25 ml glass syringe fitted with a 27 G thin-walled needle, a
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16/130 4023/50 rubber coated with FLUROTEC and a rubber needle protector FM 27. In one embodiment, the syringe is a 1 mL, 2 mL, 3 mL, 5 mL or 10 mL plastic syringe with a needle .
[0038] In certain embodiments, the present invention provides a filled syringe comprising a stable liquid pharmaceutical formulation comprising: (i) from 5 ± 0.75 mg / mL to 250 ±
37.5 mg / ml of a human antibody that specifically binds to human PD 1; (ii) histidine buffer at from 5 mM ± 1 mM to 20 ± 4 mM; (iii) polysorbate 80 in from 0.05% ± 0.025% to 0.3% ± 0.15% (w / v); (iv) sucrose in from 1% ± 0.2% to 10% ± 2% (w / v); and (v) proline from 1% ± 0.2% to 5% ± 1% at a pH 6.0 ± 0.3, where the antibody comprises an HCVR / LCVR comprising a pair of amino acid sequences SEQ ID NOs: 1/2; where the formulation has an attribute selected from the group consisting of:
(i) 98% of the antibody is in its native form after storage at 5 ° C for 12 months; (ii)> 53% of the antibody is the main loading variant after storage at 5 ° C for 12 months; (iii)> 97% of the antibody is in its native form after storage at 25 ° C for 6 months; (iv) the formulation is stable to agitation stress, where> 98% of the antibody is in its native form after 120 minutes of agitation stress in the filled syringe; (v) more than 90% of the antibodies have a molecular weight of 143 kDa ± 1 kDa; (vi) the pharmaceutical formulation has a viscosity of less than 20 cP, less than 15 cP or less than 10 cP; (vii) more than 96% of the antibodies have a native conformation when stored for 12 months at 5 ° C; and (viii) at least 97% or more of the antibodies have a native conformation when stored at ~ 80 ° C, ~ 30 ° C and / or -20 ° C for 6 months. [0039] In certain embodiments, the present invention provides a glass vial comprising a stable liquid pharmaceutical formulation comprising: (i) from 5 ± 0.75 mg / ml to 250 ±
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37.5 mg / ml of a human antibody that specifically binds to human PD1; (ii) histidine buffer at from 5 mM ± 1 mM to 20 ± 4 mM; (iii) polysorbate 80 in from 0.05% ± 0.025% to 0.3% ± 0.15% (w / v); (iv) sucrose in from 1% ± 0.2% to 10% ± 2% (w / v); and (v) proline from 1% ± 0.2% to 5% ± 1% at a pH 6.0 ± 0.3, where the antibody comprises an HCVR / LCVR comprising a pair of amino acid sequences SEQ ID Nos: 1/2; where the formulation has an attribute selected from the group consisting of:
(i) the formulation is stable to storage and stress in a glass bottle; (ii) the formulation is stable and compatible for use in IV delivery devices; (Hi) the formulation is chemically and physically stable to dilution with standard diluents known in the art (for example, 0.9% sodium chloride or 5% dextrose); (iv) the formulation is stable to IV bags made of glass or polymeric plastics (for example, polyvinyl chloride, phthalates, polyolefins or polypropylene); (v) the formulation is compatible with standard infusion pumps (for example, peristaltic pump, fluid displacement pump); (vi)> 90% of the antibodies have a molecular weight of 143 kDa ± 1 kDa; (vii) the pharmaceutical formulation has a viscosity of less than 20 cP, less than 15 cP or less than 10 cP; (viii) more than 96% of the antibodies have a native conformation when stored for 12 months at 5 ° C; and (ix) at least 97% or more of the antibodies have a native conformation when stored at -80 ° C, -30 ° C and / or 20 ° C for 6 months.
[0040] Other modalities will become evident from a review of the detailed description below.
BRIEF DESCRIPTION OF THE FIGURES
[0041] Figure 1 is a table showing the effect of pH on the stability of 150 mg / mL of mAb1 Incubated at 45 ° C for 28 days. ^a Os
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18/130 SE-UPLC and CEX-UPLC results for 'Raw Material' are the average values of Raw Material for all formulations.
[0042] Figure 2 shows the storage stability of three formulations F1, F2 and F3, in which F1 comprises 210 mg / mL of mAb1, 10 mM histidine and 3% proline at pH 6.0; F2 comprises 210 mg / mL of mAb1, 10 mM histidine and 3% sucrose at pH 6.0; and F3 comprises 210 mg / ml of mAb1, 10 mM histidine and 5% sucrose at pH 6.0. Storage stability is measured by% of high molecular weight (HMW) species generated when stored at -80 ° C (A), -30 ° C (B) and -20 ° C (C) for up to 9 months and analyzed by size exclusion chromatography (SEC).
[0043] Figure 3 shows the storage stability of three formulations F1, F2 and F3, where F1 comprises 150 mg / ml mAb1, 10 mM histidine, 9% sucrose and 0.2 polysorbate (PS80) % at pH 6.0; F2 comprises 175 mg / ml of mAb1, 10 mM histidine, 3% proline and 0.2% PS80 at pH 6.0; and F3 comprises 175 mg / ml mAb1, 10 mM histidine, 5% sucrose, 1.5% proline and 0.2% PS80 at pH 6.0. Storage stability is measured by the% of high molecular weight species (HMW) generated when stored at -80 ° C (A), -30 ° C (B) and -20 ° C (C) for up to 6 months and analyzed by size exclusion chromatography (SEC).
[0044] Figure 4 is a table showing viscosity of 150 mg / mL of mAb1 with the addition of excipients and viscosity modifiers.
[0045] Figure 5 is a table showing the effect of viscosity modifiers on the stability of 175 mg / ml of mAb1 incubated at 45 ° C for 14 days. ^a The pH, SE-UPLC and CEXUPLC results for 'Raw material' are the average values of the Raw material for
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19/130 all formulations,
DETAILED DESCRIPTION
[0046] Before the present methods are described, it should be understood that the present invention is not limited to the particular experimental methods and conditions described, since these methods and conditions may vary. It should also be understood that the terminology used here is intended to describe only particular modalities, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.
[0047] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those versed in the technique to which the present invention belongs. As used here, the term about ”, when used in reference to a specific numerical value or range of values cited, means that the value may vary from the value quoted by no more than 1%. For example, as used here, the expression about 100 includes 99 and 101 and all intermediate values (for example, 99,1,99,2, 99,3, 99,4, etc.). Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are now described. All publications mentioned here are incorporated by reference in their entirety.
[0048] As used herein, the term pharmaceutical formulation means a combination of at least one active ingredient (for example, a small molecule, macromolecule, compound, etc., which is capable of exerting a biological effect on a human or an non-human animal) and at least one inactive ingredient which, when combined with the active ingredient or one or more additional inactive ingredients, is suitable for therapeutic administration to a human or non-human animal. The term formulation, as used
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20/130 here, means pharmaceutical formulation, unless otherwise indicated. The present invention provides pharmaceutical formulations that comprise at least one therapeutic polypeptide. According to certain embodiments of the present invention, the therapeutic polypeptide is an antibody or antigen-binding fragment thereof that specifically binds to human programmed death protein-1 (PD-1). More specifically, the present invention includes pharmaceutical formulations that comprise: (i) a human antibody that specifically binds to human PD-1 (li) a histidine buffer; (iii) an organic cosolvent that is a non-ionic surfactant; (iv) a stabilizer that is a carbohydrate; and, optionally, (v) a viscosity modifier that is an amino acid. The formulations included in the present invention are described in detail below.
ANTIBODIES THAT SPECIFICALLY CONNECT TO PD-1
[0049] The pharmaceutical formulations of the present invention can comprise a human antibody or antigen-binding fragment thereof that specifically binds to human PD-1. As used here, the term PD-Γ means human programmed death protein-1. Anti-human PD-1 antibodies are described, for example, in United States Patents / Publications 8008449, 8168757, 20110008369, 20130017199, 20130022595, 20150203579 and in documents W02006121168, W02009114335, WO2012145493, WO2013 0146628, W0200910 and WO2013 EP2504028. [0050] The term antibody, as used here, generally refers to immunoglobulin molecules that comprise four polypeptide chains, two heavy (H) and two light (L) bitches interconnected by disulfide bonds, as well as multimers thereof (for example) , IgM); however, immunoglobulin molecules that consist only of heavy chains (that is, they lack light chains) are also included in the definition of the term antibody. Each chain
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21/130 heavy comprises a heavy chain variable region (abbreviated here as HCVR or Vh) and a heavy chain constant region. The heavy chain constant region comprises three domains, CH1, CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated here as LCVR or Vl) and a light chain constant region. The light chain constant region comprises a domain (CL1). The Vh and Vl regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDR), interspersed with regions that are called more conserved structural regions (FR). Each Vh and Vl is composed of three CDRs and four FRs positioned, from the amino terminal to the carboxyl terminal, in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
[0051] Unless specifically indicated otherwise, the term antibody, as used herein, is to be understood as encompassing complete antibody molecules, as well as antigen-binding fragments thereof. The term antigen binding portion or antigen binding fragment of an antibody (or simply antibody portion or antibody fragment), as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to human PD-1 or an epitope thereof.
[0052] An isolated antibody, as used here, is intended to refer to an antibody substantially free of other antibodies that have different antigenic specificities (for example, an isolated antibody that specifically binds to human PD-1 is substantially free of antibodies that specifically bind to antigens other than human PD-1).
[0053] The term specifically binds or similar means that a
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22/130 antibody or antigen-binding fragment likewise forms a complex with an antigen that is relatively stable under physiological conditions. The specific bond can be characterized by a dissociation constant of at least about 1x10 ⁸ M or greater. Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance and so on. An isolated antibody that specifically binds to human PD-1 may, however, cross-react with other antigens, such as PD-1 molecules from other species (orthologists). In the context of the present invention, multispecific (e.g., bispecific) antibodies that bind to human PD-1, as well as to one or more additional antigens, are considered to bind specifically to human PD-1. In addition, an isolated antibody can be substantially free of other cellular material or chemicals.
[0054] Examples of anti-human PD-1 antibodies that can be included in the pharmaceutical formulations of the present invention are presented in Patent Application Publications US20150203579, WO2015112800 and whose descriptions are incorporated by reference in full.
[0055] According to certain embodiments of the present invention, the human anti-PD-1 antibody or antigen binding fragment thereof comprises a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 3, an HCDR2 of SEQ ID NO: 4 and an HCDR3 of SEQ ID NO: 5. In certain embodiments, the human anti-PD-1 antibody or antigen binding fragment thereof comprises an HCVR of SEQ ID NO: 1.
[0056] In accordance with certain embodiments of the present invention, the human anti-PD-1 antibody or antigen-binding fragment
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The genus of the same comprises a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 6, an LCDR2 of SEQ ID NO: 7 and an LCDR3 of SEQ ID NO: 8. In certain embodiments, the antibody human anti-PD-1 or antigen binding fragment thereof comprises an LCVR of SEQ ID NO: 2.
[0057] According to certain embodiments of the present invention, the human anti-PD-1 antibody or antigen binding fragment thereof comprises an HCVR that has 90%, 95%, 98% or 99% sequence identity with SEQ ID NO: 1.
[0058] According to certain embodiments of the present invention, the human anti-PD-1 antibody or antigen binding fragment thereof comprises an LCVR that has 90%, 95%, 98% or 99% sequence identity with SEQ ID NO: 2.
[0059] According to certain embodiments of the present invention, the human anti-PD-1 antibody or antigen binding fragment thereof comprises an HCVR comprising an amino acid sequence of SEQ ID NO: 1 which has no more than 5 substitutions of amino acids.
[0060] In accordance with certain embodiments of the present invention, the human anti-PD-1 antibody or antigen binding fragment thereof comprises an LCVR comprising an amino acid sequence of SEQ ID NO: 2 which has no more than 2 amino acid substitutions.
[0061] Sequence identity can be measured using any method known in the art (for example, GAP, BESTFIT and BLAST).
[0062] The present invention also includes formulations that comprise anti-PD-1 antibodies, wherein the anti-PD-1 antibodies comprise variants of any of the amino acid sequences of
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HCVR, LCVR and / or CDR described here that have one or more conservative amino acid substitutions. For example, the present invention includes formulations that comprise anti-PD-1 antibodies that have HCVR, LCVR and / or CDR amino acid sequences with, for example, 10 or less, 8 or less, 6 or less, 4 or less, etc. amino acid substitutions with respect to any of the HCVR, LCVR and / or CDR amino acid sequences described here.
[0063] In certain embodiments, the anti-PD1 antibody comprises an Fc region chosen from the group consisting of the human IgG1, IgG2, IgG3 and IgG4 isotypes.
[0064] The exemplary non-limitative antibody used in the Examples here is termed as mAb1. This antibody is also cited in US 20150203579 as H2M7798N or H4H7798N and is also known as ^Si REGN2810 ^Si or cemiplimab. MAb1 (H4H7798N) comprises a pair of HCVR / LCVR amino acid sequences that have SEQ ID NOs: 1/2 and HCDR1-HCDR2HCDR3 / LCDR1-LCDR2-LCDR3 domains represented by SEQ ID NOs: 3-55 / SEQ ID NOs: 6 -7-8.
[0065] In accordance with certain embodiments of the present invention, the human anti-PD-1 antibody or antigen binding fragment thereof comprises a heavy chain of SEQ ID NO: 9 and a light bitch of SEQ ID NO: 10.
[0066] It is well known in the art that amino acid terminal dividing can occur during antibody production (see, for example, Wang et al. 2007, J. Pharma Sci 96: 1-26). Therefore, in certain embodiments, the anti-PD-1 antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 11. SEQ ID NO: 11 comprises the amino acid sequence heavy chain in which the C-terminal lysine is absent from the amino acid sequence of SEQ ID NO:
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9. In certain embodiments, the formulations of the present invention contain about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98% or more of the antibody antiPD-1 in which C-terminal lysine is absent.
[0067] The amount of antibody or antigen-binding fragment thereof contained in the pharmaceutical formulations of the present invention may vary depending on the specific properties desired from the formulations, as well as the specific circumstances and purposes for which the formulations are intended to be used. In certain embodiments, pharmaceutical formulations are liquid formulations that can contain 5 ± 0.75 mg / mL to 250 ± 37.5 mg / mL of antibody; 10 ± 1.5 mg / ml to 240 ± 36 mg / ml of antibody; 20 ± 3.0 mg / ml to 230 ± 34.5 mg / ml of antibody; 25 ± 3.75 mg / ml to 240 ± 36 mg / ml of antibody; 50 ± 7.5 mg / ml to 230 ± 34.5 mg / ml of antibody; 60 ± 9 mg / ml to 240 ± 36 mg / ml of antibody; 70 ± 10.5 mg / ml to 230 ± 34.5 mg / ml of antibody; 80 ± 12 mg / ml to 220 ± 33 mg / ml of antibody; 90 ± 13.5 mg / ml to 210 ± 31.5 mg / ml of antibody; 100 ± 15 mg / ml to 200 ± 30 mg / ml of antibody; 110 ± 16.5 mg / ml to 190 ± 28.5 mg / ml of antibody; 120 ± 18 mg / ml to 180 ± 27 mg / ml of antibody; 130 ± 19.5 mg / ml to 170 ± 25.5 mg / ml of antibody; 140 ± 21 mg / ml to 160 ± 24 mg / ml of antibody; 150 ± 22.5 mg / ml of antibody; or 175 ± 26.25 mg / ml For example, the formulations of the present invention can comprise about 5 mg / ml; about 10 mg / ml; about 15 mg / ml; about 20 mg / ml; about 25 mg / ml; about 30 mg / ml; about 35 mg / ml; about 40 mg / ml; about 45 mg / ml; about 50 mg / ml; about 55 mg / ml; about 60 mg / ml; about 65 mg / ml; about 70 mg / ml; about 75 mg / ml; about 80 mg / ml; about 85 mg / ml; about 90 mg / ml; about 95 mg / ml; about 100 mg / ml; about 105 mg / ml; about 110 mg / ml; about 115 mg / ml; about 120 mg / ml; about 125 mg / ml; about 130 mg / ml; about 135 mg / ml;
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26/130 about 140 mg / ml; about 145 mg / ml; about 150 mg / ml; about 155 mg / ml; about 160 mg / ml; about 165 mg / ml; about 170 mg / ml; about 175 mg / ml; about 180 mg / ml; about 185 mg / ml; about 190 mg / ml; about 195 mg / ml; about 200 mg / ml; about 205 mg / ml; about 210 mg / ml; about 215 mg / ml; about 220 mg / ml; about 225 mg / ml; about 230 mg / ml; about 235 mg / ml; about 240 mg / ml; about 245 mg / ml; or about 250 mg / ml of an antibody or antigen-binding fragment thereof that specifically binds to human PD-1.
EXCIPIENTS AND pH
[0068] The pharmaceutical formulations of the present invention comprise one or more excipients. The term excipient, as used herein, means any non-therapeutic agent added to the formulation to impart a desired consistency, viscosity or stabilizing effect,
[0069] In certain embodiments, the pharmaceutical formulation of the invention comprises at least one organic cosolvent in a type and in an amount that stabilizes the anti-human PD-1 antibody under conditions of rough agitation or manipulation such as, for example, vortexing. In some embodiments, what is meant by stabilization is the prevention of the formation of more than 3% of aggregated antibody of the total amount of antibody (on a molar basis) during the course of gross manipulation. In some embodiments, rough handling is to vortex a solution containing the antibody and organic co-solvent for about 60 minutes or about 120 minutes. [0070] In certain embodiments, the organic co-solvent is a non-ionic surfactant, such as an alkyl (poly) ethylene oxide. Specific nonionic surfactants that can be included in the formulations of the present invention include, for example, polysorbates, such as polysorbate 20, polysorbate 28, polysorbate 40, polysorbate 60,
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27/130 polysorbate 65, polysorbate 80, polysorbate 81, polysorbate 81 and polysorbate 85; poloxamers, such as poloxamer 181, poloxamer 188, poloxamer 407; or polyethylene glycol (PEG). Polysorbate 20 is also known as TWEEN 20, sorbitan monolaurate and polloxyethylene sorbitan monolaurate. Poloxamer 188 is also known as PLURONIC F68.
[0071] The amount of non-ionic surfactant contained in the pharmaceutical formulations of the present invention may vary depending on the specific properties desired from the formulations, as well as the specific circumstances and purposes for which the formulations are to be used. In certain embodiments, formulations may contain 0.01% ± 0.005% to 0.5% ± 0.25% surfactant. For example, the formulations of the present invention can comprise about 0.005%; about 0.01%; about 0.02%; about 0.03%; about 0.04%; about 0.05%; about 0.06%; about 0.07%; about 0.08%; about 0.09%; about 0.1%; about 0.11%; about 0.12%; about 0.13%; about 0.14%; about 0.15%; about
0.16%; about 0.17%; about 0.18%; about 0.19%; about
0.20%; about 0.21%; about 0.22%; about 0.23%; about
0.24%; about 0.25%; about 0.26%; about 0.27%; about
0.28%; about 0.29%; about 0.30%; about 0.35%; about
0.40%; about 0.45%; about 0.46%; about 0.47%; about
0.48%; about 0.49%; about 0.50%; about 0.55%; or about 0.575% polysorbate 20 or polysorbate 80.
[0072] The pharmaceutical formulations of the present invention can also comprise one or more stabilizers in a type and in an amount that stabilizes the anti-human PD-1 antibody under conditions of heat stress. In some embodiments, what is meant by stabilization is the maintenance of more than about 91% of the antibody in a native conformation when the solution containing the antibody and the
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28/130 thermal stability is maintained at about 45 ° C for up to about 28 days. In some embodiments, what is meant by stabilization is keeping less than about 6% of the antibody aggregated when the solution containing the antibody and the thermal stabilizer is kept at about 45 ° C for up to about 28 days. As used here, native means the primary form of the antibody by size exclusion, which is generally an intact antibody monomer. The native term also refers to the non-aggregated and non-degraded form of the antibody. [0073] In certain embodiments, the thermal stabilizer is a sugar, such as sucrose, the amount of which in the formulation may vary depending on the specific circumstances and the intended purposes for which the formulation is used. In certain embodiments, the formulations can contain about 1% to about 15% sugar; about 2% to about 14% sugar; about 3% to about 13% sugar; about 4% to about 12% sugar; about 5% to about 12% sugar; about 6% to about 11% sugar; about 7% to about 10% sugar; about 8% to 11% sugar; or about 9% to 11% sugar. For example, the pharmaceutical formulations of the present invention can comprise 4% ± 0.8%; 5% ±
I%; 6% ± 1.2%; 7% ± 1.4%; 8% ± 1.6%; 9% ± 1.8%; 10% ± 2%;
II% ± 2.2%; 12% ± 2.4%; 13% ± 2.6%; or about 14% ± 2.8% sugar (for example, sucrose).
[0074] The pharmaceutical formulations of the present invention can also comprise a buffer or buffer system that serves to maintain a stable pH and helps to stabilize the anti-human PD-1 antibody. The term buffer, as used herein, denotes a pharmaceutically acceptable buffer that maintains a stable pH or resists variations in the pH of the solution. In preferred embodiments, the buffer comprises histidine. In the context of the present invention, history
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29/130 dyne or buffer comprising histidine is a buffer comprising the amino acid histidine. Examples of histidine buffers include histidine chloride, histidine acetate, histidine phosphate and histidine sulfate. In a preferred embodiment, the histidine buffer is prepared by dissolving L-histidine and L-histidine hydrochloride (for example, as the monohydrate) in a defined amount and proportion. In one embodiment, the histidine buffer is prepared to titrate L-histidine (free, solid base) with dilute hydrochloric acid. The term histidine is used interchangeably with histidine buffer throughout the present invention. In some embodiments, what is meant by stabilization is where less than 4.5% ± 0.5% of the antibody is aggregated when the solution containing the antibody and the buffer is kept at about 45 ° C for up to about 28 days. In some modalities, what is meant by stabilizes is where less than 3% ± 0.5% or less than
2.5% ± 0.5% of the antibody is added when the solution containing the antibody and the buffer is kept at about 37 ° C for up to 28 days. In some modalities, what is meant by stabilizes is where at least 93% ± 0.5% or at least 94% ± 0.5% of the antibody is in its native conformation, as determined by size exclusion chromatography when the solution containing the antibody and the buffer is kept at about 45 ° C for up to about 28 days. In some modalities, what is meant by stabilizes is where at least 94% ± 0.5% or at least 95% ± 0.5% of the antibody is in its native conformation, as determined by size exclusion chromatography when the solution containing the antibody and the buffer is kept at about 37 ° C for up to about 28 days. By native or native conformation is meant the fraction of antibody that is not aggregated or degraded. This is usually determined through an assay that measures the relative size of the entity
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30/130 antibody, such as a size exclusion chromatography assay. The non-aggregated and non-degraded antibody elutes in a fraction that is equivalent to the native antibody and is usually the main elution fraction. The aggregated antibody elutes at a fraction that indicates a larger size than the native antibody. The degraded antibody elutes at a fraction that indicates a smaller size than the native antibody. [0075] In some embodiments, what is meant by stabilizes is where at least 35% ± 0.5% of the antibody is in its main charge form, as determined by means of cation exchange chromatography when the solution containing the antibody and the buffer is maintained at about 45 ° C for up to about 28 days. In some modalities, what is meant by stabilizes is where at least 46% ± 0.5% or at least 39% ± 0.5% of the antibody is in its main charge form, as determined by means of cation exchange chromatography when the solution containing the antibody and the buffer is kept at about 37 ° C for up to about 28 days. By main charge or main charge form is meant the fraction of antibody that elutes from an ion exchange resin at the main peak, which is generally flanked by more basic peaks on one side and more acidic peaks on the other side.
[0076] The pharmaceutical formulations of the present invention can have a pH of about 5.2 to about 6.4. For example, the formulations of the present invention can have a pH of about 5.5; about 5.6; about 5.7; about 5.8; about 5.9; about 6.0; about 6.1; about 6.2; about 6.3; about 6.4; or about 6.5. In some embodiments, the pH is 6.0 ± 0.4; 6.0 ± 0.3; 6.0 ± 0.2; 6.0 ± 0.1; about 6.0; or 6.0. [0077] In some embodiments, the buffer or buffer system comprises at least one buffer that has a buffering range that overlaps all or part of the pH 5.5 - 7.4 range. In
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In certain embodiments, the buffer comprises a histidine buffer. In certain embodiments, the hystidine buffer is present in a concentration of 5 mM ± 1 mM to 15 mM ± 3 mM; 6 mM ± 1.2 mM to 14 mM ± 2.8 mM; 7 mM ± 1.4 mM to 13 mM ± 2.6 mM; 8 mM ± 1.6 mM to 12 mM ± 2.4 mM; 9 m M ± 1.8 mM to 11 mM ± 2.2 mM; 10 mM ± 2 mM; or about 10 mM. In certain embodiments, the buffer system comprises 10 m M ± 2 mM hystidine at pH 6.0 ± 0.3. In preferred embodiments, the hystidine buffer comprises Lhistidine and L-hystidine monohydrate monohydrate. In one embodiment, the hystidine buffer comprises L-histidine at a concentration of 4.8 mM ± 0.96 mM. In one embodiment, the hystidine buffer comprises L-histidine monohydrate monohydrate at a concentration of 5.2 mM ± 1.04 mM. In one embodiment, the hystidine buffer comprises L-histidine at a concentration of 4.8 mM ± 0.96 mM and L-histidine monohydrate monohydrate at a concentration of 5.2 mM ± 1.04 mM.
[0078] The pharmaceutical formulations of the present invention may also comprise one or more excipients which serve to maintain a reduced viscosity or decrease the viscosity of formulations containing a high concentration of pharmaceutical anti-PD-1 antibody (for example, generally> 150 mg / ml antibody). In certain embodiments, the viscosity modifier is an amino acid. In one embodiment, the amino acid is proline. In one embodiment, the pharmaceutical formulation of the present invention contains proline, preferably as L-proline, in a concentration of 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4% , 4.5% or 5%. The term proline is used interchangeably with L-proine throughout the present invention. In some embodiments, the formulation comprises proline in an amount sufficient to maintain the viscosity of the liquid formulation at less than 20 ± 3 cPoise, less than 15 ± 2.25 cPoise or less than 11 ± 1.65
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32/130 ePoise. In some embodiments, the formulation comprises proline in an amount sufficient to maintain viscosity at or below 15 ± 2.25 cPoise. In certain embodiments, the formulations may contain about 1% to about 5% proline; about 2% to about 4% proline; or about 3% proline. For example, the pharmaceutical formulations of the present invention can comprise 1% ± 0.2% proline; 1.5% ± 0.3%; 2% ± 0.4%; 2.5% ± 0.5%; 3% ± 0.6%; 3.5% ± 0.7%; 4% ± 0.8%; 4.5% ± 0.9%; or about 5% ± 1%.
[0079] During the antibody purification process, it may be desired or necessary to exchange one buffer for another to obtain appropriate concentrations of excipient, antibody concentration, pH, etc. The buffer exchange can be carried out, for example, by means of ultrafiltration / diafiltration (UF / DF) using, for example, a semipermeable tangential flow filtration membrane. The use of such techniques, however, has the potential to cause the Gibbs-Donnan effect [Bolton ef a /., 2011, BiotechnoL Prog. 27 (1): 140-152]. The accumulation of positive charge on the product side of the membrane during protein concentration is counterbalanced electrically by the preferential movement of positive ions to the opposite side of the membrane. The potential consequence of this phenomenon is that the final concentrations of certain components (eg histidine, L-proline, etc.) may be lower than the intended target concentrations of these components due to electrostatic repulsion of the excipients from the positively charged diafiltration buffer to the antibody protein positively charged during the UF / DF step. Thus, the present invention includes formulations in which the concentration, for example, of histidine and / or L-proline, varies between the amounts or ranges cited here because of the Gibbs-Donnan effect.
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[0080] Volume exclusion describes the behavior of highly concentrated samples in which a significant portion of the total volume of the solution is absorbed by the solute, especially large molecules, such as proteins, excluding the solvent from this space. This then decreases the total volume of solvent available for other solutes to be dissolved, which can result in uneven division across the ultrafiltration membrane. Thus, the present invention includes formulations in which the concentration, for example, of histidine and / or L-proline, can vary in relation to the amounts or ranges cited here because of the volume exclusion effect.
[0081] During the manufacture of the formulations of the present invention, variations in the composition of the formulation can occur. These variations may include the concentration of the active ingredient, the concentration of the excipients and / or the pH of the formulation. Since changes in any of these parameters can potentially affect drug stability or potency, proven acceptable range (PAR) studies were conducted to assess whether variations in composition, within defined limits, would affect the stability or potency of the drug. antibody. Therefore, the present invention includes formulations that comprise anti-PD-1 antibodies that are stable and retain potency with a variation of up to 50% in the excipient concentration. For example, anti-PD-1 antibody formulations are included here in which the stability and potency of said formulations are not affected by a variation of ± 10%, ± 20%, ± 30%, ± 30%, ± 40% or ± 50% in the concentration of antibody, sucrose, histidine buffer and / or polysorbate.
STABILITY AND VISCOSITY OF PHARMACEUTICAL FORMULATIONS
[0082] The pharmaceutical formulations of the present invention typically exhibit high levels of stability. The term stable, as used here in reference to pharmaceutical formulations, means that
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34/130 antibodies in pharmaceutical formulations retain an acceptable degree of chemical structure or biological function after storage under defined conditions. A formulation can be stable even if the antibody contained in it does not retain 100% of its chemical structure or biological function after storage for a defined period of time. Under certain circumstances, maintaining about 90%, 95%, 96%, 97%, 98% or 99% of an antibody's structure or function after storage for a defined period can be considered stable ”.
[0083] Stability can be measured, inter alia, by determining the percentage of native antibody that remains in the formulation after storage for a defined period of time at a defined temperature. The percentage of native antibody can be determined, inter alia, by size exclusion chromatography (eg, ultra-performance liquid chromatography with size exclusion [SE-UPLC]), so that native means non-aggregated and not degraded. An acceptable degree of stability, as such a phrase is used here, means that at least 90% of the native form of the antibody can be detected in the formulation after storage for a defined period of time at a given temperature. In certain embodiments, at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the native form of the antibody can be detected in the formulation after storage for a defined period at a defined temperature. The defined period after which stability is measured can be at least 14 days, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months or more. THE
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35/130 defined temperature at which the pharmaceutical formulation can be stored when assessing stability can be any temperature from about ~ 80 ° C to 45 ° C, for example, storage at about -80 ° C, about -30 ° C, about -20 ° C, about 0 ° C, about 4 ° -8 ° C, about 5 ° C, about 25 ° C, about 35 ° C, about 37 ° C or about 45 ° C. For example, a pharmaceutical formulation can be considered stable if, after 6 months of storage at 5 ° C, more than about 95%, 96%, 97% or 98% of the native antibody is detected by SE-UPLC. A pharmaceutical formulation can also be considered stable if, after 6 months of storage at 25 ° C, more than about 95%, 96%, 97% or 98% of the native antibody is detected by means of SE-UPLC. A pharmaceutical formulation can also be considered stable if, after 28 days of storage at 45 ° C, more than about 89%, 90%, 91%, 92%, 93%, 94%, 95% or 96% of the native antibody are detected by means of SE-UPLC. A pharmaceutical formulation can also be considered stable if, after 12 months of storage at -20 ° C, more than about 96%, 97% or 98% of the native antibody is detected by means of SE-UPLC. A pharmaceutical formulation can also be considered stable if, after 12 months of storage at -30 ° C, more than about 96%, 97% or 98% of the native antibody is detected using SE-UPLC. A pharmaceutical formulation can also be considered stable if, after 12 months of storage at ~ 80 ° C, more than 96%, 97% or 98% of the native antibody is detected by means of SE-UPLC.
[0084] Stability can be measured, inter alia, by determining the percentage of antibody that forms in an aggregate within the formulation after storage for a defined period of time at a defined temperature, where stability is inversely proportional to the percentage of formed aggregate. The percentage of an
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36/130 aggregate antibody can be determined, inter alia, by size exclusion chromatography (eg, ultra-performance liquid chromatography with size exclusion [SE-UPLC]). An acceptable degree of stability, as this phrase is used here, means that a maximum of 5% of the antibody is in an aggregated form (also called a high molecular weight form - HMW) detected in the formulation after storage for the defined amount of time in a certain temperature. In certain embodiments, an acceptable degree of stability means that at most about 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1% of the antibody can be detected in an aggregate in the formulation after storage for a defined period of time at a specified temperature. The defined period of time after which stability is measured can be at least 2 weeks, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months or more. The temperature at which the pharmaceutical formulation can be stored when assessing stability can be any temperature from about -80 ° C to about 45 ° C, for example, storage at about -80 ° C, about - 30 ° C, about -20 ° C, about 0 ° C, about 4 ° ~ 8 ° C, about 5 ° C, about 25 ° C, about 35 ° C, about 37 ° C or about 45 ° C. For example, a pharmaceutical formulation can be considered stable if, after 12 months of storage at 5 ° C, less than 2%, 1%, 0.5% or 0.1% of the antibody is detected in aggregate form. A pharmaceutical formulation can also be considered stable if, after three months of storage at 25 ° C, less than about 4%, 3%, 2%, 1%, 0.5% or 0.1% of the antibody is detected in the aggregate form. A pharmaceutical formulation also
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37/130 can be considered stable if, after 28 days of storage at 45 ° C, less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or 0.5% of the antibody is detected in aggregate form. A pharmaceutical formulation can also be considered stable if, after three months of storage at -20 ° C, -30 ° C or -80 ° C, less than about 3%, 2%, 1%, 0.5% or 0 , 1% of the antibody is detected in an aggregated form.
[0085] Stability can be measured, inter alia, by determining the percentage of antibody that migrates in a more acidic fraction during ion exchange (acidic form) than in the main antibody fraction (main charge form), where stability it is inversely proportional to the antibody fraction in the acid form. While not wishing to be bound by theory, deactivation of the antibody can cause the antibody to be more negatively charged and therefore more acidic than the non-deamidated antibody (see, for example, Robinson, N., Protein Deamidation, PNAS, April 16, 2002, 99 (8): 5283-5288). The percentage of acidified antibody can be determined, inter alia, by means of ion exchange chromatography (for example, cation exchange ultra-performance liquid chromatography [CEX-UPLC]). An acceptable degree of stability, as such phrase is used here, means that a maximum of 45% of the antibody is in a more acidic form detected in the formulation after storage for a defined period of time at a defined temperature. In certain embodiments, an acceptable degree of stability means that at most about 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% , 0.5% or 0.1% of the antibody can be detected in an acid form in the formulation after storage for a defined period of time at a given temperature. In one embodiment, an acceptable degree of stability means that less than 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5% or
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0.1% of the antibody can be detected in an acid form in the formulation after storage for a defined period of time. The defined period after which stability is measured can be at least 2 weeks, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months or more. The temperature at which the pharmaceutical formulation can be stored when assessing stability can be any temperature from about -80 ° C to about 45 ° C, for example, storage at about 80 ° C, about -30 ° C, about -20 ° C, about 0 ° C, about 4-8 ° C, about 5 ° C, about 25 ° C or about 45 ° C. For example, a pharmaceutical formulation can be considered stable if, after three months of storage at -80 ° C, -30 ° C or -20 ° C, less than about 30%, 29%, 28%, 27%, 26 %, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1% of the antibody is in a more acidic form. A pharmaceutical formulation can also be considered stable if, after six months of storage at 5 ° C, less than 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23 %, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1% of the antibody is in a more acidic form. A pharmaceutical formulation can also be considered stable if, after six months of storage at 25 ° C, less than 43%, 42%, 41%, 40%, 39%, 38%,%, 36%, 35%, 34% , 33%, 32%, 31%, 30%, 29%, 28%, 27%,%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17% , 16%,%, 14%, 13%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1% of the antibody is in a more acidic form.
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A pharmaceutical formulation can also be considered stable if, after 28 days of storage at 45 ° C, less than about 49%, 48%, 47%, 46%, 45%, 44%, 43%, 43%, 42% , 41%, 40%, 39%,%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%,%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%,%, 15%, 14%, 13%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1% of the antibody is detected in a more acidic way.
[0086] Other methods can be used to assess the stability of the formulations of the present invention such as, for example, differential scanning calorimetry (DSC) to determine thermal stability, controlled agitation to determine mechanical stability and absorbance at about 350 nm or about 405 nm to determine the turbidity of the solution. For example, a formulation of the present invention can be considered stable if, after 6 or more months of storage at about 5 ° C to about 25 ° C, the change in OD405 of the formulation is less than about 0.05 ( for example, 0.04, 0.03, 0.02, 0.01 or less) in relation to the OD ^os of the formulation at time zero. [0087] Measurement of the biological activity or binding affinity of the antibody to its target can also be used to assess stability. For example, a formulation of the present invention can be considered stable if, after storage, for example, at 5 ° C, 25 ° C, 45 ° C, etc., for a defined period of time (for example, 1 to 12 months ), The anti-PD-1 antibody contained in the formulation binds to PD-1 with an affinity that is at least 90%, 95% or more of the antibody binding affinity prior to said storage. The binding affinity can be determined, for example, by means of ELISA or surface plasmon resonance. Biological activity can be determined through a PD-1 activity assay such as, for example, contact of a cell that expresses PD-1 with the formulation
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40/130 which comprises the anti-PD-1 antibody. The binding of the antibody to a cell can be measured directly such as, for example, via FACS analysis. Alternatively, the activity downstream of the PD-1 system can be measured in the presence of the antibody and compared with the activity of the PD-1 system in the absence of antibody. In some embodiments, PD-1 can be endogenous to the cell. In other embodiments, PD-1 can be expressed ectopically in the cell.
[0088] Additional methods for assessing the stability of an antibody in the formulation are demonstrated in the Examples presented below.
[0089] The liquid pharmaceutical formulations of the present invention may, in certain embodiments, exhibit low to moderate viscosity levels. Viscosity, as used here, can be kinematic viscosity or absolute viscosity. Kinematic viscosity is a measure of the resistive flow of a fluid under the influence of gravity. When two fluids of equal volume are placed in identical capillary viscometers and allowed to flow by gravity, a viscous fluid takes longer than a less viscous fluid to flow through the capillary. For example, if one fluid takes 200 seconds to complete its flow and another fluid takes 400 seconds, the second fluid is twice as viscous as the first on a kinematic viscosity scale. Absolute viscosity, sometimes called dynamic or simple viscosity, is the product of kinematic viscosity and fluid density (Absolute viscosity = kinematic viscosity x density). The dimension of the kinematic viscosity is L ² / T, where Léo length and T is time. In general, the kinematic viscosity is expressed in centistokes (cSt). The SI unit of kinematic viscosity is mm ² / s, which is 1 cSt. The absolute viscosity is expressed in units of centipoise (cP). The SI unit of absolute viscosity is milliPascal-second (mPa-s), where 1 cP = 1 mPa-s.
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[0090] As used here, a low viscosity level, in reference to a fluid formulation of the present invention, will exhibit an absolute viscosity of less than about 20 ePoise (cP). For example, a fluid formulation of the invention will be considered to have a low viscosity if, when measured using standard viscosity measurement techniques, the formulation exhibits an absolute viscosity of about 20 cP, about 19 cP, about 18 cP, about 15 cP, about 12 cP, about 10 cP, about 9 cP, about 8 cP or less. As used herein, a moderate level of viscosity, in reference to a fluid formulation of the present invention, will exhibit an absolute viscosity between about 35 cP and about 20 cP. For example, a fluid formulation of the invention will be considered to have moderate viscosity if, when measured using standard viscosity measurement techniques, the formulation exhibits an absolute viscosity of about 34 cP, about 33 cP, about 32 cP, about about 31 cP, about 30 cP, about 29 cP, about 28 cP, about 27 cP, about 26 cP, about 25 cP, about 24 cP, about 23 cP, about 22 cP, about from 21 cP, approximately 20 cP, about 19 cP, 18 cP, about 17 cP, about 16 cP or about 15.1 cP.
[0091] As illustrated in the examples below, the present inventors have made the surprising discovery that low viscosity liquid formulations that comprise high concentrations of an anti-human PD-1 antibody (for example, from about 50 mg / mL to 250 mg / ml) can be obtained by formulating the antibody with about 1% to about 5% proline and about 5% sucrose. Such formulations are stable to stress during handling and storage at temperatures ranging from 45 ° C to -80 ° C (shown here) and have low viscosity (have viscosity ranging from 7 to 15 cP).
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EXAMPLE FORMULATIONS
[0092] In accordance with one aspect of the present invention, the pharmaceutical formulation is a stable, low viscosity, generally physiologically isotonic liquid formulation comprising: (i) a human antibody that specifically binds to human PD-1 (e.g. H4H7798N) at a concentration of up to 250 mg / mL ± 45 mg / mL;
(ii) a histidine buffer system that provides sufficient buffering at a pH of about 6.0 ± 0.3; (iii) an organic cosolvent that protects the structural integrity of the antibody; (iv) a thermal stabilizer that is a sugar; and (iv) a viscosity modifier that is an amino acid that serves to maintain viscosity manageable for injection in a volume convenient for subcutaneous administration.
[0093] According to one embodiment, the low viscosity stable pharmaceutical formulation comprises: (i) a human IgG4 antibody that specifically binds to human PD-1 and which comprises an HCDR1 of SEQ ID NO: 3, an HCDR2 of SEQ ID NO: 4, an HCDR3 of SEQ ID NO: 5, an LCDR1 of SEQ ID NO: 6, an LCDR2 of SEQ ID NO: 7 and an LCDR3 of SEQ ID NO: 8 at a concentration of up to 200 mg / mL ± 30 mg / ml; (ii) 10 mM ± 2 mM histidine buffer that buffer at pH 6.0 ± 0.3; (iii) polysorbate at 80 to 0.2% w / v ± 0.1% w / v; (iv) 5% ± 1% w / v sucrose; and (v) 1.5% (w / v) ± 0.3% L-proline. [0094] According to one embodiment, the stable low viscosity pharmaceutical formulation comprises: (i) a human IgG4 antibody that specifically binds to human PD-1 and which comprises an HCDR1 of SEQ ID NO: 3, an HCDR2 of SEQ ID NO: 4, an HCDR3 of SEQ ID NO: 5, an LCDR1 of SEQ ID NO: 6, an LCDR2 of SEQ ID NO: 7 and an LCDR3 of SEQ ID NO: 8 at a concentration of 175 mg / mL ± 26.25 mg / ml; (ii) 10 mM ± 2 mM histidine buffer that buffer at pH 6.0 ± 0.3; (iii) polysorbate 80 at 0.2% w / v ± 0.1% w / v; (iv) 5% ± 1% w / v sucrose; and (v) 1.5% (w / v) ± 0.3% L-proline.
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[0095] According to one embodiment, the stable low viscosity pharmaceutical formulation comprises: (i) a human IgG4 antibody that specifically binds to human PD-1 and which comprises an HCDR1 of SEQ ID NO: 3, an HCDR2 of SEQ ID NO: 4, an HCDR3 of SEQ ID NO: 5, an LCDR1 of SEQ ID NO: 6, an LCDR2 of SEQ ID NO: 7 and an LCDR3 of SEQ ID NO: 8 at a concentration of 150 mg / mL ± 22.5 mg / ml; (ii) 10 mM ± 2 mM histidine buffer that buffered at pH 6.0 ± 0.3: (iii) polysorbate 80 at 0.2% w / v ± 0.1% w / v; (iv) 5% ± 1% w / v sucrose; and (v) 1.5% L-proline (w / v) ± 0.3%.
[0096] According to one embodiment, the low viscosity stable pharmaceutical formulation comprises: (i) a human IgG4 antibody that specifically binds to human PD-1 and which comprises an HCDR1 of SEQ ID NO: 3, an HCDR2 of SEQ ID NO: 4, an HCDR3 of SEQ ID NO: 5, an LCDR1 of SEQ ID NO: 6, an LCDR2 of SEQ ID NO: 7 and an LCDR3 of SEQ ID NO: 8 at a concentration of 100 mg / mL ± 15 mg / ml; (ii) 10 m M ± 2 mM histidine buffer which buffer at pH 6.0 ± 0.3; (iii) sucrose at 5% w / v ± 1% w / v; (iv) polysorbate 80 at 0.2% w / v ± 0.1%; and 1.5% (w / v) L-proline ± 0.3%.
[0097] According to one embodiment, the stable low viscosity pharmaceutical formulation comprises: (i) a human IgG4 antibody that specifically binds to human PD-1 and which comprises an HCDR1 of SEQ ID NO: 3, an HCDR2 of SEQ ID NO: 4, an HCDR3 of SEQ ID NO: 5, an LCDR1 of SEQ ID NO: 6, an LCDR2 of SEQ ID NO: 7 and an LCDR3 of SEQ ID NO: 8 at a concentration of 50 mg / mL ± 7.5 mg / ml; (ii) 10 mM ± 2 mM histidine buffer that buffer at pH 6.0 ± 0.3; (iii) sucrose at 5% w / v ± 1% w / v; (iv) polysorbate 80 at 0.2% w / v ± 0.1%; and 1.5% (w / v) L-proline ± 0.3%.
[0098] According to one embodiment, the stable low viscosity pharmaceutical formulation comprises: (i) a human IgG4 antibody that specifically binds to human PD-1 and which comprises
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44/130 an HCDR1 of SEQ ID NO: 3, an HCDR2 of SEQ ID NO: 4, an HCDR3 of SEQ ID NO: 5, an LCDR1 of SEQ ID NO: 6, an LCDR2 of SEQ ID NO: 7 and an LCDR3 of SEQ ID NO: 8 at a concentration of 25 mg / mL ± 3.75 mg / mL: (ii) 10 m M ± 2 mM histidine buffer that buffered at a pH 6.0 ± 0.3; (iii) sucrose at 5% w / v ± 1% w / v; (iv) polysorbate 80 at 0.2% w / v ± 0.1%; and 1.5% (w / v) L-proline ± 0.3%.
[0099] Additional non-limiting examples of the pharmaceutical formulations covered by the present invention are presented elsewhere here, including the Working Examples presented below.
CONTAINERS AND METHODS OF ADMINISTRATION
[0100] The pharmaceutical formulations of the present invention can be contained within any container suitable for storage of drugs and other therapeutic compositions. For example, pharmaceutical formulations can be contained in a sealed or sterilized glass or plastic container with a defined volume, such as a vial, ampoule, syringe, cartridge or bottle. Different types of bottles can be used to contain the formulations of the present invention including, for example, transparent and opaque glass or plastic bottles (for example, amber). Likewise, any type of syringe can be used to contain or administer the pharmaceutical formulations of the present invention.
[0101] The pharmaceutical formulations of the present invention can be contained within common tungsten syringes or syringes with low tungsten content. As will be appreciated by those skilled in the art, the glass syringe manufacturing process usually involves the use of a hot tungsten rod that works to pierce the glass, thereby creating a hole from which liquids can be extracted and expelled from the syringe. This process results in the deposition of traces of tungsten on the internal surface
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45/130 of the syringe. Subsequent washing and other processing steps can be used to reduce the amount of tungsten in the syringe. As used here, the term common tungsten means that the syringe contains more than or equal to 500 parts per billion (ppb) of tungsten. The term low tungsten content means that the syringe contains less than 500 ppb of tungsten. For example, a low tungsten syringe according to the present invention can contain less than about 490, 480, 470, 460, 450, 440, 430, 420, 410, 390, 350, 300, 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10 or less ppb of tungsten.
[0102] The rubber plungers used in syringes and the rubber stoppers used to close the vial openings can be coated to prevent contamination of the medical contents of the syringe or vial or to preserve its stability. Thus, the pharmaceutical formulations of the present invention, according to certain embodiments, can be contained within a syringe comprising a coated plunger or in a vial that is sealed with a coated rubber stopper. For example, the plunger or stopper can be coated with a fluorocarbon film. Examples of suitable coated stoppers and pistons for use with syringes and vials containing pharmaceutical formulations of the invention are mentioned, for example, in U.S. Patent ^Nos 4,997,423; 5,908,686; 6,286,699; 6,645,635; and 7,226,554, the content of which is incorporated herein by reference in its entirety. Specific exemplary coated rubber stoppers and plungers that can be used in the context of the present invention are commercially available under the trade name FluroTec®, available from West Pharmaceutical Services, Inc. (Lionville, PA). FluroTec® is an example of a fluorocarbon coating used to minimize or prevent the drug from adhering to rubber surfaces.
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[0103] In accordance with certain embodiments of the present invention, pharmaceutical formulations can be contained within a low tungsten syringe comprising a fluorocarbon-plated plunger.
[0104] Pharmaceutical formulations can be administered to a patient via parenteral routes, such as injections (eg, subcutaneous, intravenous, intramuscular, intraperitoneal, etc.) or percutaneous, mucous, nasal, pulmonary or oral. Numerous reusable pen or autoinjector delivery devices can be used to subcutaneously administer the pharmaceutical formulations of the present invention. Examples include, but are not limited to, AUTOPEN ™ (Owen Mumford, Inc., Woodstock, UK), the DISETRONIC ™ pen (Disetronic Medical Systems, Bergdorf, Switzerland), the HUMALOG MIX 75/25 ™ pen, the HUMALOG ™ pen, HUMALIN 70 / PEN 30 ™ pen (Eli Lilly and Co., Indianapolis, IN), NOVOPEN ™ I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR ™ (Novo Nordisk, Copenhagen, Denmark), the BD pen ™ (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN ™, OPTIPEN PRO ™, OPTIPEN STARLET ™ and OPTICLIK ™ (Sanofi-Aventis, Frankfurt, Germany). Examples of disposable pen-type or auto-injector delivery devices with applications in the subcutaneous administration of a pharmaceutical composition of the present invention include, but are not limited to, SOLOSTAR ™ (Sanofi-Aventis), FLEXPEN ™ (Novo Nordisk) and KWIKPEN ™ (Eli Lilly) pens ), the SURECLICK ™ autoinjectors (Amgen, Thousand Oaks, CA), PENLET ™ (Haselmeier, Stuttgart, Germany), EPIPEN ™ (Dey, LP) and the HUMIRA ™ pen (Abbott Labs, Abbott Park, IL)).
[0105] The use of a microinfuser to administer the pharmaceutical formulations of the present invention is also considered here. As used here, the term "micro-infuser" means a subcutaneous delivery device designed to slowly deliver large
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47/130 volumes (for example, up to about 2.5 ml or more) of a therapeutic formulation over an extended period of time (for example, about 10, 15, 20, 25, 30 or more minutes). See, e.g., U.S. Patent ^Nos 6,629,949; US 6,659,982; and Meehan et al., J, Controlled Release 46: 107-116 (1996). Microinfusers are particularly useful for administering large doses of therapeutic proteins contained in high concentration (for example, about 100, 125, 150, 175, 200 or more mg / ml) or viscous solutions.
[0106] In certain embodiments, the stable liquid pharmaceutical formulation of any of the above is contained in a sterile glass bottle and is administered as an IV infusion.
[0107] In one embodiment, the container is a 20 ml type 1 clear borosilicate glass vial. In certain embodiments, the container is a 2 ml, 5 ml or 10 ml type 1 borosilicate glass vial with a chlorobutyl stopper, with a FluroTec® coating.
[0108] In one embodiment, the liquid pharmaceutical formulation of the present invention comprising about 25 mg / ml or 50 mg / ml of mAb1 is administered intravenously and can be contained in a glass bottle.
[0109] In certain embodiments, the present invention provides an autoinjector that comprises any of the liquid formulations described here. In some embodiments, the present invention provides an autoinjector comprising a stable liquid formulation comprising about 50 mg / ml, about 100 mg / ml, about 150 mg / ml or about 175 mg / ml mAb1, histidine a about 10 mM at a pH of about 6.0, about 5% sucrose, about 1.5% proline and about 0.2% polysorbate 80.
[0110] In certain embodiments, the present invention provides
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48/130 a filled syringe comprising any of the liquid formulations described here. In some embodiments, the present invention provides a filled syringe comprising a stable liquid formulation comprising about 50 mg / ml, about 100 mg / ml, about 150 mg / ml or about 175 mg / ml mAb1, histidine at about 10 mM at a pH of about 6.0, about 5% sucrose, about 1.5% proline and about 0.2% polysorbate 80. In certain embodiments, the syringe is a 1 mL or 2.25 mL glass syringe filled with a 27 gauge thin-walled needle, a fluorocarbon-coated rubber plunger and a rubber needle protector.
[0111] In one embodiment, the liquid pharmaceutical formulation containing about 175 mg / mL ± 26.25 mg / mL of mAb1 is administered in a volume of approximately up to 2 mL in a filled syringe. In certain embodiments, the syringe is a 1 mL or 2.25 mL glass syringe filled with a 27 gauge thin-walled needle, a fluorocarbon-coated rubber plunger and a rubber needle protector. In one embodiment, the syringe is a 1 mL long glass OMPI syringe equipped with a 27 gauge needle, an FM27 rubber needle protector and a 4023/50 rubber plunger coated with FluroTec®.
[0112] In one embodiment, the liquid pharmaceutical formulation containing about 150 mg / ml ± 22.5 mg / ml antibody of anti-DP-1 is administered in a volume of up to about 2 ml in a filled syringe. In one embodiment, the syringe is a 1 mL or 2.25 mL glass syringe filled with a 27 gauge thin-walled needle, a fluorocarbon-coated rubber plunger and a rubber needle protector. In one embodiment, the syringe is a 1 mL long glass OMPI syringe fitted with a 27 gauge needle, an FM27 rubber needle protector and a 4023/50 rubber plunger
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THERAPEUTIC USES OF PHARMACEUTICAL FORMULATIONS [0113] The pharmaceutical formulations of the present invention are useful, inter alia, for the treatment, prevention or amelioration of any disease or disorder associated with PD-1 activity, including PD-1 mediated diseases or disorders. Non-limiting and exemplary diseases and disorders that can be treated or prevented by administering the pharmaceutical formulations of the present invention include viral infections, autoimmune diseases and various cancers such as, for example, brain cancer, lung cancer, prostate cancer, colorectal cancer, head and neck cancer, skin cancer, various types of blood cancer and endometrial cancer.
EXAMPLES
[0114] The following examples are presented in order to provide those skilled in the art with a complete description and disclosure of how to make and use the methods and compositions of the invention and are not intended to limit the scope of what the inventors consider their invention. Efforts have been made to ensure accuracy in relation to the numbers used (eg quantities, temperature, etc.), however, some experimental errors and deviations should be considered. Unless otherwise stated, the parts are parts per mol, the molecular weight is the average molecular weight, the temperature is in degrees centigrade and the pressure is equal to or close to atmospheric pressure.
Example 1: Development of an Anti-PD-1 Antibody Formulation [0115] The objectives of the formulation activities were to develop a formulation with the following attributes:
® A liquid formulation with an anti-PD-1 antibody concentration sufficient to deliver a dose of 250 mg or more by intravenous infusion;
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50/130 * An almost iso-osmolar formulation that is stable after dilution with commonly used diluents, for example, 0.9% sodium chloride injection or 5% dextrose injection, for intravenous infusion;
® A formulation that is compatible and stable in type 1 glass vials and standard serum stopper as a package;
«A sterile drug solution (PD) that supports long-term stability;
o A formulation that minimizes high molecular weight (HMW) antibody species when subjected to manipulation and heat stress;
o A formulation that minimizes variations in the relative distribution of antibody-loaded species when subjected to thermal stress; and o A formulation that retains biological activity when subjected to manipulation and thermal stress.
[0116] During the development of the formulation, three primary protein stress conditions (representing extreme handling conditions beyond which the antibody drug would not be subjected during handling, manufacture, transportation, storage and labeling) were employed to develop and optimize antibody formulations and assess the effects of potential real-world stresses on drug stability. These stress conditions include:
® Shaking (vortexing) the protein solution at room temperature. The vortex in glass jars exceeds the agitation that occurs during manipulation and manufacture of the protein.
«Incubation of the protein solution at an elevated temperature (37 ° C, 40 ° C or 45 ° C) in relation to the storage condition
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® Subject the protein to several freezing and thawing cycles. Since the protein will undergo at least one freeze and thaw cycle during the manufacture of DP, several freeze and thaw cycles simulate and exceed the actual stress that the protein is expected to experience.
[0117] There were four main objectives in the initial formulation development work:
[0118] 1. Buffer and pH selection: Choosing buffer and pH can have a great effect on protein stability, so deciding on the ideal buffer and pH species is an important process. Studies are presented in these sections that demonstrate the justification for choosing the ideal buffer and pH of the antibody.
[0119] 2. Selection of organic surfactant or cosolvent: An organic surfactant or cosolvent, such as polysorbate, is usually necessary to prevent protein precipitation or aggregation when agitated. Soluble protein can be subjected to stirring when manipulated, filtered, mixed, manufactured, transported and administered. The antibody drug substance in a simple buffered solution can be visibly cloudy with excessive agitation. Therefore, it was determined that protein stability when handling and shaking was important.
[0120] 3. Identification / selection of stabilizing / tonifying excipients: The addition of sugars, salts and amino acids has been examined for their ability to improve the stability of the antibody to heat stress and to increase the life span of DP. The justification for the inclusion of these thermal stabilizers, as well as studies that identify the ideal concentrations in the final formulation, are presented here.
[0121] 4. Selection of antibody concentration: The effect of antibody concentration on drug stability was examined
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[0122] Initial formulation development activities were conducted using 5-50 mg / mL of the anti-PD-1 antibody and involved screening organic cosolvents, thermal stabilizers and buffers in liquid anti-PD-1 antibody formulations to identify excipients that are compatible with the protein and increase its stability, maintaining an almost physiological osmolarity and low viscosity for intravenous and subcutaneous injection. Buffering conditions were also examined to determine the ideal pH for maximum protein stability (described in Examples 4, 6 and 7).
[0123] The results of this initial formulation development work were used to develop an initial formulation suitable for Phase 1 clinical studies. The phase 1 formulation was also a benchmark for optimizing later phase clinical and commercial formulations.
[0124] With the knowledge obtained from the initial development of the formulation, the development activities of the later phase involved the optimization of pH, concentration of surfactant and stabilizers to identify excipients that improve protein stability in low and high protein concentrations (up to 175 mg / ml mAb1) (described in Examples 5, 8, 9 and 10).
[0125] Throughout the development of the formulation, the formulations were evaluated for stress and storage stability. The methods used to assess stability in formulation development studies are described in Example 3 here. Examples 11 and 12 describe the storage and stress stability of the formulations.
[0126] Example 13 describes the stability of the formulations when excipients varied within specific ranges.
[0127] The results generated from these studies were used
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53/130 to develop stable liquid formulations suitable for clinical use for intravenous (IV) or subcutaneous (SC) administration. Example 14 describes containers used for the formulations here. Examples 15, 16 and 17 describe the compatibility and stability of formulations in glass vials, filled syringes and devices for intravenous administration. Such formulations met the objectives defined for the development of the formulation:
® The formulations developed are suitable for the doses developed;
® An iso-osmolar tonicity under physiological conditions;
o The osmolality of the formulation at 50 mg / mL is approximately 318 mOsm / kg;
® Sterile DP solution that supports long-term liquid stability;
o Minimal formation of HMW antibody species occurs after prolonged storage at 2-8 ° C;
o Little or no change in the relative distribution of species loaded with antibodies occurs after prolonged storage at 2-8 ° C; and o Minimal formation of subvisible particles was observed in the DP under accelerated storage and stress conditions and after storage at 5 ° C for 12 months.
[0128] Other attributes of the formulations will be evident from the description here.
[0129] Anti-PD-1 Antibodies: Anti-PD-1 antibodies are described in US20150203579, incorporated here in full. The exemplificative antibody used in the Examples below is a fully human antiPD-1 antibody H4H7798N (as described in US20150203579, known as REGN2810 or cemiplimab) that
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54/130 comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 9 and a female dog which comprises the amino acid sequence of SEQ ID NO: 10; a pair of HCVR / LCVR amino acid sequences comprising SEQ ID NOs: 1/2; and heavy and light chain CDR sequences comprising SEQ ID NOs: 3-8; and named here as mAbl.
Example 2: Exemplary Formulations
[0130] In certain embodiments, mAbl is formulated as an aqueous buffered formulation containing 5 mg / mL ± 0.75 mg / mL to 250 mg / mL ± 45.0 mg / mL mAbl, 10 µM histidine buffer mM ± 2 mM, polysorbate 0.2% ± 0.1% w / v, sucrose 1% ± 0.2% to 10% ± 2% w / v and 1% proline ± 0.02% to 5% ± 1% w / v at a pH 6.0 ± 0.3.
[0131] Exemplary formulations include:
® A stable, low-viscosity pharmaceutical formulation comprising: 25 mg / ml ± 3.75 mg / ml mAbl, 10 ± 2 mM histidine buffer, 0.2% ± 0.1% w / v polysorbate, sucrose 5% ± 1% w / v and L-proline 1.5% ± 0.3% w / v at a pH 6.0 ± 0.3.
* A stable low viscosity pharmaceutical formulation comprising: 50 mg / ml ± 7.5 mg / ml mAbl, 10 ± 2 mM histidine buffer, 0.2% polysorbate ± 0.1% w / v, sucrose 5% ± 1% w / v and L-proline 1.5% ± 0.3% w / v at a pH 6.0 ± 0.3.
'A stable, low viscosity pharmaceutical formulation comprising: 150 ± 23 mg / ml mAbl, 10 ± 2 mM histidine buffer, 0.2% ± 0.1% w / v polysorbate, 5% ± sucrose 1% w / v and L-proline 1.5% ± 0.3% w / v at a pH 6.0 ± 0.3.
® A stable, low-viscosity pharmaceutical formulation comprising: 175 ± 27 mg / mL mAbl, 10 ± 2 mM histidine buffer, 0.2% ± 0.1% w / v polysorbate, 5% ± sucrose 1% w / v and L-proline 1.5% ± 0.3% w / v at a pH 6.0 ± 0.3.
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Example 3: Methods Used to Assess Formulation Stability [0132] The following tests have been applied to assess formulation stability:
«Color and appearance through visual inspection« pH ® Turbidity measured by increasing OD to 405nm or nephelometry * Particle material analysis performed using microflow imaging (MFI) (reported as particle count obtained as such) and obscuring the light (HIAC) ® Protein concentration using ultra high performance reverse phase liquid chromatography (RP-UPLC) ® Purity via size exclusion ultra performance liquid chromatography (SE-UPLC) or capillary electrophoresis of Reduced and unreduced sodium dodecyl sulfate microchips (MCESDS) ® Analysis of charge variants by means of cation exchange chromatography-ultra-performance liquid chromatography (CEX-UPLC) or capillary isoelectric imaging (ICIEF) ® Power through bioassay: The relative potency of each sample is determined using a biological assay and is defined as: (ICsoda reference sample / ICsoda sample) * 100%. The measured power of the storage stability samples must be between 50% and 150% of the measured power of the reference standard.
[0133] The physical stability of a formulation refers to properties such as color, appearance, pH, turbidity and protein concentration. The presence of visible particles in the solution can be detected by visual inspection. A solution passes visual inspection if it is clear to slightly opalescent, essentially free of visible particles and colorless to light yellow. In addition, turbidity, measured
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56/130 by OD at 405 nm, can also be used to detect particulate matter in solution. An increase in OD at 405 nm may indicate the presence of particulates, an increase in opalescence or color variation in the test articles. MFI is used to measure subvisible particulates> 2 pm in size. The protein concentration of mAb1 is measured using an RP-UPLC assay and reported as a percentage of protein recovery in relation to the raw material. In the RPUPLC assay, mAb1 is eluted from the RP column as a single peak. The protein concentration is determined from the total peak area of mAb1 by comparing it to a calibration curve generated using mAb1 standards. The percentage of recovery is calculated based on the protein concentration measured in relation to the initial protein concentration.
[0134] Chemical stability refers to the formation of covalently modified forms (for example, covalent aggregates, divage products or variant charge forms) and non-covalently modified forms (for example, non-covalent aggregates) of the protein. The degradation products of higher and lower molecular weight can be separated from native mAb1 by means of SE-UPLC and MCE-SDS methods. The percentage of degraded mAb1 in the SEUPLC and MCE-SDS methods is calculated from the ratio between the area of all non-native peaks and the total area of all mAb1 peaks. The loading forms of mAb1 are decomposed using CEXUPLC and iCIEF. In the CEX-UPLC method, peaks with retention times prior to the main peak are marked as acidic peaks; peaks with retention times after those of the main peak are marked as basic peaks. In the iCIEF method, peaks focused on a pl less than that of the main peak are marked as acids, while those focused on a pl greater than that of the main peak are called basic.
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Example 4: Effect of Different Buffers and pH
[0135] The effect of buffer and pH on the thermal stability of mAb1 was examined in liquid formulations by incubating 5 mg / mL of mAb1 at 45 ° C for 28 days in a series of buffer systems in different pH ranges. The following pH and buffer systems were studied: acetate (pH 4.5, 5.0, 5.5), histidine (pH 5.5, 6.0, 6.5) and phosphate (pH 6.0, 6.5, 7.0). Based on the results of the SE ~ UPLC analysis, maximum protein stability was observed when mAb1 was formulated at a pH between 6.0 and 6.5 in histidine buffer (Table 1).
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Table 1: Effect of buffer and pH on the stability of 5 mg / mL of mAb1 incubated at 45 ° C for 28 days
Formulation 5 mg / ml mAb1, 10 mM buffer Volume offloor 0.4 mL Container 2 mL type 1 borosilicate glass bottle with a 4432/50 butyl rubber stopper coated with FluroTec® pH / buffer Color and appearance Turbidity (increase in OD at 405 nm) % of protein recovered by RP-UPLC Variation in purity by SE-UPLC ¹ > Variation in load variants by CEX-UPLC ^a) % HMW % Native % LMW % Acid % Main % Bas-sico pH 4.5, acetate Approved 0.00 87 5.2 -7.2 2.0 11.1 -15.4 4.3 pH 5.0, acetate Approved 0.00 82 5.0 -5.9 0.9 7.5 -10.9 3.4 pH 5.5, acetate Approved 0.00 90 4.7 -5.4 0.7 12.8 -13.7 0.9 pH 5.5, histidine Approved 0.00 97 5.6 -6.4 0.8 10.8 -12.4 1.6 pH 6.0, histidine Approved 0.00 86 1.9 -2.4 0.6 13.4 -12.6 -0.7 pH 6.5, histidine Approved 0.00 84 1.2 -1.8 0.7 25.4 -21.3 -4.1 pH 6.0, phosphate Approved 0.01 92 3.7 -4.3 0.6 24.8 -21.8 -3.0 pH 6.5, phosphate Approved 0.03 91 4.9 -5.8 0.9 49.6 -40.3 -9.3 pH 7, Õ, phosphate Approved 0.03 95 10.4 -11.6 1.2 56.5 42.2 -14.3
58/130 ^a Reported as a relative variation in purity relative to raw material. The raw material (without incubation) contains> 97.2% native peak by SE-UPLC and> 49.0% main peak by CEX-UPLC in all formulations.
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CEX = cation exchange; DS drug substance; HMW = high molecular weight; LMW = low molecular weight; OD = optical density; PR = reverse phase; SE - exclusion by size; UPLC = high performance liquid chromatography
[0136] Based on the results of the CEX-UPLC analysis, maximum protein stability was observed when mAb1 was formulated at a pH between 5.5 and 6.0 in histidine buffer or at a pH between 5.0 and 5, 5 in acetate buffer. These analyzes also revealed that aggregation (that is, formation of HMW species), fragmentation (that is, formation of LMW species) and formation of load variants were the main degradation pathways. The histidine buffer was selected as the formulation buffer because it provided the best overall level of protein stabilization in relation to the formation of HMW and LMW species and the formation of charge variants. A pH 6.0 was chosen for the formulation because the formation of HMW species and charge variants, which are the main degradation pathways, were minimized at this pH. Based on these results, 10 mM histidine buffer at pH 6.0 was chosen for the mAb1 formulation. Example 5: pH screening in Histidine Buffers
[0137] The effect of buffer and pH on the thermal stability of mAb1 was examined in liquid formulations of high concentration. 150 mg / mL mAb1 was incubated at 45 ° C for 28 days in a series of histidine buffers whose pH varied between 5.3, 5.5, 5.8, 6.0 and 6.3 with and without thermal stabilizers . With 9% sucrose, based on the results of the SE-UPLC analysis, maximum protein stability was observed when mAb1 was formulated at a pH between 5.8 and 6.3 in histidine buffer (Figure 1). Based on the results of the CEX-UPLC analysis, maximum protein stability was observed when mAb1 was formulated at a pH between 5.3 and 6.0 in histidine buffer (Table 2).
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Table 2: Effect of pH on the stability of mAb1 at 150 mg / mL incubated at 45 ° C for 28 days
Formulation 150 mg / ml mAbf, 10 mM histidine Filling volume 0.4 mL Container / seal 2 mL type 1 borosilicate glass bottle with 4432/50 butyl rubber stopper coated with FluroTec® pH / Stabilizing Color and appearance Turbidity (auto-OD at 405 nm) % of protein recovered by RP-UPLC Purity variation by SE-UPLC ¹ > Variation in load variants by CEX-UPLC ^a > % HMW % Native % LMW % Acid % Main% Basic pH 5.3 / none Failed ² '> N / D N / D N / D N / D N / D N / D N / D N / D pH 5.5 / none Failed ^b > N / D N / D N / D N / D N / D N / D N / D N / D pH 5.8 / none Disapproved 0.40 95 46.2 -46.4 0.3 6.4 -10.2 3.9 pH 6.0 / none Disapproved 0.51 99 41.2 -41.5 0.3 10.6 -7.3 -3.3 pH 6.3 / none Disapproved 0.65 98 35.0 -35.4 0.4 19.6 -14.3 -5.3 pH 5.3 / 9% sucrose (w / v) Approved Õ, 14 95 41.9 -42, Ϊ Õ, 3 7.9 -11.1 3.2, pH 5.5 / 9% sucrose (w / v) Approved 0.16 99 30.8 -31.2 0.4 9.5 -12.5 2.9 pH 5.8 / 9% sucrose (w / v) Approved 0.13 100 22.8 -23.3 0.5 9.7 -11.8 2.1 pH 6.0 / 9% sucrose (w / v) Approved 0.14 99 19.4 -19.9 0.5 13.5 -14.5 1.0 pH 6.3 / 9% Sucrose (w / vj Approved 0.15 98 16.9 -17.4 Õ.5 22.4 -22.1 -Õ.4
60/130 ^a Reported as a relative variation in purity relative to raw material. The raw material (without incubation) contains> 94.0% native peak by SE-UPLC and> 48.7% main peak by CEX-UPLC in all formulations.
^b Gelled sample. No further analysis was performed.
NA = Not applicable
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CEX, cation exchange; HMW, high molecular weight; LMW, low molecular weight; DO, optical density; PR, reverse phase; SE, exclusion by size; UPLC, ultra-performance liquid chromatography
[0138] These analyzes also revealed that aggregation (ie formation of HMW species) and formation of charge variants were the main degradation pathways. A pH 6.0 was chosen for the DP formulation because the formation of HMW species and charge variants, which are the main degradation pathways, were minimized at this pH. Based on these results, 10 mM histidine buffer at pH 6.0 was chosen for the DP formulation with a high concentration of mAb1.
Example 6: Selection of Agitation Stress Protectors [0139] Stabilizers, such as surfactants and organic cosolvents, are often added to antibody formulations to protect the protein against agitation-induced aggregation. The effect of co-solvents and organic surfactants on the stability under stress by agitation and on the thermal stability of mAb1 at 5 mg / mL was examined in liquid formulations. The following cosolvents and surfactants were evaluated: polysorbate 20 0.1%, polysorbate 80 0.1% and PEG3350 1.0%. The results of the stress stability studies under agitation are summarized in Table 3.
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Table 3: Effect of organic co-solvents and surfactants on the stability of mAb1 at 5 mg / mL after stirring (120 minutes of vortex)
Formulation 5 mg / mL mAb1, 10 mM histidine, pH 6.0 Filling volume 0.4 mL Container 2 mL type 1 borosilicate glass bottle with a 4432/50 butyl rubber stopper coated with FluroTec® Co-solvent, 'Ύtivo Color and appearance Turbidity (increase in OD at405 nm) pH % of protein recovered by RP-UPLC Variation in Purity by SE-UPLC Variation in load variants by CEX-UPLC ³ %HMW % At-tivo %LMW % Acid % Mainpal % Bas-sico Without cossoivente / ten-soactive Disapproved 1.69 6.0 76 15.3 -23.7 -8.4 -2.3 -1.7 3.9 5% sucrose (w / v) Disapproved 1.75 6.0 64 9.4 -12.8 3.4, -1.7 -0.1 1.8 Polysorbate 2Õ at 0.1% (w / v) ^b ) Approved 0.00 6.0 102 -0.1 -0.8 0.8 0.2 0.2 -0.3 Polysorbate 80 0.1% (w / v) ^b ) Approved 0.00 6.0 100 -0.2 -0.5 0.4 0.2 -0.1 -0.1 1% PEG3350 (w / v) ^b > Disapproveof 0.20 6.0 93 0.3 -0.5 0.2 -0.2 -0.3 0.4
62/130 ^a Reported as a relative variation in purity relative to raw material. The raw material (without incubation) contains> 98.2% native peak by SE-UPLC and> 49.1% main peak by CEX-UPLC in all 5 formulations. ^b The formulation also contains 5% sucrose.
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CEX = cation exchange; HMW = high molecular weight; LMW = low molecular weight; OD = optical density; PR = reverse phase; SE = exclusion by size; UPLC = high performance liquid chromatography [0140] mAb1 was unstable when vortexed for 120 min in the absence of an organic or surfactant co-solvent. After vortexing in the absence of cosolvent or surfactant, the solution became cloudy, exhibited a substantial increase in turbidity and had an increase of
15.3% in the aggregates, as determined using SE-UPLC, in addition to the 24% loss in protein recovery by RP-UPLC (Table 3). 1% PEG3350 did not provide sufficient stabilization of mAb1 after 120 min of vortexing. In the presence of 1% PEG3350, the solution became cloudy and exhibited an increase in turbidity (Table 3). In contrast, 0.1% polysorbate 20 and 0.1% polysorbate 80 protected mAb1 against instability induced by agitation to the same extent (Table 3). [0141] However, the formulation containing 0.1% polysorbate 80 exhibited a decrease in the amount of aggregates compared to the formulation containing 20% 0.1% polysorbate when incubated at 45 ° C (Table 4). Polysorbate 80 0.1% was chosen as a surfactant for the formulation of DP mAb1 because it stabilized the protein under stirring stress, had less negative effect on the thermal stability of the protein than polysorbate 20 (as determined by SE-UPLC analyzes and CEX-UPLC) and has a safe history of use in monoclonal antibody formulations.
Example 7: Selection of Heat Stress Protectors [0142] Stabilizers, such as sucrose, are often added to antibody formulations to increase the thermal stability of the protein in liquid formulations. mAb1 at five (5) mg / mL in a liquid formulation exhibited improved stability when formulated with 5% sucrose and incubated under accelerated conditions (Table 4).
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Table 4: Effect of organic co-solvents and surfactants on the stability of mAb1 at 5 mg / mL incubated at 45 ° C for days
Formulation 5 mg / mL mAb1, 10 mM histidine, pH 6.0 Filling volume 0.4 mL Container / seal 2 mL type 1 borosilicate glass bottle with 4432/50 butyl rubber stopper coated with FluroTec® Co-solvent / stresstivo Color and Appearancereference Turbidity (auto-ment of OD a405 nm) pH % of protein recovered by RP-UPLC Change in Purity by SE-UPLC ¹ > Change in load variants by CEX-UPLC ^a > %HMW % At-tivo %LMW % Acid % Mainpal % Bas-sico Without cosolvent / ten-soactive Approved 0.00 6.1 96 2.8 -3.2 0.5 10.8 -10.7 -0.2 5% sucrose (w / v) Approved 0.01 6.1 99 1.6 -2.0 0.3 12.6 -11.7 -0.9 0.1% poisorbate 2Õ (w / v) ^b ) Approved 0.00 6.0 92 27.6 -28.4 0.7 0.1 -24.0 23.9 Poisorbate 80 to 0.1% (w / v) Approved 0.00 6.1 96 12.8 -14.1 0.9 4.4, -20.4 15.9 1% PEG3350 (w / v) Approved 0.00 6.1 90 8.4 -8.0 -0.4 0.4 -25.0 24.5
64/130 ^a Reported as a relative variation in purity relative to raw material. The raw material (without incubation) contains> 98.2% native peak by SE-UPLC and> 49.1% main peak by CEX-UPLC in all 5 formulations ^b The formulation also contains 5% sucrose
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CEX = cation exchange; HMW = high molecular weight; LMW = low molecular weight; OD = optical density; PR = reverse phase; SE - exclusion by size; UPLC = high performance liquid chromatography
[0143] After incubation at 45 ° C for 29 days, the relative amount of HMW species increased by 1.7% in the formulation containing 5% sucrose compared to a 2.8% increase in the control formulation without sucrose. For this reason, sucrose was chosen as a thermal stabilizer. To make the formulation isotonic and maximize thermal stability, the sucrose concentration was increased to 10% for the mAb1 formulation.
Example 8: Stabilizer Optimization
[0144] The objective of optimizing thermal stabilizers was to identify the stabilizing components that could be used to develop a DP formulation that supports an antibody concentration of up to 200 mg / mL. 10% sucrose was selected in the initial formulation. It was found that, with 10% sucrose, the viscosity of mAb1 was about 20 cP at 20 ° C and was considered to be very high for a robust and later phase commercial product. Therefore, a modified mAb1 formulation was needed which exhibited both favorable stability and lower viscosity.
[0145] Sucrose was chosen as a thermal stabilizer for mAb1 during the development of the low concentration formulation. For the development of the formulation in high concentration, different concentrations of sucrose and L-proline were evaluated for the stability and viscosity of mAb1 at concentrations of 150 and 175 mg / mL at 25 ° C (Table 5) and at 40 ° C for 1 month. The formation of HMW species decreased with increasing sucrose concentrations when the formulations were incubated at 40 ° C for 28 days. 3% L-proline
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66/130 conferred a stabilization similar to 5% sucrose and maximum stabilization was observed with 9% sucrose.
[0146] Although 9% sucrose gave slightly better stabilization compared to 3% L-proline, it also increased the formulation's viscosity. At 175 mg / mL, the mAb1 formulation with 9% sucrose has a viscosity of 27 centipoise, which represents manufacturing and administration challenges. The 175 mg / mL mAb1 formulation with 3% proline has a viscosity of approximately 20 centipoise, which is manageable with the current manufacturing process.
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Table 5: Accelerated stability of mAb1 in high concentration with thermal stabilizers at 25 ° C for 1 month
Formulation 210 mg / mL mAb1, 10 mM histidine, pH 6.0 Filling volume 0.8 mL Container / seal 5 mL polycarbonate bottle with silicone-coated polypropylene screw cap Excipients Color and appearance Turbidity(Increase of OD at 405 nm) pH % mAb1 recovered by RP-UPLC Purity by SE-UPLC Load variants byCEX-UPLC %HMW % At-tivo %LMW %Acid % Main % Bas-sico Raw material ¹⁾ Approved 0.00 6.0 1ÕÕ 2.9 96.6 0.5 25.3 47.4 27.3 3% proline Approved 0.01 6.0 106 4.1 95.3 0.6 25.2 47.1 27.8 3% Sucrose Approved 0.00 6.0 107 4.7 94.8 0.6 25.2 46.5 28.3 5% Sucrose Approved 0.00 6.0 104 4.7 94.8 0.6 25.1 46.1 28.8 Formulation SDS / DP: 150 mg / ml or 175 mg / ml mAb1, 10 mM histidine, pH 6.0, 0.1% PS 80 Filling volume 0.4 mL Container / Sealing 2 mL type 1 borosilicate glass bottle with 4432/50 butyl rubber stopper coated with FluroTec® Feedstock Approved 0.00 6.1 100 2.3 97.4 0.4 24.9 50.2 24.9 9% sucrose, 0.1% PS 80 Approved 0.00 6.1 101 3.7 95.7 0.7 28.4 47.7 23.9 3% proline, PS 80 a0.1% Approved 0.00 6.1 101 3.7 95.7 0.7 26.5 47.7 25.8
67/130 ^a The pH, SE-UPLC and CEX-UPLC results for 'Raw material' are the average values of the initial formulations
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[0147] However, based on the storage stability at -20 ° C, -30 ° C and -80 ° C, the 3% proline formulation was found not to be as stable as sucrose formulations (Figure 2). As shown in Figure 2, formulation F3 (with 5% sucrose) was stable at -20 ° C, -30 ° C and -80 ° C with <3% HMW species.
[0148] The effect of L ~ proline on mAb1 stabilization was examined with the 50 mg / mL formulation. After incubation at 45 ° C for 28 days, the formulation with L-proline showed lower levels of HMW species compared to the formulation without L-proline, showing that L-proline stabilizes the antibody at a concentration of 50 mg / mL ( Table 6). In addition, the impact of L-proline on the antibody protein structure was examined using biophysical techniques (Fourier transform infrared spectroscopy, CD spectroscopy, fluorescence emission spectroscopy and differential scanning calorimetry). The results showed that L-proline did not disturb the secondary and tertiary structure of the antibody.
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Table 6: Effect of stabilizers on the stability of 50 mg / mL mAbl after incubation at 45 ° C for 28 days
Formulation 50 mg / ml mAbl, 10 mM L-histidine, 5% sucrose, pH 6.0, 0.2% polysorbate 80 Volume offloor 0.6 mL Container / seal 2 mL type 1 glass bottle with 4432/50 chlorobutyl stopper coated with FluroTec® Stabilizer(% in w / v) Color and Appearancereference Turbidity (increase in OD at 405 nm) pH % of protein recovered by RP-UPLC Variation in purity by SE-UPLC ^a Variation in load variants by CEX-UPLC ^a %HMW % mono-mere %LMW % Acid % Mainpal % Basic - Approved 0.02 6.0 96 12.1 -12.7 Õ, 6 iò, 8 -11.9 Ϊ .0 1.5% L-proline Approved 0.02 6.1 96 11.3 -11.9 0.6 11.0 -11.8 0.8 3.0% L-proline Approved 0.01 6.0 96 10.9 -11.5 0.6 12.8 -12.9 0.1
69/130 ^a) Reported as a change in purity from raw material. The raw material (without incubation) contains> 98.5% of monomer peak by SE-UPLC and> 52.8% of main peak by CEX-UPLC in the three formulations.
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CEX, cation exchange; DS, drug substance; HMW, high molecular weight; LMW, low molecular weight; OD, optical density; PR, reverse phase; SE, exclusion by size; UPLC, ultra-performance liquid chromatography
[0149] The effect of different stabilizers on the thermal stability of high concentrations (150 and 175 mg / mL) of mAb1 was further examined in liquid formulations. The stabilizers evaluated were sucrose 9% (w / v), L-proline 3% (w / v) and sucrose 5% (w / v) with L-proline 1.5% (w / v). The results of the accelerated stability study are summarized in Table 7.
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Table 7: Effect of stabilizers on the stability of mAb1 after incubation at 25 ° C and 40 ° C
Formulation 150 or 175 mg / mL mAb1, 10 mM histidine, pH 6.0, 0.1% polysorbate 80 Filling volume 0.5 mL Container / Sealing 2 mL type 1 borosilicate glass bottle with 4432/50 butyl rubber stopper coated with FluroTec® Incubation 25 ° C for 3 months Stabilizer (% in w / v) mAb1Cone.(mg / mL) Color and appearance Turbidity (increase in OD at 405 nm) pH % of protein recovered by RPUPLC Variation in purity by SE-UPLC ¹ > Variation in load variants by CEX-UPLC ^a % HMW % Native %LMW % Acid % Main % Basic 9% Sucrose 150 Approved 0.02 6.2 110 2.6 -2.8 0.2 6.0 -4.2 -1.9 3% L-proline 175 Approved 0.00 6.2 112 2.4 -2.5 0.2 6.5 -4.1 -2.4 5% Sucrose, 1.5% Lproline 175 Approved 0.00 6.2 110 2.3 -2.6 0.2 6.0 -3.7 -2.4 Incubation 40 ° C for 28 days Stabilizer (% in w / v) mAb1Cone.(mg / mL) Color and appearance Turbidity (increase in OD at 405 nm) pH % of protein recovered by RPUPLC Variation in purityby SE-UPLC * Variation in load variants by CEX-UPLC ^a % HMW % Native %LMW % Acid % Main % Basic 9% Sucrose 150 Approved 0.04 6.1 102 6.9 -7.5 0.5 9.4 -9.7 0.3 3% L-proline 175 Approved 0.03 6.2 103 11.7 -12.3 0.6 12.1 -10.7 -1.4 5% Sucrose, 1.5% Lproline 175 Approved 0.03 6.1 103 8.8 -9.4 0.7 11.4 -10.2 -1.2
71/130 ^a Reported as change in purity in relation to raw material. The raw material (without incubation) contains> 97.3% of native peak by SE-UPLC and> 49.6% of main peak by CEX-UPLC in the three formulations.
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CEX, cation exchange; HMW, high molecular weight; LMW, low molecular weight; OD, optical density; PR, reverse phase; SE, exclusion by size; UPLC, ultra-performance liquid chromatography
[0150] After an incubation at 40 ° C for 28 days, 9% sucrose provided the best stabilization and had the highest viscosity among high concentration formulations. The formulation of 5% sucrose / 1.5% L-proline ranked second for stability after 28 days at 40 ° C. After incubation at 25 ° C for three months, the stability of mAb1 was almost the same in all formulations examined; however, the formulation with 5% sucrose / 1.5% L-proline was slightly better than the other two formulations. However, after incubation at -20 ° C, -30 ° C and -80 ° C, sucrose at 5% and 9% provided better stability than 3% proline (Figure 3). 175 mg / ml mAb1 with 5% sucrose / 1.5% L-proline has a viscosity of 14 cP at 20 ° C. To provide a formulation that produces an isotonic solution and achieves a better balance between stability and viscosity, 5% sucrose / 1.5% L-proline was selected for the development of a later stage DP formulation.
Example 9: Selection of Viscosity Modifiers
[0151] The viscosity of protein formulations increases exponentially as the protein concentration increases. When the viscosity starts to exceed about 10 to 15 cP at 20 ° C, the viscosity of the formulation must be taken into account when developing a formulation: this is simply because the viscosity correlates with the ease of injection through a filled syringe (PFS) or other needle-based delivery device; the most important thing is that maintaining a reasonably low viscosity is critical to the development of a device
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73/130 of administration, such as an autoinjector. The effect of excipients on the formulation's viscosity was examined in a liquid formulation with the following potential viscosity modifiers: proline, arginine.HCI, histidine.HCI, magnesium acetate and NaCI. Figure 4 summarizes the viscosity of mAb1 at 150 mg / ml with viscosity modifiers. Arginine.HCI, histidine.HCI, magnesium acetate and 25-100 mM NaCI decrease the viscosity of mAb1 formulations to 150 mg / mL.
[0152] The impact of viscosity modifiers on the stability of the mAb1 formulation was also examined. Formulations of 150 mg / ml mAb1 with viscosity modifiers, such as arginine.HCI, histidine.HCI, magnesium acetate and NaCI, were prepared and incubated at 45 ° C for 28 days. The results are shown in Figure 5. It was found that maximum protein stability was observed when mAb1 was formulated without viscosity modifiers; all of these viscosity modifying salts negatively affect the stability of mAb1. Therefore, salts were not included in the final formulation.
[0153] L-proline, as a stabilizer, minimized the viscosity of the solution to antibody concentrations equal to or greater than 50 mg / mL. The results of the accelerated stability studies for antibodies in high concentration with varying amounts of L-proline, with and without sucrose, are summarized in Table 7. After incubation at 25 ° C for three months, the formulation with 5% / L sucrose -1.5% proline provided slightly better stability compared to the other two formulations with regard to the formation of HMW species. After an incubation at 40 ° C for 28 days, the formulation containing 9% sucrose provided better stabilization and the formulation with 5% sucrose / 1.5% L-proline was second. The 9% sucrose formulation has a viscosity of 20 cP to 175
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74/130 mg / ml of antibody, while the viscosity of a formulation at 175 mg / ml with 5% sucrose / 1.5% L-proline has a viscosity of 14 cP at 20 ° C. The addition of L-proline to the formulation was important to decrease the viscosity at high concentrations of protein, as well as to stabilize the antibody.
[0154] In short, 5% sucrose / 1.5% L-proline was selected for both 50 mg / mL formulations of antibody and antibodies in high concentration. This combination of excipients achieved an isotonic formulation with acceptable stability and viscosity at all tested antibody concentrations (up to 175 mg / mL).
Example 10: Optimization of Polysorbate Concentration (PS)
[0155] During the development of the formulation, a formation of species of higher molecular weight and an increase in turbidity was observed when the mAb1 formulation was stirred without surfactant. The protein was stabilized under agitation by adding polysorbate 80 (PS 80). During the development of the high concentration liquid mAb1 formulation, instability to agitation was observed as an increase in high molecular weight species. A study was conducted to determine the minimum amount of polysorbate 80 needed to protect mAb1 up to 175 mg / mL against agitation-induced instability. The formulations in this study contained 5% sucrose and 1.5% L-proline, so that the effect of polysorbate 80 could be studied with a formulation composition more representative of the final formulation. The nominal concentrations of polysorbate 80 included in the study were 0%, 0.02%, 0.04%, 0.06%, 0.08%, 0.1%, 0.15% and 0.2% (p / v). In the absence of polysorbate 80, the solution became cloudy and exhibited a significant increase in turbidity after vortexing. A reduction dependent on the concentration of polysorbate 80 in the amount% HMW was observed after 120 minutes of stirring.
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75/130, a concentration of 0.15-0.2% of polysorbate 80 was found to be sufficient to stabilize mAb1 at 150 mg / ml and 175 mg / ml against agitation-induced aggregation (Table 8). The addition of polysorbate 80 at 0.2% (w / v) (nominal value) completely prevented the formation of HMW species after stirring for 120 minutes.
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Table 8: Stability of mAb1 at 150 mg / mL and 175 mg / mL with PS 80 after 120 minutes of shaking
Filling volume 0.4 mL Container / Sealing 2 ml type 1 borosilicate glass bottle with a 4432/50 butyl rubber stopper coated withFluroTec® Formulation [PS 80] Color and Appearancereference Turbidity(Increase of OD at 405 nm) pH % of mAb1 recovered by RP-UPLC Increase in HMW% by SE-UPLC 150 mg / ml mAb1,10 mM histidine, pH 6.0 9% sucrose 0.00 Disapproved 0.38 6.0 Not applicable 0.02 Approved 0.00 6.0 97 4.7 0.04 Approved 0.00 6.0 100 0.3 0.06 Approved 0.00 6.0 102 0.3 0.08 Approved 0.04 6.0 99 Õ, Õ 0.10 Approved 0.01 6.0 101 o, 1 0.15 Approved 0.00 6.0 98 ο, ϊ 0.20 Approved 0.00 6.0 106 0.1 175 mg / ml mAb1,10 mM histidine, pH 6.0,3% proline 0.00 Disapproved 0.38 6.0 Not applicable 0.02 Approved 0.06 6.1 100 7.0 0.04 Approved 0.00 6.0 97 2.4 0.06 Approved 0.01 6.1 700 2.3 0.08 Approved 0.03 6.1 98 0.9 0.10 Approved 0.00 6.1 102 0.4
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Filling volume 0.4 mL Container / Sealing 2 ml type 1 borosilicate glass bottle with a 4432/50 butyl rubber stopper coated withFluroTec® Formulation [PS 80] Color and Appearancereference Turbidity(Increase of OD at 405 nm) pH % of mAb1 recovered by RP-UPLC Increase in HMW% by SE-UPLC0.15 Approved 0.00 6.1 102 o, 1 0.20 Approved 0.00 6.1 10Ϊ ο, ϊ 175 mg / ml mAb1,10 mM histidine, pH 6.0, 5% sucrose1.5% proline 0.00 Disapproved 0.36 6.1 Not applicable 0.02 Approved 0.01 6.1 97 3.5 0.04 Approved 0.01 6.1 98 2.0 0.06 Approved 0.01 6.1 100 1.3 0.08 Approved 0.01 6.1 99 1.0 0.10 Approved 0.01 6.0 102 0.3 0.15 Approved 0.00 6.1 101 0.2 0.20 Approved 0.00 6.1 98 0.0
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Ό156] Table 9 details the effect of polysorbate 80 concentration on the stability of mAb1 at 175 mg / mL after stirring (120 minutes of vortex).
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Table 9: Effect of polysorbate 80 concentration on mAb1 stability at 175 mg / mL after stirring (120 minutes of vortex)
Formulation 175 mg / mL mAb1, 10 mM histidine, pH 6.0, 5% (w / v) sucrose, 1.5% (w / v) L-proline Volume offloor 0.4 mL Container / seal 2 mL type 1 borosilicate glass bottle with a 4432/50 butyl rubber stopper coated with FluroTec® PS 80Cone, nominal(% w / v) Color and appearance Turbidity (increase in OD at 405 nm) pH % of protein recovered by RP-UPLC Variation in Purity by SE-UPLC) Variation in load variants by CEX-UPLC ³ⁱ %HMW % At-tivo %LMW % Acid % Mainpal % Basic 0.00% Disapproved 0.36 6.0 N / D N / D N / D N / D N / D N / D N / D 0.02% Approved Õ, ÕÍ 6.0 97 3.5 -3.5 0.0 -0.6 0.8 -0.1 0.04% Approved 0.01 6.0 98 2.0 -2.0 0.0 0.1 -0.2 -0.2 0.06% Approved 0.01 6.0 100 1.3 -1.3 0.0 -0.2 0.3 -0.1 0.08% Approved 0.01 6.0 99 1.0 -1.0 0.0 -0.2 0.0 0.0 0.10% Approved 0.02 6.0 102 0.3 -Õ, 3 0.0 Õ, 2 0.0 0.0 0.15% Approved 0.00 6.1 101 0.2 -0.2 0.0 0.2 -0.3 0.1 0.20% Approved 0.00 6.1 98 0.0 0.0 0.0 0.0 -0.1 0.2
78/130 ^a Reported as a relative variation in purity relative to raw material. The raw material (without incubation) contains> 97.4% native peak by SE-UPLC and> 48.2% main peak by CEX-UPLC in all formulations.
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CEX, cation exchange; HMW, high molecular weight; LMW, low molecular weight; NA, not available; OD, optical density; PR, reverse phase; SE, exclusion by size; UPLC, high performance liquid chromatography
[0157] The ability of polysorbate 80 to 0.2% (w / v) to protect mAb1 against instability induced by agitation was confirmed by another study with the final formulation at 50 mg / ml (Table 10).
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Table 10: Effect of PS80 concentration on the stability of mAb1 at 50 mg / mL after stirring (120 minutes of vortex)
Formulation 50 mg / mL mAbt, 10 mM L-histidine, pH 6.0, 5% (w / v) sucrose, 1.5% (w / v) L-proline Filling volumement 0.6 mL Container / seal 2 mL type 1 glass bottle with a 4432/50 chlorobutyl stopper coated with FluorTec® Polysorbate 80Cone. (% in w / v) Color and appearanceco Turbidity (increase in OD at 405 nm) pH % of protein recovered by RPUPLC Purity variation by SE-UPLC ³ Variation in load variants by CEX-UPLC ³ %HMW % mo-name-ros %LMW % Acid % Mainpal % Bas-sico 0.0% Approved 0.01 6.1 99 8.0 -8.0 0.0 1.7 -1.8 -0.3 0.2% Approved 0.0Q 6.1 99 Õ, Õ Õ, 1 -Hi -Õ, 2 Õ, 1 -Õ, 2
80/130 ^a Reported as a relative variation in purity relative to raw material. The raw material (without incubation) contains> 98.5% of monomer peak by SE-UPLC and> 52.8% of main peak by CEX-UPLC in all five formulations.
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CEX, catholic exchange; DS, drug substance; HMW, high molecular weight; LMW, low molecular weight; NA, not available; OD, optical density; PR, reverse phase; SE, exclusion by size; UPLC, ultra-performance liquid chromatography [0158] Based on these results, polysorbate 80 at 0.2% (w / v) was selected as the surfactant, as it provided sufficient stabilization to prevent the formation of HMW species under stress by agitation . Example 11: Storage and Stress Stability of Exemplary Formulations
[0159] The storage stability of mAb1 formulations at 50 mg / ml and 175 mg / ml in glass bottles is shown in Table 11 and Table 12 and the accelerated stability and stress data for the two formulations are shown in Table 13 and Table 14, respectively. Stability research studies have shown that the 50 mg / mL and 175 mg / mL mAb1 formulations in glass bottles are stable for at least 24 months when stored at 2 ° C to 8 ° C. In addition, the 50 mg / mL mAb1 formulation also exhibited excellent stability under accelerated and stress conditions. The formulation is stable when stored at 25 ° C for at least 3 months and 40 ° C for at least 7 days, demonstrating the compatibility of the formulation at 50 mg / mL with the main sealing components of the container. There were no appreciable changes in color or appearance, turbidity, particulate matter, pH, protein concentration, purity measured by SE-UPLC or CEX-UPLC and iCIEF, and the potency was maintained under these conditions.
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Table 11: Stability survey of the formulation at 50 mg / mL stored at 2 - 8 ° C
Formulation 50 mg / mL mAb1, 10 mM L-hystidine, 5% (w / v) sucrose, 1.5% (w / v) L-proline and 0.2% (w / v) polysorbate , pH 6.0 Filling volume 1.2 mL Container / Sealing 3 ml type 1 glass vials with a 13 mm 4432/50 chlorobutyl stopper coated withFluroTec® Test Storage life at 2-8 ° C (months) 0 1 3 6 9 12 18 24 36 Color and appearance Approved Approves-of Approves-of Approved Approved Approves-of Approved ApprovedTurbidity (increase in OD at 405 nm) 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00Turbidity (NTU by nephelometry) 7.41 6.01 6.15 6.61 6.03 6.48 6.29 5.83pH 6.0 6.0 6.0 6.1 6.0 6.0 6.0 6.0Analysis of subvisible individuals by HIAC (N / mL) > 10 pm 2 NR 21 8 NR 11 15 21> 25 pm 0 NR 2 00 NR 1 1 1Analysis of subvisible individuals by IMF (N / mL) 2 to 10 pm 248 NR 887 1875 NR 607 125 4 776> 10 pm 15 NR 26 44 NR 15 19 8> 25 pm 4 NR 3 17 NR Ϊ 3 1% of protein recovered by RP-UPLC 100 100 98 103 101 105 98 98Purity by MCE-SDS Not reduced; Main peak% 99.2 NR NR 98.7 NR 98.9 99.2 99.2Reduced; % jail 100 NR NR 1 00 NR 99.6 99.8 99.9
82/130
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heavy + lightPurity by SE-UPLC % HMW 0.5 0.3 0.4 0.4 0.4 0.5 0.5 0.5% monomers 99.2 99.1 99.2 99.2 99.1 99.2 99.2 99.1% LMW 0.4 0.5 0.4 0.4 0.5 0.3 0.4 0.4Analysis of load variants by CEX-UPLC % Acid 18.9 19.0 18.7 19.1 1’9.4 Ϊ9.1 2Õ, 7 20.5% Main 53.9 53.5 54.5 53.7 53.6 55.8 53.8 53.4% Basic 27.3 27.6 26.8 27.2 27.1 25.1 25.5 26.0Analysis of load variants poriCIEF % Acid 31.9 NR NR 32.3 NR 33.9 33.1 34.0% Main 54.7 NR NR 54.2 NR 54.0 ’5’4.0 53.9% Basic 13.5 NR NR 13.5 NR 12.1 12.9 12.1% relative power (bioassay) 126 NR NR 127 NR 125 110 104
CEX, cation exchange; DS, drug substance; HMW, high molecular weight; iCIEF, isoeietric imaging
83/130 by capillary focus, LMW, low molecular weight; IMF, microflow; monomer, intact antibody; NR, it is not mandatory; OD, optical density; PR, reverse phase; SE, exclusion by size; UPLC, high performance liquid chromatography
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Table 12: Formulation stability survey at 175 mg / mL stored at 2 - 8 ° C
Formulation 175 mg / ml mAbl, 10 mM L-histidine, 5% (w / v) sucrose, 1.5% (w / v) L-proline and 0.2% (w / v) polysorbate , pH 6.0 Filling volume 1.2 mL Container / Sealing 3 ml type 1 glass vials with a 13 mm 4432/50 chlorobutyl stopper coated withFluroTec® Test Storage life at 5 ° C (months) 0 1 3 6 9 12 18 24 36 Color and appearance Approved Approved Approved Approved Approved Approved Approved Approves-ofTurbidity (increase in OD at 405 nm) 0.00 0.01 0.00 0.00 0.00 0.00 0.00 0.00Turbidity (NTU by nephelometry) 6.72 6.95 6.57 6.54 6.62 7.01 6.67 6.87pH 6.0 6.0 6.0 6.0 6.0 6.0 6.0 6.1Analysis of subvisible particles by HIAC (N / mL) > 10 pm Õ NR 28, 131 NR 3Õ 35 55> 25 pm 0 NR 11 56 NR 1 2 3Analysis of subvisible particulates by IMF (N / mL) 2 to 10 pm 144 NR 441 244 NR 265 319 117> 10 pm 22 NR 36, 22 NR 29 28 8> 25 pm Ϊ NR 11 7 NR 3 Õ 1% of protein recovered by RP-UPLC 100 95 97 98 97 99 104 101Purity by MCE-SDS Not reduced; Main peak% 97.9 NR 98.1 98.0 NR 97.9 98.0 97.9Reduced; % jail 100 NR 99.6 100 NR 99.8 99.8 99.8
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heavy + lightPurity by SE-UPLC % HMW 0.6 0.6 0.7 0.7 0.8 0.9 1.0 1.0% monomer 99.1 98.9 99.1 98.7 98.7 98.7 98.6 98.6% LMW 0.4 0.5 0.3 0.5 0.5 0.5 0.4 0.4Analysis of load variants by CEX-UPLC % Acid 18.7 18.3 18.1 18.7 19.1 Í9.Õ 19.0 20.3% Main 53.6 55.0 54.6 53.3 53.6 55.5 54.3 53.8% Basic 27.8 26.7 27.4 28.1 27.3 25.5 26.8 25.9Analysis of load variants poriCIEF % Acid 31.8 NR 32.4 31.9 NR 33.9 32.3 32.0% Main 54.6 NR 54.6 54.9 NR 54.8 54.6 54.3% Basic 13.6 NR 13.1 13.2 NR 11.3 13.1 13.7% relative power (bioassay) 102 NR NR 118 NR 110 91 107
CEX, catholic exchange; DS, drug substance; HMW, high molecular weight; iCIEF, isoelectric imaging
85/130 by capillary focus, LMW, low molecular weight; IMF, microflow; monomer, intact antibody; NR, it is not mandatory; OD, optical density; PR, reverse phase; SE, exclusion by size; UPLC, high performance liquid chromatography
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Table 13: Stability survey of the formulation at 50 mg / mL stored under accelerated and stress conditions
Formulation 50 mg / ml mAb1, 10 mM L-histidine, 5% (w / v) sucrose, 1.5% (w / v) L-proline, 0.2% (w / v) polysorbate , pH 6.0 Filling volume 1.2 mL Container / Sealing 3 ml type 1 glass vials with a 13 mm 4432/50 chlorobutyl stopper coated with FluroTec® Storage at 25 ° C / 60% RH (months) Storage at 40 ° C (days) Test 0 1 3 6 7 14 28 Color and appearance Approved Approved Approved Approved Approved Approved Approved Turbidity (increase in OD at 405 nm) 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Turbidity (NTU by Nephiometry) 7.41 6.05 6.54 6.90 6.10 6.60 6.93 pH 6.0 6.0 6.0 6.1 6.0 6.0 6.0 Analysis of subvisible particulates by HIAC (# / mL) > 10 pm 2 NR 17 21 NR NR 18 > 25 pm 00 NR 00 1 NR NR 1 Analysis of subvisible particulates by MFI (# / mL) 2-10 pm 248 NR 1618 1531 NR NR 1543 > 10 pm 15 NR 29 47 NR NR 41 > 25 pm 4 NR 2 15 NR NR 15 % of protein recovered by RP-UPLC 100 100 100 103 103 102 98 Purity by MCE-SDS Not reduced; Main peak% 99.2 NR 99.1 98.8 NR NR 98.9 Reduced; % jail 100 NR 99.5 100 NR NR 99.5
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heavy + light Purity by SE-UPLC % HMW 0.5 0.4 0.6 0.7 0.7 0.9 1.4 % monomers 99.2 99.0 99.1 98.8 98.9 98.5 97.9 % LMW 0.4 0.5 0.3 0.5 0.5 0.6 0.7 Analysis of load variants by CEX-UPLC % Acid 18.9 19.3 21.8 27.0 19.9 22.5 27.2 % Main 53.9 53.4 52.1 48.3 52.3 50.1 46.8 % Basic 27.3 27.4 26.1 24.8 27.8 27.4 26.0 Analysis of load variants poriCIEF % Acid 31.9 NR 38.1 43.7 NR NR 45.4 % Main 54.7 NR 48.6 44.3 NR NR 42.1 % Basic 13.5 NR 13.3 12.0 NR NR 12.5 % relative power per bioassay 126 NR NR 120 NR NR 99
CEX, cation exchange; DS, drug substance; HMW, high molecular weight; iCIEF, isoelectric imaging
87/130 by capillary focus, LMW, low molecular weight; IMF, microflow; monomer, intact antibody; NR, it is not mandatory; OD, optical density; PR, reverse phase; SE, exclusion by size; UPLC, high performance liquid chromatography
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Table 14: Stability survey of the formulation at 175 mg / mL stored under accelerated and stress conditions
Formulation 175 mg / mL mAb1, 10 mM L-histidine, 5% (w / v) sucrose, 1.5% (w / v) L-proline, 0.2% (w / v) polysorbate 80 , pH 6.0 Filling volume 1.2 mL Container / Sealing 3 ml type 1 glass vials with a 13 mm 4432/50 chlorobutyl stopper coated withFluroTec® Storage at 25 ° C / 60% RH (months) Storage at 40 ° C (days) Test 0 1 3 6 7 14 28 Color and appearance Approved Approved Approved Approved Approved Approved Approved Turbidity (increase in OD at 405 nm) 0.00 0.00 0.00 0.00 0.00 0.00 0.01 Turbidity (NTU by Nephiometry) 6.72 6.77 6.82 6.99 6.90 7.30 7.56 pH 6.0 6.0 6.0 6.1 6.0 5.9 6.0 Analysis of subvisible particulates by HIAC (# / mL) > 10 pm Õ NR 28 58 NR NR 97 > 25 pm 00 NR 13 16 NR NR 44 Analysis of subvisible particulates by MFI (# / mL) 2-10 pm 144 NR 623 277 NR NR 1131 > 10 pm 22 NR 25 8 NR NR 531 > 25 pm Ϊ NR 3 2 NR NR 3 % of protein recovered by RP-UPLC 100 95 97 99 98 99 95 Purity by MCE-SDS Not reduced;Main peak% 97.9 NR NR 97.4 NR NR 97.5 Reduced; 100 NR NR 99.7 NR NR 99.4
88/130
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% heavy chain+ light Purity by SE-UPLC % HMW 0.6 0.9 1.2 1.5 1.7 2.6 3.9 % monomers 99.1 98.6 98.5 98.0 97.7 96.8 95.5 % LMW 0.4 0.5 0.3 0.6 0.6 0.7 0.6 Analysis of load variants byCEX-UPLC % Acid 18.7 18.9 21.3 26.2 19.7 220 23.9 % Main 53.6 54.4 52.3 48.3 51.7 50.1 49.4 % Basic 27.8 26.7 26.4 25.5 28.6 27.9 26.7 Analysis of load variants by iCIEF % Acid 31.8 NR 37.0 45.0 NR NR 47.3 % Main 54.6 NR 50.3 43.3 NR NR 39.2 % Basic 13.6 NR 12.7 11.7 NR NR 13.5 % relative power per bioenergy 102 NR NR 123 NR NR 86
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CEX, cation exchange; DS, drug substance; HMW, high molecular weight; iCIEF, isoelectric imaging by capillary focus, LMW, low molecular weight; IMF, microflow; Monomer, antibody intact; NR, it is not mandatory; OD, optical density; PR, reverse phase; SE, exclusion by size; UPLC, high performance liquid chromatography
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Example 12: Stability of mAb1 formulations comprising histidine, sucrose and polysorbate buffer
[0160] Tables 15-24 summarize the storage stability of exemplary mAb1 formulations that comprise 10 mM histidine buffer at pH 6.0, sucrose and polysorbate.
Table 15: Stability survey of the mAb1 formulation stored at -80 ° C
Formulation 72.2 mg / mL mAb1, 10 mM histidine, pH 6.0, 5% (w / v) sucrose Filling volume 1.0 mL Container / Sealing 5 mL polycarbonate bottle with silicone-coated polypropylene screw capStorage life at -80 ° C (months) Test 0 1 3 6 9 12 Color and appearance Approvego-of Approvego-of Approvego-of Approvego-of Approvego-of Approvego-of Turbidity (increase in OD at 405 nm) 0.00 0.00 0.00 0.00 0.00 0.00 pH 6.2 6.3, 6.2 6.1 6.2 6.1 % of protein recovered by RP-UPLC 100 94 95 98 95 94 Purity byMCE-SDS Not reduced;% Main peak 99.5 NR NR 99.5 NR 99.2 Reduced;% Heavy chain +Light 100 NR NR 100 NR 99.8 Purity by SE-UPLC % HMW 0.7 0.7 0.6 0.6 0.7 0.6 % Native 98.7 98.8 98.9 98.7 98.9 98.8 % LMW 0.6 0.6 0.5 0.6 0.4 0.6 Analysis of load variants by CEX-UPLC % Acid 22.2 22.2 22.6 23.2 24.1 23.5 % Main 49.7 49.8 48.9 46.8 45.2 44.1 % Basic 28, Ϊ 23.0 28.5 30.0 30.7 32.4 Analysis of load variants by ICIEF % Acid 38.9 NR NR 39.1 NR 37.5 % Main 56.5 NR NR 56.2 NR 57.2 % Basic 4.6 NR NR 4.7 NR 5.3 % relative power (bioassay) 95 NR NR 81 NR 120 ’
CEX ™ cation exchange; HMW · - high molecular weight;
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91/130 iCIEF Isoelectric imaging by capillary focus; LMW · low molecular weight; MCE-SDS = Capillary electrophoresis in sodium microchip-dodecyl sulfate; IMF ··· microflow imaging; NR ··· It is not mandatory; OD = optical density; RH = relative humidity; PR = reverse phase; SE = exclusion by size; UPLC = high performance liquid chromatography
Table 16: Stability survey of the mAb1 formulation stored at -30 ° C
Formulation 72.2 mg / mL mAb1, 10 mM histidine, pH 6.0, 5% (w / v) sucrose Filling volume 1.0 mL Container / Sealing 5 mL polycarbonate bottle with silicone-coated polypropylene screw capStorage life at -3Õ ° C (months) Test 0 1 3 6 9 12 Color and appearance Approvevadas Approvevadas Approvevadas Approvego-of Approvevadas Approvevadas Turbidity. (ÕD increase at 405 nmj 0.00 0.00 0.00 0.00 0.00 Õ, ÕÍ pH 6.2 6.3 6.2 6.1 6.2 6.1 % of protein recovered by RP-UPLC 100 93 95 100 96 96 Purity byMCE-SDS Not reduced;% Main peak 99.5 NR NR 99.1 NR 99.2 Reduced;% Heavy + light chain 100 NR NR 100 NR 99.8 Purity by SE-UPLC % HMW 0.7 0.7 0.6 0.7 0.7 0.6 % Native 98.7 98.8 99.0 98.7 98.9 98.8 % LMW 0.6 0.5 0.4 0.6 0.4 0.6 Analysis of load variants by CEX-UPLC % Acid 22.2 22.5 22.5 23.4 24.2 23.4 % Main 49.7 49.6 48.9 46.6 45.6 44.1 % Basic 28.1 28.0 28.6 30.0 30.2 32.5 Analysis of load variants poriCIEF % Acid 38.9 NR NR 38.2 NR 37.8 % Main 56.5 NR NR 56.3 NR 56.6 % Basic 4.6 NR NR 5.5 NR 5.7
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Table 17: Stability survey of the mAb1 formulation stored at -20 ° C
Formulation 72.2 mg / mL mAb1, 10 mM histidine, pH 6.0, 5% (w / v) sucrose Filling volume 1.0 mL Container / Sealing 5 mL polycarbonate bottle with silicone-coated polypropylene screw capStorage duration a - 20 ° C (months) Test 0 1 3 6 9 12 Approve Approve Approve Approve Approve Approve Color and appearancego- go- go- go- go- go- of of of of of of Turbidity (increase in OD at 405 nm) 0.00 0.00 0.01 0.01 0.00 Õ, ÕÍ pH 6.2 6.3 6.2 6.1 6.1 6.2 % of protein recovered by RP-UPLC 100 95 97 101 98 98 Purity by MCE- Not reduced;% Main peak 99.5 NR NR 99.5 NR 99.3 SDS Reduced;% Chainoutlet + light 100 NR NR 100 NR 99.9 Purity by SE- % HMW 0.7 0.7 0.7 0.7 0.8 0.7 UPLC % Native 98.7 98.8 98.9 98.6 98.8 98.8 % LMW Õ, 6 Õ, 5 Õ, 5 Õ, 7 Õ, 4 Õ, 6 Variant analysis % Acid 22.2 22.4 22.3 22.4 24.6 23.4 load by CEX- % Main 49.7 49.7 49.2 47.6 45.5 44.2 UPLC % Basic 28.1 27.9 28.5 30.0 30.0 32.5 Variant analysis % Acid 38.9 NR NR 38.6 NR 38.6 of loading per ICEEF % Main 56.5 NR NR 56.8 NR 56.2 % Basic 4.6 NR NR 4.6 NR 5.3 % relative power (bioassay) 95 NR NR 96 NR 111
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Table 18: Stability survey of the mAb1 formulation - Effect under accelerated conditions
Formulation 72.2 mg / mL mAb1, 10 mM histidine, pH 6.0, 5% (w / v) sucrose Filling volume 1.0 mL Container / Sealing 5 mL polycarbonate bottle with silicone-coated polypropylene screw cap Storage at 5 ° C (days) Storage at 25 ° C / 60% RH (days) Storage a40 ° C / 75% RH (days) Test T = 0 28 56 14 28 14 28 Color and appearance Approved Approved Approved Approved Approved Approved Approved Turbidity (increase in OD at 405 nm) 0.00 0.01 0.01 0.02 0.01 0.03 0.05 pH 6.2 6.2 6.1 6.2 6.2 6.2 6.1 % of protein recovered by RP-UPLC 100 96 100 97 100 105 113 Purity by MCE-SDS Not reduced;% Main peak 99.5 NR 99.1 NR 99.5 NR 99.1 Reduced;% Heavy + light chain 100 NR 100 NR 99.2 NR 98.7 Purity by SE-UPLC % HMW Õ.7 0.9 1.1 1.4 1.7 3.1, 4.6 % Native 98.7 98.5 98.4 98.0 97.7 96.1 94.6 % LMW 0.6 0.6 0.6 0.7 0.7 0.8 0.9 Analysis of load variants by CEX-UPLC % Acid 22.2 22.2 22.3 22.0 22.6 25.6 30.8 % Main 49.7 49.8 48.9 49.6 49.1 46.0 41.7 % Basic 28.1 28.1 28.8 28.4 28.3 28.3 27.5 Analysis of load variants by iCIEF % Acid 38.9 NR 39.0 NR 41.1 NR 58.2 % Main 56.5 NR 56.8 NR 53.8 NR 36.3 % Basic 4.6 NR 4.2 NR 5.1 NR 5.5 % relative power (bioassay) 95 NR 95 NR 84 NR 87
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Table 19: Stability survey of the mAb1 formulation stored at - 80 ° C
Formulation 25 mg / mL mAb1, 10 mM histidine, pH 6.0, 10% (w / v) sucrose, 0.1% polysorbate 80 Filling volume 1.0 mL Container / Sealing 5 mL polycarbonate bottle with silicone-coated polypropylene screw capStorage life at -80 ° C (months) Test 0 1 3 6 9 12 Color and appearance Approved Approved Approved Approved Approved Approved Turbidity (increase in OD at 405 nm) 0.00 0.00 0.0Q 0.00 0.01 0.01 pH 6.1 6.1 6.1 6.0 6.0 6.0 % of protein recovered by RP-UPLC 100 100 97 95 100 99 Purity by MCE-SDS Not reduced; % Main peak 99.5 NR NR 99.4 NR 99.5 Reduced; % Heavy + light chain 100 NR NR WÕ NR 100 Purity by SE-UPLC % HMW 0.8 0.8 0.8 0.8 0.8 0.8 % Native 98.6 98.7 98.6 98.6 98.8 98.7 % LMW 0.6 0.5 0.6 0.6 0.5 0.5 Analysis of load variants by CEX-UPLC % Acid 22.8 23.0 23.3 23.5 23.9 25.4 % Main 47.3 47.3 46.2 45.3 44.2 44.2 % Basic 30.0 29.8 30.6 31.2 31.9 30.4 Analysis of load variants by iCIEF % Acid 40.1 NR NR 38.1 NR 41.3 % Main 56.1 NR NR 57.4 NR 54.1 % Basic 3.8 NR NR 4.6 NR 4.6 % relative power (bioassay) 112 NR NR 130 NR 107
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Table 20: Stability survey of the mAb1 formulation stored at - 30 ° C
Formulation 25 mg / mL mAb1, 10 mM histidine, pH 6.0, 10% (w / v) sucrose, 0.1% polysorbate 80 Filling volume 1.0 mL Container / Sealing 5 mL polycarbonate bottle with silicone-coated polypropylene screw capStorage life at -30 ° C (months) Test 0 1 3 6 9 12 Color and appearance Approved Approved Approved Approved Approved Approved Turbidity (increase in OD at 405 nm) 0.00 0.00 0.00 0.00 0.01 0.01 pH 6.1 6.1 6.1 6.0 6.1 6.0 % of protein recovered by RP-UPLC 100 100 102 97 100 102 Purity byMCE-SDS Not reduced; % Main peak 99.5 NR NR 99.1 NR 99.5 Reduced; % Heavy + light chain 100 NR NR 100 NR 100 Purity bySE-UPLC % HMW 0.8 0.8 0.8 0.8 0.8 0.8 % Native 98.6 98.7 98.6 98.7 98.8 98.7 % LMW 0.6 0.5 0.6 0.6 0.4 0.6 Analysis of load variants by CEX-UPLC % Acid 22.8 23.1 22.9 23.5 23.7 25.1 % Main 47.3 47.1 46.5 45.3 44.4 44.4 % Basic 30.0 29.8 30.6 31.2 31.9 30.5 Analysis of load variants poriCIEF % Acid 40.1 NR NR 38.0 NR 41.3 % Main 56.1 NR NR 57.3 NR 53.3 % Basic 3.8 NR NR 4.7 NR 5.4
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Table 21: Stability survey of the mAb1 formulation stored at - 20 ° C
Formulation 25 mg / mL mAb1, 10 mM histidine, pH 6.0, 10% (w / v) sucrose, 0.1% polysorbate 80 Filling volume 1.0 mL Container / Sealing 5 mL polycarbonate bottle with silicone-coated polypropylene screw capStorage life at -20 ° C (months) Test 0 1 3 The 9 12 Color and appearance Approved Approved Approved Approved Approved Approved Turbidity (increase in OD at 405 nm) 0.00 0.00 0.00 0.00 0.01 0.00 pH 6.1 6.1 6.1 6.0 6.1 6.0 % of protein recovered by RP-UPLC 100 99 102 97 106 102 Purity by MCE-SDS Not reduced; % Main peak 99.5 NR NR 99.4 NR 99.4 Reduced; % Heavy + light chain 100 NR NR 100 NR 99.0 Purity by SE-UPLC % HMW 0.8 0.8 0.8 0.8 0.7 0.8 % Native 98.6 98.7 98.6 98.7 98.8 98.7 % LMW 0.6 0.5 0.6 0.6 0.4 0.6 Analysis of load variants by CEX-UPLC % Acid 22.8 22.9 23.5 23.6 24.3 25.2 % Main 47.3 47.3 46.0 45.2 43.8 43.9 % Basic 30.0 29.8 30.6 31.2 31.9 30.8 Analysis of load variants by iCIEF % Acid 40.1 NR NR 38.4 NR 41.6 % Main 56.1 NR NR 57.9 NR 53.3 % Basic 3.8 NR NR 3.7 NR 5.1 % relative power (bioassay) 112 NR NR 120 NR 131
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Table 22: Stability survey of the mAbl formulation - Effect under accelerated conditions
Formulation 25 mg / mL mAbl, 10 mM histidine, pH 6.0, 10% (w / v) sucrose, 0.1% polysorbate 80 Filling volume 1.0 mL Container / Sealing 5 mL polycarbonate bottle with silicone-coated polypropylene screw capNo storage Storage at 5 ° C (days) Storage at 25 ° C / 60% RH (days) Storage at 40 ° C / 75% RH (days) Test T = 0 28 56 14 28 14 28 Color and appearance Approved Approved Approved Approved Approved Approved Approved Turbidity (increase in OD at 405 nm) 0.00 0.00 0.00 0.00 0.00 0.01 0.03 pH 6.1 6.1 6.0 6.1 6.1 6.0 6.0 % of protein recovered by RP-UPLC 100 101 102 106 107 113 114 Purity byMCE-SDS Not reduced; % Main peak 99.5 NR 99.1 NR 99.5 NR 99.3 Reduced; % Heavy + light chain 100 NR 100 NR 100 NR 99.1 Purity by SE-UPLC % HMW 0.8 0.7 0.7 0.9 0.9 3.4 5.0 % Native 98.6 98.8 98.7 98.6 98.6 95.7 93.7 % LMW 0.6 0.5 0.6 0.5 0.6 0.9 1.3 Analysis of load variants by CEX-UPLC % Acid 22.8 22.8 23.3 22.6 22.8 29.6 32.5 % Main 47.3 47.3 46.1 47.2 47.0 39.5 36.4 % Basic 30.0 29.9 30.6 30.2 30.2 30.9 31.1 Analysis of load variants poriCIEF % Acid 40.1 NR 39.8 NR 40.2 NR 53.7 % Main 56.1 NR 57.0 NR 55.9 NR 42.2 % Basic 3.8 NR 3.2, NR 3.9 NR 4.0 % relative power (bioassay) 112 NR 103 NR 91 NR 79
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Table 23: Stability survey of the mAb1 formulation stored at 5 ° C
Formulation 25 mg / ml mAb1, 10 mM histidine, 10% (w / v) sucrose, 0.1% (w / v) polysorbate, pH 6.0 Filling volume 1.0 mL Container / Sealing 2 mL type 1 borosilicate glass vials with 13 mm West S2-451 4432/50 GRY B2-40 stoppers coated with FiuroTec®Storage life at 5 ° C (months) Test 0 1 3 6 9 12 Color and appearance Approved Approved Approved Approved Approved Approved Turbidity (increase in OD at 405 nm) 0.00 0.00 0.00 0.01 0.01 0.00 pH 6.2 6.1 6.1 6.1 6.0 6.1 Analysis of individuals by MFI (particles / mL) 2 to 10 pm 823 NR NR 2169 NR 29331 > 10 pm 15 NR NR 10 NR 56 > 25 pm 00 NR NR 3 NR 5 % of protein recovered by RP-UPLC 100 100 98 101 102 90 Purity by MCE-SDS Not reduced; Main peak% 99.4 NR NR 99.5 NR 99.2 Reduced; % heavy + light chain 100 NR NR 100 NR 100 Purity by SE-UPLC % HMW 0.7 0.7 0.7 0.8 0.9 0.9 % Native 98.7 98.8 98.7 98.6 98.6 98.5 % LMW 0.6 0.5 0.6 0.6 0.5 0.6 Analysis of load variants by CEX-UPLC % Acid 23.0 22.8 23.5 22.4 24.1 22.4 % Main 48.5 48.6 46.2 47.2 44.4 45.5 % Basic 28.6 28.6 30.4 30.4 31.5 32.1 Analysis of load variants by iCIEF % Acid 39.8 NR NR 39.2 NR 40.1 % Main 56.7 NR NR 56.5 NR 55.7 % Basic 3.4 NR NR 4.3 NR 4.1 % relative power (bioassay) 111 NR NR 132 NR 124
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Table 24: Stability survey of the mAb1 formulation stored under accelerated conditions
Formulation 25 mg / ml mAb1, 10 mM histidine, 10 7o (w / v) sucrose, 80 to 0.17o (w / v) polysorbate, pH 6.0 Filling volume 1.0 mL Container / Sealing 2 mL type 1 borosilicate glass vials with 13 mm West S2-451 4432/50 GRY B2-40 stoppers coated with FluroTec® Storage at 25 ° C / 60 ° RH (months) Storage at 45 ° C (days) Test 0 0.5 1 3 7 14 28 Color and appearance Approved Approved Approved Approved Approved Approved Approved Turbidity (increase in OD at 405 nm) 0.00 0.01 0.01 0.00 0.01 0.00 0.02 pH 6.2 6.1 6.1 6.1 6.2 6.1 6.1 Particle analysis by MFI (particles / mL) 2 to 10 pm 823 NR NR 1056 NR NR 521 > 10 pm 15 NR NR 23 NR NR 83 > 25 pm 0 0 NR NR 3 NR NR 4 % of protein recovered by RP-UPLC 100 99 100 99 99 100 99 Purity byMCE-SDS Not reduced; Main peak% 99.4 NR NR 99.4 NR NR 99.2 Reduced; % heavy + light chain 100 NR NR 100 NR NR 98.6 Purity by SE-UPLC % HMW 0.7 0.8 0.9 1.1 1.6 3.2, 8.5 % Native 98.7 98.6 98.5 98.1 97.6 95.9 89.7 % LMW 0.6 0.6 0.6 0.8 0.8 1.0 1.8 Analysis of load variants by CEX-UPLC % Acid 23.0 22.6 23.1 25.8 24.7 27.3 34.0 7th Main 48.5 48.7 48.1 44.5 46.2 44.1 35.9 7th Basic 28.6 28.8 28.9 29.8 29.1 28.6 30.2 Analysis of load variants poriCIEF 7th Acid 39.8 NR NR 47.1 NR NR 66.9 7th Main 56.7 NR NR 49.5 NR NR 30.2 7th Basic 3.4 NR NR 3.5 NR NR 3.0 % relative power per bioassay 111 NR NR 94 NR NR 63
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[0161] Tables 25-27 summarize the stress stability of exemplary formulations.
Table 25: Stability survey of the mAb1 formulation - Effect of stress conditions
Formulation 72.2 mg / mL mAb1, 10 mM histidine, pH 6.0, 5% (w / v) sucrose Filling volume 1.0 mL Container / Sealing 5 mL polycarbonate bottle with silicone-coated polypropylene screw cap Agitation (min) Freeze / Thawment (cycles) Test T = 0 10 15 4 8 Color and appearance Approved Approved Approved Approved Approved Turbidity (increase in OD at 405 nm) 0.00 0.00 0.09 0.01 0.00 pH 6.2 6.2 6.2 6.3 6.3 % of protein recovered by RP-UPLC 100 100 99 95 95 Purity by MCE-SDS Not reduced; % Main peak 99.5 NR 99.2 NR 99.4 Reduced; % Heavy + light chain 100 NR 100 NR 100 Purity by SE-UPLC % HMW 0.7 0.8 4.6 0.6 0.7 % Native 98.7 98.7 94.9 98.9 98.8 % LMW 0.6 0.5 0.5 0.5 0.5 Analysis of load variants by CEX-UPLC % Acid 22.2 22.2 21.9 22.0 22.2 % Main 49.7 49.6 49.9 49.7 49.6 % Basic 28.1 28.2 28.2 28.3 28.2 Analysis of load variants by iCIEF % Acid 38.9 NR 40.8 NR 39.0 % Main 56.5 NR 54.7 NR 56.5 % Basic 4.6 NR 4.6 NR 4.5 % relative power (bioassay) 95 NR 87 NR 89
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Table 26: Stability survey of the mAb1 formulation - Effect of stress conditions
Formulation 25 mg / mL mAbl, 10 mM histidine, pH 6.0, 10% (w / v) sucrose, 0.1% (w / v) polysorbate Filling volume 1.0 mL Container / Sealing 5 mL polycarbonate bottle with silicone-coated polypropylene screw capNo stress Agitation (minutes) Freezing / De-freezing (cycles) Test T = 0 60 120 4 8 Color and appearance Approved Approved Approved Approved Approved Turbidity (increase in OD at 405 nm) 0.00 0.00 0.00 0.00 0.00 pH 6.1 6.1 6.1 6.1 6.1 % of protein recovered by RP-UPLC 100 101 100 99 100 Purity by MCE-SDS Not reduced; % Main peak 99.5 NR 99.5 NR 99.6 Reduced; % Heavy + light chain 100 NR 100 NR 100 Purity by SE-UPLC % HMW 0.8 0.8 0.7 0.8 0.8 % Native 98.6 98.7 98.7 98.7 98.6 % LMW 0.6 0.6 0.6 0.5 0.6 Analysis of load variants by CEX-UPLC % Acid 22.8 22.8 22.8 22.9 22.7 % Main 47.3 47.2 47.3 47.1 47.2 % Basic 30.0 30.0 29.2 30.1 30.1 Analysis of load variants by iCIEF % Acid 40.1 NR 39.6 NR 40.0 % Main 56.1 NR 56.9 NR 56.3 % Basic 3.8 NR 3.5 NR 3.6 % relative power (bioassay) 112 NR 106 NR 137
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Table 27: Stability survey of the mAb1 formulation - Effect of stress conditions
Formulation 25 mg / ml mAb1, 10 mM histidine, 10% (w / v) sucrose, 0.1% (w / v) polysorbate, pH 6.0 Filling volume 1.0 mL Container / Sealing 2 mL type 1 borosilicate glass vials with 13 mm West S2-451 4432/50 GRY B2-40 stoppers coated with FluroTec® Agitation (min) Freeze / Thaw (cycles) Test 0 60 120 4 8 Color and appearance Approved Approved Approved Approved Approved Turbidity (increase in OD at 405 nm) 0.00 0.00 0.00 0.00 0.00 pH 6.2 6.2 6.2 6.2 6.2 Particle analysis by MFI (particles / mL) 2 to 10 pm 823 NR 1170 NR 1404 > 10 pm 15 NR 38, NR 67 > 25 pm 0 0 NR 00 NR 2 % of protein recovered by RP-UPLC 100 100 101 101 101 Purity by MCE-SDS Not reduced; Main peak% 99.4 NR 99.4 NR 99.3 Reduced; % heavy + light chain 100 NR 100 NR 100 Purity by SE-UPLC % HMW 0.7 0.6 0.7 0.7 0.7 % Native 98.7 98.8 98.7 98.7 98.6 % LMW 0.6 0.6 0.6 0.6 0.7 Analysis of load variants by CEX-UPLC % Acid 23.0 22.9 22.8 22.8 22.7 % Main 48.5 48.3 48.4 48.4 48.5 % Basic 28.6 28.8 28.8 28.8 28.8 Analysis of load variants by iCIEF % Acid 39.8 NR 39.6 NR 39.6 % Main 56.7 NR 56.9 NR 56.9 % Basic 3.4 NR 3.4 NR 3.6 % relative power per bioassay 111 NR 90 NR 129
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Table 28: Accelerated and stressed stability of mAb1 formulations at 50 mg / mL and 25 mg / mL
Formulation 50 mg / ml mAb1, 10 mM histidine, 10% (w / v) sucrose, 0.1% (w / v) polysorbate, pH 6.0 25 mg / ml mAb1, 10 mM histidine, 10% (w / v) sucrose, 0.1% (w / v) polysorbate, pH 6.0 Filling volume 1.0 mL 1, Õ mL Container / Sealing 2 mL type 1 borosilicate glass vials with 13 mm West S2-451 4432/50 GRY B2-40 stoppers coated with FluroTec® 2 mL type 1 borosilicate glass vials with 13 mm West S2-451 4432/50 GRY B2-40 stoppers coated with FluroTec® Test T = 0 25 ° C / 60% RH1 month 45 ° C28 days T = 0 25 ° C / 60% RH1 month 45 ° C28 days Color and appearance Approved Approved Approved Approved Approved Approved Turbidity (increase in OD at405 nm) 0.00 0.01 0.02 0.00 0.01 0.02 pH 6.0 6.0 6.1 6.2 6.1 6.1 % of protein recovered by RP-UPLC 100 103 113 100 100 99 Purity by SE-UPLC % HMW 1.9 4.0 10.8 0.7 0.9 8.5 % mo-name-ros 97.5 95.2 88.0 98.7 98.5 89.7 % LMW 0.6 0.8 1.2 0.6 0.6 1.8 Analysis of load variants by CEX-UPLC % Acid 24.6 29.0 37.1 23.0 23.1 34.0 % Main 49.9 42.5 33.2 48.5 48.1 35.9 % Basic 25.6 28.5 29.7 28.6 28.9 30.2
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Example 13: Study of proven acceptable range (PAR)
[0162] During the manufacture of the drug mAb1 (DP), variations in the composition of DP can occur. These variations may include the concentration of the active ingredient, the concentration of the excipients and / or the pH of the formulation. Since changes in any of these parameters can potentially affect drug stability or potency, proven acceptable range (PAR) studies were performed to assess whether variations in DP composition, within defined limits, would affect stability or potency of DP mAb1.
[0163] Two experiment design studies (DOE) were used to evaluate the effect of each formulation parameter, as well as the interactions on formulation stability:
® A pre-PAR fractional factorial design study with accelerated and stress stability assessment to identify critical formulation parameters that may affect the stability of DP mAb1;
® A complete PAR study of factorial design, including critical formulation parameters identified in the PréPAR study, with long-term stability to demonstrate acceptable ranges of formulation parameters.
Pre-PAR Study Design
[0164] To assess critical parameters and / or interaction of formulations in the PD composition that may be important for product quality, a fractional factorial DOE was applied to examine the accelerated and stressed stability of the formulations by varying all formulation parameters , including protein concentration (± 10%), buffer and stabilizer concentrations (± 20%), surfactant concentration (± 50%) and pH (± 0.3 units). The tested formulation parameter ranges were defined as equal to or greater than the specification acceptance and manufacturing experience criteria.
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The study was designed with statistical software using a fractional factorial IV experiment of 2 ^fator (6-2) resolutions. Together with four target formulations as central points, the study included 20 runs, as shown in Table 29.
Table 29: Formulations tested in the pre-PAR study
Formulation [mAb1] mg / ml [histidine]mM % sucrose (w / v) % L-proline (w / v) % polysorbate 80 (w / v) pH Ϊ 45 12 4 1.2 Õ.3 6.3 2 45 12 4 1.8 0.3 5.7 3 45 8 6 1.8 0.3 6.3 4 50 10 5 1.5 0.2 6.0 5 45 8 4 1.8 0.1 6.3 6 55 8 6 1.2 o, 1 6.3 7 55 8 4 1.2 0.3 6.3 8 50 10 5 1.5 0.2 6.0 9 55 12 6 1.8 0.3 6.3 10 45 12 6 1.2 0.1 6.3 11 55 8 4 1.8 0.3 5.7 12 55 8 6 1.8 Õ, 1 5.7 13 45 12 6 1.8 0.1 5.7 14 55 12 6 1.2 0.3 5.7 15 50 10 5 1.5 0.2 6.0 16 45 8 6 1.2 0.3 5.7 17 55 12 4 1.8 o, 1 6.3 18 45 8 4 1.2 0.1 5.7 19 50. 10 5 1.5 0.2 6.0 20 55 12 4 1.2 0.1 5.7
[0165] All 20 formulations under accelerated and stress conditions (25 ° C, 37 ° C, freeze / thaw [F / T] and agitation) were characterized and evaluated for physico-chemical properties and stability, including visual inspection , pH, turbidity, osmolarity, conductivity, purity, concentration and recovery of pro
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Results of the Pre-PAR Study
[0166] All 20 formulations showed no change after agitation or stress by F / T. The results of the incubation at 25 ° C and 37 ° C were analyzed using a regression model (JMP fit model with standard quadratic personality and emphasis on the leverage effect). The statistical analysis of the main variables and interaction of all experimental formulations in relation to the critical attributes of quality revealed that pH, protein concentration and sucrose concentration were important for the quality of the product. The two product quality attributes that had an impact were the HMW species and acid charge variants. It was shown that other formulation parameters, including concentrations of histidine, proline or polysorbate 80, within the tested ranges had no statistically significant impact on product quality. Under accelerated conditions, there were no secondary or higher interactions that had an impact on the stability of the formulation. The results of the pre-PAR study indicated that pH, mAb1 concentration and sucrose concentration were critical for the stability of the mAb1 formulation and the formulation's critical parameters for the 50 mg / mL mAb1 formulation were considered
[0167] Although pH, mAb1 concentration and sucrose concentration have been identified as the critical formulation parameters, the impact of these three factors on quality attributes was minimal. Based on the statistical analysis, the variation in the pH range 5.76.3, 45-55 mg / mL mAb1 and / or 4-6% sucrose probably had an impact <15% on the formation of HMW species and variants acid charge.
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[0168] To confirm the impact on long-term storage stability under the recommended storage condition for DP, the following three critical parameters of the formulation: pH, mAbl concentration and sucrose concentration, were evaluated in a PAR study with long-term storage stability. PAR Study Design
[0169] A DOE of full factorial design was applied to examine the long-term storage stability of formulations with variable pH (± 0.3 units), protein concentration (± 10%) and sucrose concentration (± 20 %), resulting in eight experimental runs (Table 30); a reference formulation (formulation 3, the target formulation in Table 30) was included as the center point formulation.
Table 30: Formulations tested in PAR studies
Formu-lation [mAbl]mg / mL [histidine] mM % of bagsrose (p / v) % L-proline(p / v) % polysorbate 80 (w / v) pH 1 55 10 6 1.5 0.2 5.7 2 45 10 6 1.5 0.2 5.7 3 5Õ 1Õ 5 Ϊ, 5 Õ, 2 6.0 4 55 10 6 1.5 0.2 6.3 5 55 10 4 1.5 0.2 6.3 6 55 10 4 1.5 0.2 5.7 7 45 10 4 1.5 Õ, 2 6.3 8 45 10 4 1.5 0.2 5.7 9 45 10 6 1.5 0.2 6.3
[0170] The complete factorial design study allows estimating all the terms of the main effect, as well as the terms of the interaction. The tested formulation parameter ranges, defined as equal to or greater than the specification acceptance and manufacturing experience criteria, remained the same as in the pre-PAR study.
[0171] The stability of the experimental formulations was compared with the stability of a reference formulation at pH 6.0 that
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108/130 contains all components of the formulation in their nominal concentrations (F3). PAR studies used a batch of DS mAb1 manufactured using the representative commercial manufacturing process. DP formulations were filled into 10 ml Schott type 1 borosilicate glass vials with 20 mm West S2-451 4432/50 GRY B2-40 stoppers coated with FluroTec® (DP commercial representation) and evaluated for stability long-term storage at 2-8 ° C. The formulations were studied according to the analysis plan in Table 31.
Table 31: Analysis plan for the PAR study
Test Samples analyzed Quality attributes Appearance All Color, visible particles Turbidity (increase in OD at 405 nm) All Color, particulates, clarity Nephelometry turbidity t = 0, 6, 12, 18, 24 and36 months at 5 ° C Clarity (Precipitation of the solution, Particulate, Opalescence) pH All pH Total protein content perRP-UPLC t = 0 Protein concentration Osmolality t = 0 Solute concentration Conductivity t = 0 Conductive property Purity by SE-UPLC All Molecular weight variants:%HMW,% monomer,% LWM Analysis of load variants by CIEF t = 0, 6, 12, 18, 24 and36 months at 5 ° C Loading isoforms:% acid,% main,% basic Particulate matter (Light dimming) t = 0, 6, 12, 18, 24 and36 months at 5 ° C Subvisible particulates.Acceptance criteria established at USP <788> (<6000 particles / particle container> 10 pm and <600 particles / particle container> 25 pm) Particulate matter (Microflow Imaging, MFI) t = 0, 6, 12, 18, 24 and 36 months at 5 ° C Subvisible particulates (For information only) Bioassay t = 0, 6, 12, 18, 24 and 36 months at 5 ° C WattageAcceptance criteria: 50-150% of the reference standard
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PAR Study Results
Effect on HMW Species Formation
[0172] There was no significant increase in the% HMW, as measured by SE-UPLC in up to 12 months at 2-8 ° C for all 9 formulations. All values were well below the maximum specification and no significant increase in% HMW was observed over time.
[0173] Formation of HMW species for DP mAb1 at 50 mg / mL at 2-8 ° C was found to be extremely slow. For up to 12 months, the maximum variation in the relative amount in% HMW in the 9 formulations was ~ 0.2%. Since the variation in the% of HMW was minimal and the concentration of monomers could be considered constant, the aggregation of monomers for HMW species could be simplified as a reaction of zero magnitude. Therefore, a simplified linear model was used to analyze the% HMW stability data. By linearly adjusting the% HMW over time, the rate of formation of HMW species was derived for each formulation.
[0174] The rate was analyzed in relation to the main factors, as well as all terms of interaction, using a regression model (JMP adjustment model with standard least squares personality and emphasis on the leverage effect). The resulting regression model was statistically significant, with an R ² of 0.74. The concentration of mAb1, pH and time were statistically significant, however, the effect on the% formation of HMW species was statistically insignificant, contributing only 0.1%.
[0175] Therefore, these factors, pH 5.7 to 6.3, mAb1 concentration from 45 to 55 mg / mL and sucrose concentration of 4-6%, had no practical relevance for the stability of HMW% between 2 -8 ° C.
[0176]% HMW in all 9 formulations at the time point
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110/130 up to 12 months was well below the defined acceptance criteria of 4% and, therefore, within the release and specifications at the end of the useful life period. In addition, the linear model predicted that after 24 months of storage, the shelf life at 2 ° C - 8 ° C, the% HMW, which varied from 0.6% to 0.8%, would also be fine below the specification limit.
[0177] Based on long-term storage stability data, it was found that variations in the critical parameters of the formulation within the studied ranges had no significant impact on the stability of the mAb1 formulation. The 50 mg / mL mAb1 formulation was robust in relation to the formation of HMW species within the tested formulation composition range.
Effect on Formation of Acid Load Variants
[0178] There was no significant increase in the% of acid load variants measured up to 12 months at 2-8 ° C for all 9 formulations. All values were below the maximum specification and there was no significant increase in the% of acid charge variants over time.
[0179] The mAb1 formulation was considered to be robust with respect to the formation of acid charge variants within the composition range of the tested formulation.
Effect on General Quality Attributes
[0180] The effect of pH, concentration of mAb1 and sucrose, as well as storage time, on other general quality attributes of DP, including appearance, pH, turbidity, subvisible particles, protein recovery,% monomer and% LMW per SEC, main% and% basic load variants by iCIEF and bioactivity, was studied. All values were within specifications and no significant variation over time or difference between PAR formulations was observed:
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111/130 * No visible precipitate or particulate was detected by visual inspection or turbidity measurements (OD at 405 nm and nephelometry);
® There were no statistically significant changes in protein recovery (RP-UPLC);
® The pH of the formulations was stable;
® There were no significant increases in subvisible particulates and no significant differences were observed in subvisible particle counts between the formulations of the PAR study.
o For subvisible particles measured by HIAC, all values were below the acceptable limits established by USP <788>, and no significant variation in subvisible particles between formulations was observed.
o In addition, subvisible particles were also measured by IMF. There was no significant variation in the subvisible particles between the formulations.
® The results of the bioassay were within the specification limit for all formulations during storage.
[0181] The results demonstrate that variations in the critical formulation parameters (pH, mAb1 concentration and sucrose concentration) in the studied ranges do not have a significant impact on the stability of the mAb1 formulation. The 50 mg / mL mAb1 formulation is robust with respect to general quality attributes within the tested formulation composition range.
Effect of Freezing and Thawing on the Stability of PAR Formulations
[0182] The physical and chemical stability of the 50 mg / mL mAb1 formulation, examined following two cycles of freezing and thawing, was not affected by the variation in the critical parameters of
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112/130 formulation, that is, a change of ± 0.3 pH units from the reference mAb1, a variation of ± 10% in the concentration of mAb1 and / or a variation of ± 20% in sucrose.
[0183] The following effects have been observed:
® No precipitate was detected by visual inspection or turbidity measurements (OD at 405 nm);
® No protein loss (RP-UPLC) was observed;
«The pH of the formulations remained constant;
® There were no significant differences in subvisible particle counts, determined by light obscuration (HIAC) or microflow imaging (MFI) between the study formulations. There was a slight increase in the subvisible particles after 2 cycles of F / T, which can be removed by filtration through a 0.22 pm filter before filling the DP.
® There were no appreciable changes in purity, as determined by SE-UPLC, in all formulations after two cycles of freezing and thawing;
«There was no appreciable variation in the distribution of the load variants, as determined by iCIEF, in all formulations after two cycles of freezing and thawing.
® The results of the bioassay showed that the activity of mAb1 was maintained in all formulations submitted to 2 cycles of freezing and thawing.
Conclusions
[0184] Pre-PAR and PAR studies based on Experimental Design (DOE) were used to evaluate the effect of formulation parameters, as well as interactions on formulation stability. The pre-PAR study under accelerated stability and stress identified pH, mAb1 concentration and sucrose concentration as critical parameters of the formulation. A complete factorial PAR study with
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113/130 long-term stability demonstrated that the variation in critical formulation parameters, within the study ranges, does not affect the quality of DP mAb1.
[0185] Specifically, the stability and potency of DP mAb1 at 50 mg / mL stored at 5 ° C for 12 months were not affected by a ± 10% variation in protein concentration, a ± 20% variation in concentration of sucrose, L-proline and / or histidine and / or variation of ± 50% in the concentration of polysorbate 80 and / or variation of pH ± 0.3 units.
[0186] The robustness of the mAb1 formulation was demonstrated by the PAR study. In general, the results of the pre-PAR and PAR study held that the variability in the compositions of the mAb1 formulation within the studied ranges would not adversely affect the stability of DP mAb1 under the recommended storage conditions (2 to 8 ° C).
[0187] Samples of SDS mAb1 at 50 mg / mL were stable after two cycles of freezing and thawing (- 30 ° C freezing and thawing at room temperature). The stability of FDS mAb1 to Freeze / Thaw stress was not affected by a ± 10% change in mAb1 concentration, a ± 20% change in sucrose and / or a change in pH ± 0.3 units compared to FDS control mAb1 (50 mg / ml). The results of these freeze / thaw studies provide support that the 50 mg / mL mAb1 SDS can be frozen and thawed during the manufacture of DP mAb1 without adversely affecting the stability of the SDS.
Example 14: Containers
[0188] The mAb1 formulations were developed in glass vials (for delivery via intravenous infusion). The mAb1 drug container for later clinical development and
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114/130 product marketing is also a filled syringe, which is presented as an independent syringe for self-injection or incorporated into an automatic injection device for self-administration. Example 15: Stability of the mAb1 formulation in glass bottles [0189] Tables 32-35 summarize the stability of exemplary mAb1 formulations in 10 ml glass bottles.
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Table 32: Stability survey of the mAb1 formulation stored at 2 - 8 ° C
Formulation 50 mg / mL mAb1, 10 mM L-histidine, 5% (w / v) sucrose, 1.5% (w / v) L-proline and 0.2% (w / v) polysorbate , pH 6.0 Filling volume 5.5 mL Container / Sealing 10 mL type 1 glass vials with a 20 mm chlorobutyl stopper 4432/50 coated with FluroTec® Test Storage duration at 5 “C (months) 0 1 3 6 9 12 18 24 Color and appearance Approved Approved Approved Approved Approved Approved ApprovedTurbidity (increase in OD at 405 nm) 0.00 0.00 0.00 0.00 0.00 0.00 0.00PH 6.0 6.0 6.0 6.0 6.0 6.0 6.0Analysis of subvisible particulates by IMF (N / mL) 2 to 10 pm 175 NR 62 772 NR 1730 NR> 10 pm 8 NR 3 144 NR 18 NR> 25 pm 0 NR •1 19 NR 4 NR% of protein recovered by SE-UPLC 100 102 103 101 105 103 104Purity by MCE-SDS Not reduced; Main peak% 99.1 NR NR 99.1 NR 99.2 NRReduced; % heavy + light chain 100 NR NR 100 NR 100 NRPurity by SE-UPLC % HMW 0.3 0.3 0.3 0.4 0.4 0.4 0.4 I -: -: -: -: -: -: -: -: -: -: -: -: -: -: -: % monomers 99.1 99.3 99.3 99.3 99.2 99.2 99.2% LMW 0.6 0.4 0.4 0.4 0.4 0.4 0.4Analysis of load variants by CEX-UPLC % Acid 18.9 18.9 18.9 19.8 20.5 19.8 18.9% Main 53.5 53.3 53.4 54.7 54.3 54.0 52.9% Basic 27.5 27.8 27.7 25.6 25.2 26.2 28.2Load Variant Analysis poriCIEF % Acid 33.8 NR 34.3 34.2 NR 34.9 NR% Main 54.8 NR 54.2 54.3 NR 54.3 NR% Basic 11.4 NR 11.4 11.4 NR 10.8 NR% relative power (bioassay) 92 NR NR 122 NR 112 NR
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CEX, cation exchange; DS, drug substance; HMW, high molecular weight; ICIEF, isoelectric imaging by capillary focus, LMW, low molecular weight; IMF, microflow; NR, it is not mandatory; OD, optical density; PR, reverse phase; SE, exclusion by size; UPLC, high performance liquid chromatography
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Table 33: Research on the stability of the mAb1 formulation under accelerated and stress conditions
Formulation 50 mg / mL mAb1, 10 mM L-histidine, 5% (w / v) sucrose, 1.5% (w / v) L-proline and 0.2% (w / v) polysorbate , pH 6.0 Filling volume 5.5 mL Container / Sealing 10 ml type 1 glass vials with a 20 mm 4432/50 chlorobuty stopper coated with FluroTec® 25 ° C / 60% RH storage (months) 40 ° C Storage (days) Test THE 1 3 6 7 14 28 Color and appearance Approved Approved Approved Approved Approved Approved Approved Turbidity (increase in OD at 405 nm) 0.00 0.00 0.00 0.00 0.00 0.00 0.00 pH 6.0 6.0 6.0 6.0 6.0 6.0 6.0 Analysis of subvisible particulates by MFI (# / mL) 2-10 pm 175 NR 326 2083 NR NR 288 > 10 pm 8 NR 4 109 NR NR 14 > 25 pm 0 NR 3 3 NR NR 1 % of protein recovered by SE-UPLC 100 102 104 101 100 101 102 Purity by MCE-SDS Not reduced; Main peak% 99.1 NR NR 98.8 NR NR 98.6 Reduced;% heavy + light chain 100 NR NR 99.7 NR NR 99.7 Purity by SE-UPLC % HMW 0.3 0.4 0.5 0.6 0.5 0.8 1.2 % monomers 99.1 99.1 99.1 99.0 98.7 98.5 98.1 % LMW 0.6 0.5 0.4 0.4 0.7 0.7 0.7 Analysis of load variants by CEX-UPLC % Acid 18.9 19.4 22.1 28.3 20.0 22.0 27.1 % Main 53.5 52.7 51.2 48.6 51.9 50.2 46.7 % Basic 27.5 27.9 26.8 23.2 27.1 27.7 26.2 Analysis of load variants by ICIEF % Acid 33.8 NR NR 41.9 NR NR 46.4 % Main 54.8 NR NR 46.5 NR NR 40.9 % Basic 1 i, 4 NR NR 11.6 NR NR 12.7 % relative power per bioassay 92 NR NR 91 NR NR 83
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CEX, cation exchange; DS, drug substance; HMW, high molecular weight; iCIEF, imaging
Petition 870190096716, of 9/27/2019, p. 146/174 isoelectric by capillary focus, LMW, low molecular weight; IMF, microflow; NR, it is not mandatory; OD, optical density; PR, reverse phase; SE, exclusion by size; UPLC, high performance liquid chromatography
Table 34: Stability survey of the mAb1 formulation stored at 2 - 8 ° C
Formulation 50 mg / mL mAb1, 10 mM L-histidine, 5% (w / v) sucrose, 1.5% (w / v) L-proline and polysorbate80 to 0.2% (w / v), pH 6.0 Filling volume 7.44 mL Container / Sealing 1 Õ mL type i glass vials with a 2Õ mm 4432 / 5Õ chlorobutyl stopper coated withFluroTec® Test Storage life at 2-8 ° C (months) 0 1 3 6 9 12 18 24 Color and appearanceTurbidity (increase in OD at 405 nm) pH Approved”Õ: õõ~ 3 ..............................“Õ“ Ϊ83~ 3~ TÕÕ99.2~ TÕÕ Approved”Õ: õõnr"Nr"Nrnrnr”99nrnr Approved”Õ7ÓÕ ~ 6jõ ~ 2"THE~ 2Õ7~ 7~ 3TÕ299.4TÕÕ Approved~ õ7õc>77th ”4.............................'”7044”7s ..........................~ 5TÕ2"9972So Approved“Õ7ÕÕ"G‘q"Nr"Nr"Nrnrnr~ 94 .........................."Nrnr ---------- Analysis of subvisible particles by HIAC (N / mL)Analysis of subvisible particulates by IMF (N / mL) > Õ one> 25 pm2 to 10 um> 10 pm> 25 um % of protein recovered by RP-UPLC Purity by MCE-SDS Not reduced; Main peak%Reduced; % heavy + light chain Purity by SE-UPLC % HMW 0.7 0.6 0.5 0.5 0.6 % monomers 98.8 98.8 98.9 99.0 98.9 % LMW 0.6 0.6 0.5 0.4 0.5
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Formulation 50 mg / mL mAb1, L-histidine a80 to 0.2% (w / v), pH 6.0 10 mM, 5% (w / v) sucrose, 1.5% (w / v) L-proline and polysorbate Filling volume 7.44 mL 1 Õ mL type i glass vials with a 2Õ mm 4432 / 5Õ coriander stopper coated with Container / SealingFluroTec®Storage life at 2-8 ° C (months)Test 0 1 3 6 9 12 18 24 Analysis of load variants % Acid 21.2 21.2 22.3 i 9.6 21.5 - % Main 52.0 52.1 52.0 51.9 52.0 -------------, by CEX-UPLC % Basic 26.8 26.8 25.7 28.6 26.6 Acid 34.3 "Nr ”34.7 ”3476 ~ NR - Analysis of load variants -------------------------------------------------_ % Main 52.1 NR 51.9 51.9 NR - by iCiEF% Basic 13.7 NR 13.4 13.4 NR % relative power (bioassayTÍ3 nr "Ϊ05 ~ Ϊ28 ~ NR -------------------------------------------------
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CEX, cation exchange; DS, drug substance; HMW, high molecular weight; iCIEF, isoelectric imaging by capillary focus, LMW, low molecular weight; IMF, microflow; NR, it is not mandatory; OD, optical density; PR, reverse phase; SE, exclusion by size; UPLC, high performance liquid chromatography
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Table 35: Stability survey of the mAb1 formulation under accelerated and stress conditions
Formulation 50 mg / mL mAb1, 10 mM L-histidine, 5% (w / v) sucrose, 1.5% (w / v) L-proline and 80 a polysorbate0.2% (w / v), pH 6.0 Filling volume 7.44 mL Container / Sealing 10 ml type 1 glass vials with a 20 mm chlorobutyl stopper 4432/50 coated with Fiuro-Tec® 25 ° C / 60% RH storage (months) 40 ° C Storage (days) Test 0 1 3 6 7 14 28 Color and appearance Approved Approved Approved Approved Approved Approved Approved Turbidity (increase in OD at 405 nm) 0.00 0.00 0.00 0.00 0.00 0.00 0.00 pH 6.0 6.0 6.0 6.0 6.0 6.0 6.0 Analysis of subvisible particulatesby HIAC (N / mL) > 10 pm 3 NR 4 11 NR NR 5 > 25 pm 0 NR 0 2 NR NR 0 Analysis of subvisible particulatesby MFI (# / mL) 2-10 pm 183 NR 540 1439 NR NR 799 > 1Õ pm 3 NR 18 16 NR NR 92 > 25 pm 1 NR 4NR NR i % protein recovered by SE-UPLC ions ions 1 õi 1 õi ion 99 100 Purity by MCE-SDS Not reduced; Main peak% 99.2 NR 99.3 98.7 NR NR 98.6 Reduced; % heavy + light chain 100 NR 100 100 NR NR 99.5 Purity by SE-UPLC % HMW 0.7 0.6 0.7 0.8 0.6 0.8 1.3 % monomers 98.8 98.8 98.8 98.7 98.7 98.5 97.9 % LMW 0.6 0.7 Õ, 6 Õ, 6 Õ, 7 0.8 0.8 Analysis of load variants by % Acid 21.2 21.7 25.3 24.4 20.5 23.2 25.8
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Formulation 50 mg / mL mAb1, 10 mM L-histidine, 5% (w / v) sucrose, 1.5% (w / v) L-proline and 80% polysorbate0.2% (w / v), pH 6.0 Filling volume 7.44 mL Container / Sealing Ion mi type i glass vials with a 2O mm 4432/50 chlorobutyl stopper coated with Fluro-TecC © 25 ° C / 60% RH storage (months) 40 ° C Storage (days) Test 0 1 3 6 7 14 28 CEX-UPLC % Main 52.0 52.1 51.4 50.5 52.4 51, Õ 49.7 % Basic 26.8 26.2 23.3 25.1 27.0 25.9 24.6 Analysis of load variants by iCIEF % Acid 34.3 NR 40 0 45 4 NR NR 46.9 % Main 52.1 NR 47, í 43.7 NR NR 39.7 % Basic 13.7 NR 12.9 11.0 NR NR 13.3 % relative power per bioassay 113 NR 143 99 NR NR 88
CEX, cation exchange; DS, drug substance; HMW, high molecular weight; iCIEF, imaging
121/130 isoelectric by capillary focus, LMW, low molecular weight; IMF, microflow; NR, it is not mandatory; OD, optical density; PR, reverse phase; SE, exclusion by size; UPLC, high performance liquid chromatography
[0190] The two formulations in different fill volumes were found to be stable to stress (40 ° C / 75% RH) (data not shown).
Example 16: Stability of the mAb1 formulation in filled syringes
[0191] Tables 36-38 summarize the stability of mAb1 formulations in high concentration in filled syringes.
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Table 36: Stability survey of the drug product mAb1 in filled syringes (PFS) stored at ° C
Formulation 175 mg / mL mAb1, 10 mM histidine, 5% (w / v) sucrose, 1.5% (w / v) L-proline, polysorbate80 to 0.2% (w / v), pH 6.0 Filling volume 1.2 mL Container / Sealing Nuova Ompi EZ Fill glass syringe of 2.25 mL with a 27G 1/2 thin-walled needle and FM30 needle protector closed with a 4023/50 rubber plunger coated with FluroTec®Storage life at 5 ° C (months) Test 0 1 3 6 9 12 Color and appearance Approved Approved Approved Approved Approved Approved Turbidity (increase in OD at 405 nm) 0.00 0.01 0.00 0.00 0.00 0.00 pH 6.0 6.0 6.0 6.0 6.0 6.0 Analysis of subvisible particulates by HI AC (# / mL) > 10 pm 35 NR 17 59 NR 13 > 25 pm 0 NR 4 Ϊ NR Ϊ Analysis of particulates byMFI (particles / mL) 2 to 10 pm 21326 NR 8613 N / D NR N / D > 10 pm 82 NR 303 N / D NR N / D > 25 pm 3 NR 2 N / D NR N / D % of protein recovered by RP-UPLC 100 96 97 100 98 100 Purity by MCE-SDS Not reduced;Main peak% 97.6 NR NR 97.7 97.1 N / D Reduced; % heavy + light chain 99.6 NR NR 99.8 99.8 N / D
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Formulation 175 mg / mL mAb1, 10 mM histidine, 5% (w / v) sucrose, 1.5% (w / v) L-proline, polysorbate80 to 0.2% (w / v), pH 6.0 Filling volume 1.2 mL Container / Sealing Nuova Ompi EZ Fill glass syringe of 2.25 mL with a 27G 1/2 thin-walled needle and FM30 needle guard closed with a 4023/50 rubber plunger coated with FluroTec®Storage life at 5 ° C (months) Test 0 1 3 6 9 12 Purity by SE-UPLC % HMW 0.6 0.6 0.7 0.7 0.8 0.9 % Native 99.1 98.9 99.1 98.7 98.7 98.6 % LMW 0.3 0.5 0.2 0.5 0.5 0.5 Analysis of load variants by CEX-UPLC % Acid 18.6 <8.5 <8.2 18.6 <9.3 <8.8 % Main 53.4 54.8 54.3 53.2 53.5 55.5 % Basic 27.9 26.8 27.5 25.3 25.6 24.3 Analysis of load variants poriCIEF % Acid 30.2 NR 30.2 32.9 NR N / D % Main 55.9 NR 55.9 53.4 NR N / D % Basic 13.9 NR 13.9 13.7 NR N / D % relative power (bioassay) 87 NR NR 141 NR N / D
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Table 37: Stability survey of the drug mAbl in a filled syringe (PFS) stored under accepted conditions
Formulation 175 mg / mL mAbl, 10 mM histidine, 5% (w / v) sucrose, 1.5% (w / v) L-proline, 0.2% (w / v) polysorbate, pH 6.0 Filling volume 1.2 mL Container / Sealing Nuova Ompi EZ Fill glass syringe of 2.25 mL with a 27G 1/2 thin-walled needle and FM30 needle guard closed with a 4023/50 rubber plunger coated with FiuroTec® Storage at 25 ° C / 60% RH (months) Storage at 40 ° C (days) Test 0 1 3 6 7 14 28 Color and appearance Approved Approved Approved Approved Approved Approved Approved Turbidity (increase in OD at 405 nm) 0.00 0.00 0.00 0.00 0.00 0.00 0.00 pH 6.0 6.0 6.0 6.1 6.0 6.0 6.0 Analysis of subvisible particulates by HIAC (# / mL) > 10 pm 3 NR 13 30 NR NR 14 > 25 pm 1 NR 1 4 NR NR 3 Analysis of particulates byMFI (particles / mL) 2-10 pm 21326 NR 4605 N / D NR NR 3717 > 10 pm 82 NR 225 N / D NR NR 51 > 25 pm 3 NR 2 N / D NR NR 8 % of protein recovered per RP-UPLC 100 98 98 100 100 100 98 Purity by MCE-SDS Not reduced;Main peak% 97.6 NR 97.7 96.5 NR NR 96.8 Reduced; % heavy + light chain 99.6 NR 99.2 99.7 NR NR 99.4 Purity by SE-UPLC % HMW 0.6 0.9 1.2 1.6 1.6 2.5 3.9 % Native 99.1 98.6 98.5 97.7 97.8 96.9 95.5
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Formulation 175 mg / mL mAb1, 10 mM histidine, 5% sucrose (w / vj, 1.5% (w / v) L-proline, 0.2% (w / v) polysorbate), pH 6 , 0 Filling volume i, 2 mL Container / Sealing Glass syringe Nuova Ompi ÉZ Fili of 2.25 mL with a 27G 1/2 thin-walled needle and FM3Õ needle protectorclosed with a 4023/50 rubber plunger coated with FluroTec® Storage at 25 ° C / 60% RH (months) Storage at 40 ° C (days) Test 0 1 3 6 7 14 28% LMW Õ, 3 Õ, 5 0.3 0.6 0.6 0.7 Õ, 6 Analysis of load variants by CEX-UPLC % Acid i 8.6 i 9.0 21.3 25 5 20, Õ 22.1 24.3 % Main 53.4 54.3 52.0 46.5 51.8 49.8 48.9 % Basic 27.9 26.8 26.6 28.1 28.3 28, i 26.8 Analysis of load variants by ICIEF % Acid 30.2 NR 36.8 44.9 NR NR 45.6 % Main 55.9 NR 49.6 42.8 NR NR 40.5 % Basic 13.9 NR 13.6 12.3 NR NR 13.9 % relative power per bioassay 87 NR NR 137 NR NR 83
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Table 38: Stability survey of the drug mAb1 in a filled syringe (PFS) - Effect of stress conditions
Formulation 1/5 mg / mL mAb1, 10 mM histidine, 5% (w / v) sucrose, 1.5% (w / v) L-proline, 0.2% (w / v) polysorbate 80 , pH 6.0 Filling volume 1.2 mL Container / Sealing Glass syringe Nuova Ompi EZ Fili of 2.25 mL with a 27G 1/2 thin-walled needle and FM30 needle protector closed with a 4023/50 rubber plunger coated with FluroTec® Agitation (min) Test 0 60 120 Color and appearance Approved Approved Approved Turbidity (increase in OD at 405 nm) 0.00 0.00 0.00 pH 6.0 6.0 6.0 Analysis of subvisible subjects by HIAC (# / mL) > 10 um 3 NR 5 > 25 um 1 NR 5 Analysis of individuals by MFI (particles / mL) 2 to 10 pm 21326 NR 13896 > 10 pm 82 NR 55 > 25 pm 3 NR 3 % of protein recovered by RP-UPLC 100 100 100 Purity by MCE-SDS Not reduced; Main peak% 97.6 NR 97.9 Reduced; % heavy + light chain 99.6 NR 99.5 Purity by SE-UPLC % HMW 0.6 0.6 0.6 % Native 99.1 98.9 98.8 % LMW 0.3 0.6 0.6 Analysis of load variants by CEX-UPLC % Acid 18.6 18.8 19.0 % Main 53.4 53.2 53.5 % Basic 27.9 28.1 27.5 Analysis of load variants by iCIEF % Acid 30.2 NR 30.4 % Main 55.9 NR 55.5 % Basic 13.9 NR 14.1 % relative power per bioassay 87 NR 100
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Example 17: Compatibility of mAb1 formulations in intravenous (IV) delivery devices
[0192] For compatibility assessment, the 50 mg / mL mAb1 formulation was added to a 100 mL IV administration bag containing 0.9% sodium chloride or 5% dextrose for injection to assess whether mAb1 it is stable when delivered intravenously. To support the variability in patient weight, two mixture concentrations, mAb1 at 1.0 mg / mL and mAb1 at 25 mg / mL, were examined in this study to reflect low and high dosage conditions. The following IV mixing components were used during the compatibility studies:
® Drug Product o 50 mg / mL anti-PD mAb1 ® Diluents o 0.9% sodium chloride for injection o 5% dextrose for injection «Bags IV o Bags IV made of polyvinyl chloride (PVC) with phthalate di- (2-ethylhexyl) (DEHP) filled with 0.9% sodium chloride for injection o Bags IV made of polyvinyl chloride (PVC) with DEHP filled with 5% Dextrose for injection o Bags IV of polyolefin (PO) filled with 0.9% sodium chloride for injection o Bags IV made of polyolefin (PO) filled with 5% Dextrose for injection o Polypropylene bags IV filled with 0.9% sodium chloride for injection o Polypropylene intravenous bottles filled with 0.9% sodium chloride for injection
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128/130 * Pumps IV o Peristaltic pump o Fluid displacement pump ® Infusion Sets IV o Set IV of PVC with DEHP o Set IV of PVC with dioctyl terephthalate (DEHT) o Set IV of PVC with trioctyl trimellitate (TOTM ) Set IV of polyethylene coated PVC o Set IV of polyurethane ® Filters o 0.2 pm polyethersulfone in-line filter o 1.2 pm polyethersulfone in-line filter o 5 pm polyethersulfone in-line filter o Filter in 15 pm polyethersulfone line
[0193] The DPs used in this study were GMP manufactured using a representative commercial DP manufacturing process. The IV administration bags containing the mixture were initially kept for 24 hours at 5 ° C; the bags were then incubated for at least 8 hours at 25 ° C. After these incubations, each of the infusion sets was connected to the intravenous bag, prepared with the mixture and kept for 1 hour at room temperature. Each mixture was then pumped through the respective infusion sets at rates of 25 mL / h and 500 mL / h.
Methods used to assess the compatibility of the mixture:
[0194] The compatibility of the mAb1 mixture with the materials used in the IV delivery device was assessed using the following tests:
® Color and appearance by visual inspection * pH ® Turbidity measured by increasing optical density (OD)
Petition 870190096716, of 9/27/2019, p. 157/174
129/130 at 405 nm ® Analysis of subvisible particles in the mixture by obscuring the light (HIAC) ® Protein concentration of mAb1 by means of high performance liquid chromatography in reverse phase (RP-UPLC) ® Purity by SE-UPLC ® Analysis of load variants by CEX-UPLC ® Power, per bioassay: The relative power of each sample is determined using the bioassay and is defined as: (Reference sample IC / Sample sample) * 100%. The measured power of the samples in relation to the storage stability must be within 50-150% of the measured power of the reference standard.
Results and Conclusions:
[0195] The 50 mg / mL mAb1 formulation diluted in either 0.9% sodium chloride or 5% dextrose for injection in concentrations of 1.0 mg / mL or 25 mg / mL, being physically and chemically stable in all conditions tested within the proposed dose ranges and administration conditions. These data support the following conclusions regarding dose preparation and IV administration of DP mAb1:
® The bags of 0.9% sodium chloride for injection and 5% dextrose for invention, made of PVC with DEHP, PO and polypropylene, are compatible with IV administration of mAb1.
® DP mAb1 can be diluted to concentrations as low as 1.0 mg / mL in IV administration bags of PVC, PO or polypropylene containing 0.9% sodium chloride or 5% dextrose for IV administration.
® DP mAb1 can be diluted to concentrations as high as 25.0 mg / mL in IV, PVC, PO or polypropylene administration bags that contain 0.9% sodium chloride or 5% dextrose for IV administration.
Petition 870190096716, of 9/27/2019, p. 158/174
130/130 * The mixture of mAbl in 0.9% sodium chloride or 5% dextrose remained stable after incubation in an IV, PVC, PO or polypropylene administration bag for periods of up to 24 hours at 5 ° C and 8 hours at 25 ° C. The diluted DP mAbl can be administered within 6 hours of preparation.
® The diluted mAbl can be administered using a standard infusion pump.
«The diluted mAbl can be administered with an infusion set consisting of PVC containing DEHP, PVC containing TOTM, polyethylene or polyurethane.
* MAbl is compatible with the use of a 0.2 pm - 5 pm inline polyethersulfone filter.
® The diluted mAbl can be administered at a rate that varies from 25 to 500 mL / hour.
[0196] The scope of the present invention should not be limited by the specific modalities described here. In fact, several modifications of the invention, in addition to those described here, will become evident to those skilled in the art from the previous description and the accompanying figures. Such modifications must fall within the scope of the attached claims.

权利要求:
Claims (72)
[1]
1. Liquid pharmaceutical formulation characterized by the fact that it comprises:
(a) an antibody that specifically binds to human programmed death protein-1 (PD-1), wherein the antibody comprises three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2 and HCDR3) contained in a region heavy chain variable (HCVR) of SEQ ID NO: 1 and three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained in a light chain variable region (LCVR) of SEQ ID NO: 2;
(b) a buffer comprising histidine;
(c) an organic solvent that comprises polysorbate;
(d) a stabilizer comprising a sugar; and (e) a viscosity modifier that comprises an amino acid;
wherein the formulation has a pH 6.0 ± 0.3.
[2]
2. Pharmaceutical formulation according to the claim
1, characterized by the fact that the concentration of antibodies is from 5 mg / mL ± 0.75 mg / mL to 250 mg / mL ± 37.5 mg / mL.
[3]
3. Pharmaceutical formulation according to claim
2, characterized by the fact that the concentration of antibodies is 25 mg / mL ± 3.75 mg / mL.
[4]
4. Pharmaceutical formulation according to claim 2, characterized by the fact that the concentration of antibodies is 50 mg / mL ± 7.5 mg / mL.
[5]
5. Pharmaceutical formulation according to claim 2, characterized by the fact that the concentration of antibodies is 150 mg / ml ± 22.5 mg / ml.
[6]
6. Pharmaceutical formulation according to claim 2, characterized by the fact that the antibody concentration is 175
Petition 870190096716, of 9/27/2019, p. 160/174
2/13 mg / ml ± 26.25 mg / ml.
[7]
Pharmaceutical formulation according to any one of claims 1 to 6, characterized in that the concentration of the histidine buffer is from 5 mM ± 1 mM to 20 mM ± 4 mM.
[8]
8. Pharmaceutical formulation according to claim 7, characterized by the fact that the concentration of the histidine buffer is 10 mM ± 2 mM.
[9]
Pharmaceutical formulation according to claim 7 or 8, characterized in that the histidine buffer comprises L-histidine and L-histidine monohydrate monohydrate.
[10]
10. Pharmaceutical formulation according to claim 9, characterized by the fact that the concentration of L-histidine is 4.8 mM ± 0.96 mM and the concentration of L-histidine monohydrate monohydrate is 5.2 mM ± 1.04 mM.
[11]
11. Pharmaceutical formulation according to any one of claims 1 to 10, characterized by the fact that the concentration of polysorbate is from 0.01% ± 0.005% to 0.5% ± 0.25% w / v.
[12]
12. Pharmaceutical formulation according to claim 11, characterized by the fact that the concentration of polysorbate is 0.1% ± 0.05% w / v.
[13]
13. Pharmaceutical formulation according to claim 11, characterized by the fact that the concentration of polysorbate is 0.2% ± 0.1% w / v.
[14]
Pharmaceutical formulation according to any one of claims 11 to 13, characterized in that the organic solvent is polysorbate 80.
[15]
Pharmaceutical formulation according to any one of claims 1 to 14, characterized in that the stabilizer is sucrose and the concentration of sucrose is 0% to 20% ± 4% w / v.
[16]
16. Pharmaceutical formulation according to claim
Petition 870190096716, of 9/27/2019, p. 161/174
3/13
15, characterized by the fact that the concentration of sucrose is 1% ± 0.2% to 10% ± 2% w / v.
[17]
17. Pharmaceutical formulation according to claim
16, characterized by the fact that the concentration of sucrose is 5% ± 1% w / v.
[18]
18. Pharmaceutical formulation according to claim
17, characterized by the fact that the viscosity modifier is proline.
[19]
19. Pharmaceutical formulation according to claim
18, characterized by the fact that the proline concentration is 0 to 5% ± 1% w / v.
[20]
20. Pharmaceutical formulation according to claim
19, characterized by the fact that the concentration of proline is 1.5% ± 0.3% w / v.
[21]
21. Pharmaceutical formulation according to claim
18, characterized by the fact of understanding:
(a) 175 mg / mL ± 26.25 mg / mL of antibody, (b) 5 mM ± 1 mM to 20 mM ± 4 mM histidine buffer, (c) 0.1% ± 0.05% polysorbate at 0.5% ± 0.25% w / v, (d) 1% ± 0.2% sucrose at 10% ± 2% w / v, and (e) 1% ± 0.2% proline at 5% ± 1% w / v;
at a pH 6.0 ± 0.3.
[22]
22. Pharmaceutical formulation according to claim
21, characterized by the fact of understanding:
(a) 175 mg / mL ± 26.25 mg / mL antibody, (b) 10 mM ± 2 mM histidine buffer, (c) 0.2% ± 0.1% w / v polysorbate, (d ) 5% ± 1% w / v sucrose, and (e) 1.5% ± 0.3% w / v proline;
at a pH 6.0 ± 0.3.
Petition 870190096716, of 9/27/2019, p. 162/174
4/13
[23]
23. Pharmaceutical formulation according to claim
22, characterized by the fact of understanding:
(a) 175 mg / ml ± 26.25 mg / ml of antibody, (b) 4.8 mM L-histidine ± 0.96 mM, (c) L-histidine monohydrate, 5.2 mM ±
1.04 mM, (d) 0.2% ± 0.1% w / v polysorbate, (e) 5% ± 1% w / v sucrose, and (f) 1.5% ± 0 proline, 3% w / v;
at a pH 6.0 ± 0.3.
[24]
24. Pharmaceutical formulation according to claim
18, characterized by the fact of understanding:
(a) 150 mg / ml ± 22.5 mg / ml antibody, (b) 5 mM ± 1 mM 20 mM ± 4 mM histidine buffer, (c) 0.1% ± 0.05% polysorbate at 0.5% ± 0.25% w / v, (d) 1% ± 0.2% sucrose at 10% ± 2% w / v, and (e) 1% ± 0.2% proline at 5% ± 1% w / v;
at a pH 6.0 ± 0.3.
[25]
25. Pharmaceutical formulation according to claim
24, characterized by the fact of understanding:
(a) 150 mg / ml ± 22.5 mg / ml antibody, (b) 10 mM ± 2 mM histidine buffer, (c) 0.2% ± 0.1% w / v polysorbate, (d ) sucrose at 5% ± 1% w / v, and (e) proline at 1.5% ± 0.3% w / v, at a pH 6.0 ± 0.3.
[26]
26. Pharmaceutical formulation according to claim
25, characterized by the fact of understanding:
(a) 150 mg / ml ± 22.5 mg / ml antibody, (b) 4.8 mM L-histidine ± 0.96 mM,
Petition 870190096716, of 9/27/2019, p. 163/174
5/13 (c) L-histidine monohydrate monohydrate at 5.2 mM ±
1.04 mM, (d) 0.2% ± 0.1% w / v polysorbate, (e) 5% ± 1% w / v sucrose, and (f) 1.5% ± 0 proline, 3% w / v; at a pH 6.0 ± 0.3.
[27]
27. Pharmaceutical formulation according to claim
18, characterized by the fact of understanding:
(a) 50 mg / ml ± 7.5 mg / ml antibody, (b) 5 mM ± 1 mM 20 mM ± 4 mM histidine buffer, (c) 0.1% ± 0.05% polysorbate at 0.5% ± 0.25% w / v, (d) sucrose at 1% ± 0.2% at 10% ± 2% w / v, and (e) proline at 1% ± 0.2% at 5% ± 1% w / v, at a pH 6.0 ± 0.3.
[28]
28. Pharmaceutical formulation according to claim
27, characterized by the fact of understanding:
(a) 50 mg / ml ± 7.5 mg / ml antibody, (b) 10 mM ± 2 mM histidine buffer, (c) 0.2% ± 0.1% w / v polysorbate, (d ) sucrose at 5% ± 1% w / v, and (e) proline at 1.5% ± 0.3% w / v, at a pH 6.0 ± 0.3.
[29]
29. Pharmaceutical formulation according to claim
28, characterized by the fact of understanding:
(a) 50 mg / ml ± 7.5 mg / ml antibody, (b) 4.8 mM L-histidine ± 0.96 mM, (c) 5.2 mM L-histidine monohydrate ± 1.04 mM, (d) 0.2% ± 0.1% w / v polysorbate, (e) 5% ± 1% w / v sucrose, and
Petition 870190096716, of 9/27/2019, p. 164/174
6/13 (f) 1.5% ± 0.3% w / v proline; at a pH 6.0 ± 0.3.
[30]
30. Pharmaceutical formulation according to claim
18, characterized by the fact of understanding:
(a) 25 mg / mL ± 3.75 mg / mL antibody, (b) 5 mM ± 1 mM 20 mM ± 4 mM histidine buffer, (c) 0.1% polysorbate ± 0.05% at 0.5% ± 0.25% w / v, (d) 1% ± 0.2% sucrose at 10% ± 2% w / v, and (e) 1% ± 0.2% proline at 5% ± 1% w / v, at a pH 6.0 ± 0.3.
[31]
31. Pharmaceutical formulation according to claim
30, characterized by the fact of understanding:
(a) 25 mg / mL ± 3.75 mg / mL of antibody, (b) 10 mM ± 2 mM histidine buffer, (c) 0.2% ± 0.1% w / v polysorbate, (d ) sucrose at 5% ± 1% w / v, and (e) proline at 1.5% ± 0.3% w / v, at a pH 6.0 ± 0.3.
[32]
32. Pharmaceutical formulation according to claim
31, characterized by the fact of understanding:
(a) 25 mg / mL ± 3.75 mg / mL antibody, (b) 4.8 mM L-histidine ± 0.96 mM, (c) 5.2 mM L-histidine monohydrate ± 1.04 mM, (d) 0.2% ± 0.1% w / v polysorbate, (e) 5% ± 1% w / v sucrose, and (f) 1.5% ± 0 proline , 3% w / v; at a pH 6.0 ± 0.3.
[33]
33. Pharmaceutical formulation according to any one of claims 1 to 32, characterized by the fact that the formulation
Petition 870190096716, of 9/27/2019, p. 165/174
7/13 has a viscosity of less than 20 cP.
[34]
34. Pharmaceutical formulation according to any one of claims 1 to 33, characterized in that at least 90% of the antibody has a native conformation after 28 days at 45 ° C.
[35]
35. Pharmaceutical formulation according to any one of claims 1 to 34, characterized in that at least 35% of the antibody is the main loading variant of the antibody after 28 days at 45 ° C.
[36]
36. Pharmaceutical formulation according to any one of claims 1 to 35, characterized by the fact that at least 94% of the antibody has a native conformation after three months at 25 ° C.
[37]
37. Pharmaceutical formulation according to any one of claims 1 to 36, characterized by the fact that at least
44% of the antibody is the main loading variant of the antibody after three months at 25 ° C.
[38]
38. Pharmaceutical formulation according to any one of claims 1 to 37, characterized by the fact that at least 96% of the antibody has a native conformation after 12 months at 5 ° C.
[39]
39. Pharmaceutical formulation according to any one of claims 1 to 38, characterized by the fact that at least
45% of the antibody is the main loading variant of the antibody after 12 months at 5 ° C.
[40]
40. Pharmaceutical formulation according to any one of claims 1 to 39, characterized in that at least 96% of the antibody has a native conformation after 12 months at -20 ° C, -30 ° C and / or -80 ° Ç.
[41]
41. Pharmaceutical formulation according to any one of claims 1 to 40, characterized in that at least 40% of the antibody is the main loading variant of the antibody after
Petition 870190096716, of 9/27/2019, p. 166/174
8/13
12 months at -20 ° C, -30 ° C and / or -80 ° C.
[42]
42. Pharmaceutical formulation characterized by the fact that it comprises:
(a) 175 mg / ml ± 26.25 mg / ml of an antibody that specifically binds to PD-1, wherein the antibody comprises an HCVR of SEQ ID NO: 1 and an LCVR of SEQ ID NO: 2, ( b) 10 mM ± 2 mM histidine buffer, pH 6.0 ± 0.3, (c) 0.2% polysorbate ± 0.1% w / v, (d) 5% ± 1% sucrose w / v; and (e) proline at 1.5% ± 0.3% w / v; on what:
(i)> 90% of the antibodies have a molecular weight of 143 kDa ± 1 kDa;
(ii) the pharmaceutical formulation has a viscosity of less than 20 cP; and (iii) at least 97% or more of the antibodies have a native conformation after storage at -80 ° C, -30 ° C and / or -20 ° C for 6 months.
[43]
43. Pharmaceutical formulation according to claim
42, characterized by the fact that it consists of:
(a) 175 mg / ml ± 26.25 mg / ml antibody, (b) 4.8 mM L-histidine ± 0.96 mM, (c) L-histidine monohydrate, 5.2 mm ± 1.04 mM, (d) polysorbate 80 at 0.2% ± 0.1% w / v, (e) sucrose at 5% ± 1% w / v; and (f) 1.5% ± 0.3% w / v proline, in water at pH 6.0 ± 0.3.
[44]
44. Pharmaceutical formulation characterized by the fact that it comprises:
(a) 150 mg / ml ± 22.5 mg / ml of an antibody that specifically binds to PD-1, where the antibody comprises an HCVR
Petition 870190096716, of 9/27/2019, p. 167/174
9/13 SEQ ID NO: 1 and an LCVR of SEQ ID NO: 2, (b) 10 mM ± 2 mM histidine buffer, pH 6 ± 0.3, (c) 0.2% polysorbate 80 0.1% w / v, (d) 5% ± 1% w / v sucrose; and (e) proline at 1.5% ± 0.3% w / v; on what:
(i)> 90% of the antibodies have a molecular weight of 143 kDa ± 1 kDa;
(ii) the pharmaceutical formulation has a viscosity of less than 15 cP; and (iii) at least 97% or more of the antibodies have a native conformation when stored at -80 ° C, -30 ° C or -20 ° C for 6 months.
[45]
45. Pharmaceutical formulation according to claim 44, characterized by the fact that it consists of:
(a) 150 mg / ml ± 22.5 mg / ml of the antibody, (b) 4.8 mM L-histidine ± 0.96 mM, (c) L-histidine monohydrate, 5.2 mm ± 1.04 mM, (d) polysorbate 80 at 0.2% ± 0.1% w / v, (e) sucrose at 5% ± 1% w / v; and (f) 1.5% ± 0.3% w / v proline, in water at pH 6.0 ± 0.3.
[46]
46. Pharmaceutical formulation characterized by the fact that it comprises:
(a) 50 mg / ml ± 7.5 mg / ml of an antibody that specifically binds to PD-1, wherein the antibody comprises an HCVR of SEQ ID NO: 1 and an LCVR of SEQ ID NO: 2, ( b) 10 mM ± 2 mM histidine buffer, pH 6 ± 0.3, (o) 0.2% polysorbate ± 0.1% w / v, (d) 1.5% ± 0 proline, 3% w / v, and (e) 5% ± 1% w / v sucrose; on what:
Petition 870190096716, of 9/27/2019, p. 168/174
10/13 (i)> 90% of antibodies have a molecular weight of 143 kDa ± 1 kDa;
(ii) more than 96% of the antibodies have a native conformation when stored for 12 months at 5 ° C; and (iii) the pharmaceutical formulation has a viscosity of less than 15 cP.
[47]
47. Pharmaceutical formulation according to claim
46, characterized by the fact that it consists of:
(a) 50 mg / ml ± 7.5 mg / ml of the antibody, (b) 4.8 mM L-histidine ± 0.96 mM, (c) L-histidine monohydrate monohydrate at 5.2 mm ± 1.04 mM, (d) polysorbate 80 to 0.2% ± 0.1% w / v, (e) 5% sucrose ± 1% w / v; and (f) 1.5% ± 0.3% w / v proline, in water at pH 6.0 ± 0.3.
[48]
48. Pharmaceutical formulation characterized by the fact that it comprises:
(a) 25 mg / ml ± 3.75 mg / ml of an antibody that specifically binds to PD-1, wherein the antibody comprises an HCVR of SEQ ID NO: 1 and an LCVR of SEQ ID NO: 2, ( b) 10 mM ± 2 mM histidine buffer, pH 6.0 ± 0.3, (c) 0.2% polysorbate ± 0.1% w / v, (d) 5% ± 1% sucrose w / v; and (e) proline at 1.5% ± 0.3% w / v; on what:
(a)> 90% of antibodies have a molecular weight of 143 kDa ± 1 kDa;
(b) the pharmaceutical formulation has a viscosity of less than 10 cP; and (c) at least 96% of the antibodies have a native conformation when stored for 12 months at 5 ° C.
Petition 870190096716, of 9/27/2019, p. 169/174
11/13
[49]
49. Pharmaceutical formulation according to claim
48, characterized by the fact that it consists of:
(a) 25 mg / ml ± 3.75 mg / ml antibody, (b) 4.8 mM L-histidine ± 0.96 mM, (c) L-histidine monohydrate 5.2 mm ± 1.04 mM, (d) polysorbate 80 at 0.2% ± 0.1% w / v, (e) sucrose at 5% w / v ± 1%; and (f) proline at 1.5% w / v ± 0.3%, in water at a pH 6.0 ± 0.3.
[50]
50. Pharmaceutical formulation according to any one of claims 1 to 49, characterized in that the antibody comprises an HCDR1 of SEQ ID NO: 3, an HCDR2 of SEQ ID NO: 4, an HCDR3 of SEQ ID NO: 5 , an LCDR1 of SEQ ID NO: 6, an LCDR2 of SEQ ID NO: 7 and an LCDR3 of SEQ ID NO: 8.
[51]
51. Pharmaceutical formulation according to claim 50, characterized in that the antibody comprises an HCVR of SEQ ID NO: 1 and an LCVR of SEQ ID NO: 2.
[52]
52. Pharmaceutical formulation according to any one of claims 1 to 51, characterized in that the antibody comprises an HCVR which has 90% sequence identity with SEQ ID NO: 1.
[53]
53. Pharmaceutical formulation according to any one of claims 1 to 51, characterized in that the antibody comprises an LCVR which has 90% sequence identity with SEQ ID NO: 2.
[54]
54. Pharmaceutical formulation according to any one of claims 1 to 51, characterized in that the antibody comprises an HCVR that has 90% sequence identity with SEQ ID NO: 1 and an LCVR that has 90% identity of sequence with SEQ ID NO: 2.
Petition 870190096716, of 9/27/2019, p. 170/174
12/13
[55]
55. Pharmaceutical formulation according to any one of claims 1 to 51, characterized in that the antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 and 11.
[56]
56. Pharmaceutical formulation according to any one of claims 1 to 51, characterized in that the antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence of SEQ ID NO: 9.
[57]
57. Pharmaceutical formulation according to any one of claims 1 to 51, characterized in that the antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence of SEQ ID NO:
11.
[58]
58. Pharmaceutical formulation according to any one of claims 1 to 51, characterized in that the antibody comprises a heavy chain and a light chain, wherein the light chain comprises the amino acid sequence of SEQ ID NO: 10.
[59]
59. Pharmaceutical formulation according to any one of claims 1 to 51, characterized in that the antibody comprises a heavy chain / light chain comprising amino acid sequences selected from the group consisting of SEQ ID NOs: 9/10 and 11/10.
[60]
60. Pharmaceutical formulation according to any one of claims 1 to 51, characterized in that the antibody comprises a heavy chain / light chain comprising amino acid sequences of SEQ ID NOs: 9/10.
[61]
61. Pharmaceutical formulation according to any one of claims 1 to 51, characterized by the fact that the antibody
Petition 870190096716, of 9/27/2019, p. 171/174
13/13 comprises a heavy chain / chain had to comprise amino acid sequences of SEQ ID NOs: 11/10.
[62]
62. Pharmaceutical formulation according to any one of claims 1 to 61, characterized in that said formulation is contained in a container.
[63]
63. Pharmaceutical formulation according to claim
62, characterized by the fact that the container is a bottle.
[64]
64. Pharmaceutical formulation according to claim
63, characterized by the fact that the vial is a 10 ml Type 1 clear glass vial.
[65]
65. Pharmaceutical formulation according to claim
62, characterized by the fact that the container is a syringe.
[66]
66. Pharmaceutical formulation according to claim 65, characterized by the fact that the syringe is glass with a low tungsten content.
[67]
67. Pharmaceutical formulation according to claim 62, characterized in that the container is a filled syringe.
[68]
68. Pharmaceutical formulation according to claim 62, characterized in that it is contained in an autoinjector.
[69]
69. Kit characterized by the fact that it comprises a pharmaceutical formulation as defined in any one of claims 1 to 61, a container and instructions.
[70]
70. Kit according to claim 69, characterized in that the container is a glass bottle.
[71]
71. Kit according to claim 69, characterized in that the container is a filled syringe.
[72]
72. Kit according to claim 69, characterized by the fact that the container is an auto-injector.

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法律状态:
2021-10-19| B350| Update of information on the portal [chapter 15.35 patent gazette]|

优先权:

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申请号 | 申请日 | 专利标题

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US201762482270P| true| 2017-04-06|2017-04-06|

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US62/482,270|2017-04-06|

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PCT/US2018/024032|WO2018187057A1|2017-04-06|2018-03-23|Stable antibody formulation|

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