improved methods for increasing antibody productivity in mammalian cell culture and minimizing aggre

专利摘要:
The present invention relates to an efficient antibody formulation and production platform that provides: i) cell culture process with improved feeding strategy resulting in high antibody titration between 2 g / l and 5 g / l; ii) improved purification process showing optimum percent recovery, high purity monomer content, minimal particulate aggregation / formation, minimum impurity levels; and iii) high concentration stable liquid formulation with optimum osmolality and low viscosity at different temperatures and without aggregation. Preferred antibodies include dengue virus epitope-specific monoclonal antibody igg1 in protein domain iii and rabies virus surface glycoprotein g-specific monoclonal antibody igg1.
公开号:BR112019011900A2
申请号:R112019011900
申请日:2017-12-20
公开日:2019-11-26
发明作者:Rajeev Mhalasakant Dhere；Sambhaji Shankar Pisar；Reddy Srinivas Reddy Peddi；Digamber Chahar Singh；Leena Ravindra Yeolekar；Pankaj Singh Chouhan；Nikhil Dattatray Avalaskar
申请人:Serum Institute Of India Private Limited；
IPC主号:

专利说明:

Invention Patent Descriptive Report for IMPROVED METHODS FOR INCREASING ANTIBODY PRODUCTIVITY IN MAMMALIAN CELL CULTURE AND MINIMIZING AGGREGATION DURING DOWNSTREAM PROCESSES, FORMULATION PROCESSES, AND FORMULATIONS OF STABLE ANTIBODIES OBTAINED FROM THE STABLE ANTIBODIES OBTAINED.
Background of the Invention [001] The development of upstream, downstream and formulation processes can often be the limiting step in the early introduction of biopharmaceutical products in clinical trials. For example, dengue is the most important mosquito-borne viral disease that affects humans. Half of the world's population lives in areas at risk for dengue, resulting in approximately 390 million infections a year worldwide. Currently, no antiviral agent is approved for the treatment of dengue. Recent vaccine tests have fallen short of expectations. The leading candidate vaccine recently demonstrated limited effectiveness, estimated to be between 30% and 60%, with limited protection to no significant protection against DENV-2. Recently, a non-immunodominant, but functionally relevant, epitope in domain III of protein E has been identified, and subsequently, an engineered antibody, Ab513, is being developed that has high affinity binding to, and neutralizes, widely various genotypes within all the four serotypes (Refer to Ram Sasisekharan et al Cell 162, 1-12, July 30, 2015, Samir Bhatt et al, Nature, 2013, April 25; 496-746: 504-507). Thus, if we consider the global medical demand for a monoclonal dengue anticorpol, recent estimates indicate that up to 390 million dengue infections occur worldwide each year with> 90 million presenting the disease, making DENV a major global threat. If we assume that 30% of the 16 million
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2/60 of diagnosed dengue cases go to the hospital, so about 5 million will need the referred Dengue monoclonal antibody, which implies that purified antibody above 4 g / L becomes a prerequisite to meet the global demand for this saving antibody of lives. In addition, the burden of dengue is high in developing countries where the availability of electricity and refrigeration are often inadequate and, therefore, antibody stability through temperature fluctuations assumes greater relevance for these regions.
[002] In fact, if it were possible to have a platform process that could be used to manufacture and formulate all candidates for monoclonal antibodies (mAb), there would be a great reduction in the time and resources needed to develop the process. This can have a significant impact on the number of clinical candidates that can be introduced into clinical trials. In addition, processes developed for early-stage clinical trials, including those developed using a platform, may not be ideal with respect to process economics, throughput, overall volumes, throughput and may not be suitable for producing the quantities needed for marketing campaigns. final stage or commercial. Another important consideration is the speed of the process development since the process development needs to occur before the introduction of a therapeutic candidate in clinical trials. (Refer to Abhinav A. Shukla et al. Journal of Chromatography B, 848 (2007) 28-39).
[003] Typically, mammalian cell culture media are based on commercially available media formulations, including, for example, Ham's DMEM or F12. Media formulations are often not sufficiently enriched to support increases in cell growth and in the biological expression of
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3/60 protein. There remains a need for better cell culture media, supplements and cell culture methods to improve protein production. Increases in cell culture antibody titers to> 2 g / L have been reported previously. (See F. Wurm, Nat. Biotechnol. 22 (2004) 1393). In addition, in perfusion reactors, cells can reach cell densities much higher than in conventional batch or batch feed reactors. (See Sven Sommerfeld and others Chemical Engineering and Processing 44 (2005) 1123-1137). However, perfusion-based processes are complex, costly and can also result in problems of sterility and unwanted heterogeneity in the glycosylation pattern. The addition of hydrolysates free of animal components (Bacto TC Yeastolate, Phytone Peptona) to chemically defined media is a common approach to increase cell density, culture viability and productivity in a timely manner. Hydrolysates are digested proteins composed of amino acids, small peptides, carbohydrates, vitamins and minerals that provide nutritional supplements to the media. Hydrolysates derived from non-animals from soy, wheat and yeast are commonly used in cell culture media and fed to improve the antibody titer (see US9284371). However, due to its complexity of composition, variations from batch to batch, an undesirable attribute of making the culture viscous, yeast extract and hydrolysates can be a significant source of variability in the medium. Due to the complexity of antibody products that include isoforms and microheterogeneities, the performance of the cell culture process can have significant effects on the quality and potency of the product, especially with regard to glycosylation, post-transcriptional modifications and impurity profiles.
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4/60 [004] At higher concentrations, proteins, particularly antibodies, often exhibit characteristic problems including aggregation, precipitation, gelation, reduced stability and / or increased viscosity.
[005] It is recognized that antibodies have characteristics that tend to form aggregates and particles in solution as they undergo degradation or aggregation or denaturation or chemical modifications resulting in the loss of biological activity over time during the manufacturing process and / or during storage . During cell culture expression, downstream purification, formulation and storage, aggregates of antibodies can be formed. Cell culture harvesting usually contains the highest level of aggregates in the process (See Deqiang Yu Journal of Chromatography A, 1457 (2016) 66-75). Degradation routes for proteins may involve chemical instability (for example, any process that involves modifying the protein by forming or dividing the bond resulting in a new chemical entity) or physical instability (for example, changes in the higher-order structure of the protein). The three most common protein degradation pathways are protein aggregation, deamidation and oxidation. Cleland et al. Critical Reviews in Therapeutic Drug Carrier Systems 10 (4): 307-377 (1993). In addition, proteins are also sensitive to, for example, pH, ionic strength, thermal stress, shear and interfacial stresses, which can lead to aggregation and result in instability. For a protein to remain biologically active, a formulation must therefore preserve the conformational integrity of at least one nuclear sequence of the protein's amino acids intact while, at the same time, protecting the protein's multiple functional groups from degradation.
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5/60 [006] An important problem caused by the formation of aggregates is that during administration the formulation can block syringes or pumps and make them unsafe for patients. These protein modifications can also make them immunogenic, resulting in the generation of anti-drug antibodies by the patient, which can reduce the availability of the drug during subsequent injections or worse induce an autoimmune reaction. A main objective in the development of antibody formulations is to maintain the solubility, stability and bioactivity of the protein.
[007] The first suggestions on how to resolve the instability problems of therapeutic protein formulations included lyophilization of the drug, followed by reconstitution immediately or shortly before administration. However, lyophilized formulations of antibodies have several limitations, including a lengthy process for lyophilization resulting in high manufacturing costs. In addition, a lyophilized formulation must be reconstituted aseptically and accurately by healthcare professionals prior to administration to patients. The reconstitution step itself requires certain specific procedures, that is, (1) a sterile diluent (ie, water for intravenous administration and 5% dextrose in water for intramuscular administration) is added to the vial containing the lyophilized antibody, slowly and aseptically , and the bottle should be rotated very gently for 30 seconds to avoid foaming; (2) the reconstituted antibody may need to remain at room temperature for at least 20 minutes until the solution is clear; and (3) the reconstituted preparation must be administered within six (6) hours after reconstitution. This reconstitution procedure is complicated and the limited time after reconstitution can cause great inconvenience in administering the formulation to patients, leading to significant waste, if not properly reconstituted, or if the dose
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Reconstituted 6/60 is not used within six (6) hours and must be discarded. Therefore, a liquid formulation is desirable due to factors of clinical and patient convenience, as well as ease of manufacture. However, liquid pharmaceutical formulations of protein therapeutic agents, that is, antibodies must be stable over the long term and contain a safe and effective amount of the pharmaceutical compound.
[008] Removing aggregates is more difficult than removing impurities related to the process due to the biophysical similarities between aggregate and monomer, the multiple sources and types of aggregates and less understanding of the aggregation mechanism.
[009] One of the most recent challenges encountered during the development of high concentration monoclonal antibody dosage form formulations is the formation of subvisible and visible protein particles during manufacture and long-term storage. The level of protein and non-protein particles in IgG formulations is an increasingly important part of the development of formulation and purification. (See Klaus Wuchner et al., Journal of Pharmaceutical Sciences, vol. 99, No. 8, August 2010). In addition, the liquid formulation must be stable at different temperatures, that is, temperatures between 2 and 8 ° C, 25 ° C, 40 ° C and 55 ° C. [0010] Many antibody preparations intended for human use require stabilizers to prevent denaturation, aggregation and other alternations in proteins before using the preparation. Previously reported liquid antibody formulations (Lucentis, Avastin) had mannitol, trehalose as stabilizers. (See Susumu Uchiyama and others Biochimica Biophysica Acta 1844 (2014) 20412052; US20160137727; W02009120684; US8568720). However, trehalose is expensive and unviable in economizing large-scale processes.
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In addition, IV antibody administration is generally administered as an infusion rather than a bolus, and thus requires dilution of the mAb formulation, including excipients in appropriate fluids suitable for IV administration. The resulting dilution of excipients, especially surfactants, which may decrease below the concentration necessary to prevent aggregation during agitation, thus resulting in the generation of aggregates and subvisible particles after gentle agitation after dilution in PVC and PO IV bags containing 0% saline. , 9%.
[0012] Hydrophobic interaction chromatography, hydroxyapatite ceramics and cation exchange resins have been used to remove aggregates, but none is ideal. Most of the antibody purification processes previously reported have been heavily based on the use of hydrophobic interaction chromatography in combination with protein A chromatography, anion exchange chromatography, cation exchange chromatography as a three or four step process (see WO2010141039, WO 2014/207763, W02013066707, WO2015099165, W02014102814, WO2015038888, W02004087761). However, hydrophobic interaction chromatography resins require large amounts of salts which are expensive, have low binding capacity, can be difficult to remove and may not be compatible with the materials of building buffer and product tanks. In addition, the difference in density between the buffers used for an HIC step can cause bed stability problems. Hydroxyapatite ceramics can also be used to separate the monomer aggregate, but the ceramic resin can be very difficult to unpack without damaging the resin. Therefore, storing the resin outside the column for reuse in a subsequent manufacturing campaign may not be possible (See Suzanne Aldington Journal of Chromatography B, 848 (2007) 64-78).
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8/60 [0013] It has been found that combinations of three cation exchange flow steps, anion exchange, hydrophobic interaction chromatography and mixed mode cation exchange chromatography properly release the host cell's protein contaminants to a CHO-derived monoclonal antibody . However, these purification schemes generally did not fit into commercial downstream operations due to the need to design the purification sequence separately for each mAb.
[0014] Thus, there is an urgent unmet need for an efficient platform process for manufacturing and formulating antibodies that meets multiple criteria, including robustness, reliability and scalability, in particular a platform that provides i) antibody titre of at least 2 g / L; ii) minimal aggregation / particulate formation through cell culture, purification and formulation processes; iii) improvement of purification, showing excellent percentage recovery, high monomer content and minimum levels of impurity; and iv) high concentration antibody formulation showing low viscosity, without aggregation and subvisible particles; thus presenting long-term stability.
Brief description of the invention [0015] The applicant has surprisingly discovered [0016] A feeding composition and feeding strategy that takes into account the consumption of nutrients, the accumulation of by-products and the balance between promoting growth versus volumetric productivity, in which, in particular, mammalian cell culture process parameters, such as use of particular basal medium, use of concentrated basal medium as a feeding solution, use of different feeding solutions together with a defined feeding strategy, maintaining lower concentrations of lactate and ammonia, have been shown to increase cell growth, longevity
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9/60 cells and protein expression; thus resulting in an increase in antibody titer.
[0017] Specific saline concentration as part of the buffer during the Protein A affinity and cation exchange steps that minimize aggregation; thus obtaining a monomer content greater than 99% with a recovery greater than 80%.
[0018] Liquid formulations of particles-free antibodies containing sucrose in combination with Histidine, Arginine, Polysorbate-80, Sodium chloride which provide greater potency and stability, reduce the viscosity of highly concentrated antibody solutions at 2-8 ° C by at least at least 9 months, at 25 ° C for at least 1 month, at 40 ° C for at least 42 days, at 55 ° C for at least 2 days, compared to sucrose-free formulation.
List of Figures:
[0019] Figure 1: Flowchart - Downstream processing for purification of monoclonal antibody [0020] Figure 2: Flowchart - Monoclonal antibody formulation process.
Detailed Description of the Invention [0021] Therapeutic proteins of the present invention include, but are not limited to, antigen binding protein, humanized antibody, chimeric antibody, human antibody, bispecific antibody, multivalent antibody, multispecific antibody, antigen binding protein fragments , polyclonal, monoclonal, diabody and nanobody, monovalent, heteroconjugate, multispecific, autoantibody, single chain antibodies, Fab fragments, F (ab) '2 fragments, fragments produced by a Fab expression library, anti-idiotypic antibodies (anti- ld), epitope-binding fragments and fragments containing CDR or combinations thereof.
[0022] In one embodiment of the present invention, the tera protein
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10/60 therapeutic is an antigen-binding protein or immunoglobulin; more preferably it is an IgG molecule and most preferably it is an IgG1 molecule. In a first aspect of the present modality, immunoglobulin / antibody is a human IgG1 (allotype G1m3) with a human kappa light chain specific for the epitope of the Dengue virus in domain III of protein E. In a second aspect of the present modality, the antibody is a fully human IgG1 monoclonal antibody specific for the glycoprotein G on the surface of the rabies virus. In a third aspect of the present modality, the therapeutic protein can be selected from the group comprising CTP19, CR57, CR4098, RVFab8, MabJA, MabJB-1, Mab 57, 17C7, 2B10, Ab513 / VIS513, N297Q-B3B9, Mab2E8, 2D22. , DMScHuMab, 3CH5L1, HMB DV5, HMB DV6, HMB DV8, DB32-6, D88, F38, A48, C88, F108, B48, A68, A100, C58, C78, C68, D98, D188, C128, C98, A11, B11, R17D6, R14B3, R16C9, R14D6, R18G9, R16F7, R17G9, R16E5, antibodies derived from the 4E11A modification, adatacept, abciximab, adalimumab, aflibercept, alefacept, alentuzumab. trastuzumab, basiliximab, bevacizumab, belatacepte, bectumomab, certolizumab, cetuximab, daclizumab, eculizumab, efalizumab, entanercept, gentuzumab, ibritumomaumbe, infliximab, ranimumuma, tumizumabuma, CD, omalizumabuma zanolimab, nivolumab, pembrolizumab, hA20, AME-I33, IMC-3G3, zalutumumab, nimotuzumab, matuzumab, ch *) ^A , KSB-102, MR1-1, SC100, SC101, SC103, muromonabe-CD3, OKT4A, gentibrumabe , motavizumab, infliximab, pegfilgrastim, CDP-571, etanercept, ABX-CBL, ABX-IL8, ABX-MAI, panitumumab, Therex, AS1405, natalizumab, HuBC-I, IDEC-131, VLA-I; CAT-152; J695, CAT-192, CAT-213, BR3-Fc, LymphoStat-B, TRAIL-RImAb, bevacizumab, omalizumab, efalizumab, MLN-02, HuMax-IL 15, HuMaxPetição 870190053787, 12/06/2019, p. 121/197
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Inflam, Hu Max-Cancer, HuMax-Lymphoma, HuMax-TAC, clenoliximab, * lumiliximab, BEC2, IMC-ICI1, DCIOI, labetuzumab, arcitumomab, epratuzumab, tacatuzumab, cetuximab, MyelomaCide, LkoCide, PrimoCide, LkoCide, MDX-070, MDX-018, MDX-1106, MDX-1103, MDX-1333, MDX-214, MDX-1100, MDX-CD4, MDX-1388, MDX-066, MDX-1307, HGS-TR2J, FG- 3019, BMS-66513, SGN-30, SGN-40, tocilizumab, CS-1008, IDM-I, golimumab, CNTO 1275, CNTO 95, CNTO 328, mepolizumab, MORIOI, MORI 02, MOR201, visilizumab, HuZAF, volocixmabe, ING-I, MLN2201, daclizumab, HCD 122, CDP860, PR0542, C 14, oregovomab, edrecolomab, etaracizumab, atezolizumab, jplimumab, mogamulizumab, lintuzumab, HulDIO, Lym-1, efalizumabe, LDP-ΟΙ, huA33, WX-G250, sibrotuzumab, ofatumumab. Chimeric KW-2871, hu3S193, huLK26; bivatuzumab. raxibacumab, CI4.18, 3F8, BC8, huHMFGI, MORAb003, MORAb-004, MORAb-009, denosumab, PRO-140, 1D09C3, huMikbeta-1, NI-0401, NI-501, cantuzumab, HuN90N, 8H9, chH B, bavituximab, huJ591, HeFi-1, Pentacea, abagovomab, tositumomab, ustequinumab, 105AD7, GMAI 61, GMA321.
[0023] In another aspect of this modality, the therapeutic protein is an antibody having binding affinity to epitopes present in the Dengue virus, rabies virus, RSV, MPV, influenza virus (influenza), zika virus, West Nile virus , Yellow fever virus, chikungunya virus. HSV, CMV, MERS, Ebola virus, Epstein-Barr virus, Varicella-Zoaster virus, mumps virus, measles virus, polio virus, Rhino virus, adenovirus, hepatitis A virus, hepatitis B virus, hepatitis C virus , Norwalk virus, Togavirus, alpha virus, rubella virus, HIV virus, Marburg virus, Ebola virus, human papilloma virus, polyomavirus, metapneumovirus, coronavirus, VSV and VEE.
[0024] In another aspect of this modality, the isoelectric point (pl)
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12/60 of said antigen binding protein is 7.0 to 8.5, more preferably, from about 7.4 to about 8.2.
[0025] In particular, the antigen binding protein is a therapeutic, prophylactic or diagnostic antibody as described in WO2014025546, WO2015122995, WO2015123362, W02006084006, WO2017027805 and WO2017165736, the contents of which are incorporated herein by reference in their entirety. More preferably, the therapeutic protein is an antibody having 80% similarity to VIS513 (Seq ID 1 or Seq ID 2). In another preferred aspect of the present embodiment, the therapeutic protein is an antibody having more than 80% similarity to the rabies monoclonal antibody (Seq ID 3 and Seq ID 4).
[0026] It is well understood that any host can be used for the expression of therapeutic protein in the methods described here. The cells can be wild-type or genetically modified to contain a recombinant nucleic acid sequence, for example, a gene, which encodes a polypeptide of interest (for example, an antibody).
[0027] In a second embodiment of the present invention, cell line used for the expression of therapeutic proteins is selected from the group including, but not limited to CHO, CHOK1SV GS-KO, GSCHO, CHO DUX-B11, CHO- K1, BSC-1, NS0 myeloma cells, CV-1 in Origin carrier SV40 cells (COS), COS-1, COS-7, P3X3Ag8.653, C127, 293 EBNA, MSR 293, COIo25, U937, SP2 cells , L cell, human embryonic kidney cells (HEK 293), baby hamster kidney cells (BHK 21), African green monkey kidney cells VERO-76, HELA, VERO, BHK, MDCK cells, WI38, NIH-3T3 cells , W138, BT483, Hs578T, HTB2, BT20, T47D, NS0 (a murine myeloma cell line that does not endogenously produce any immunoglobulin chains),
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CRL7O3O, HsS78Bst cells, PER.C6, SP2 / 0-Ag14, a myeloma cell line, a hybrid cell line, human lung cells (W138), retina cells, human hepatoma line (Hep G2) and hybridoma cells.
[0028] In another aspect of the second embodiment, animal or mammalian host cells include but are not limited to Chinese hamster ovary (CHO) cells, such as CHO-K1 (ATCC CCL61), DG44 (Chasin et al., 1986 , Som. Cell Molec. Genet., 12: 555-556; and Kolkekar et al., 1997, Biochem., 36: 10901-10909), SH87 celICHODXB11 (G. Urlaub and LA Chasin, 1980, Proc. Natl. Acad Sci., 77: 42164220, LH Grafe LA Chasin 1982, Molec. Cell. Biol., 2: 93-96), CHO-K1 Tet-On cell line (Clontech), CHO designated ECACC 85050302 (CAMR, Salisbury, Wiltshire, UK), CHO clone 13 (GEIMG, Genova, IT), CHO clone B (GEIMG, Genova, IT), CHO-K1 / SF designated ECACC 93061607 (CAMR, Salisbury, Wiltshire, United Kingdom), RR-CHOK1 designated ECACC 92052129 (CAMR, Salisbury, Wiltshire, United Kingdom), CHOKIsv (Edmonds et al., Mol. Biotech. 34: 179-190 (2006)), CHO-S (Pichler et al., Biotechnol. Bioeng. 108: 38694 (2011)), CHO cells negative for dihydrofolate reductase (CHO / DHFR, Urlaub and Chasin, 1 980, Proc. Natl. Acad. Know. USA, 77: 4216) and dp12.CHO cells (U.S. Pat. No. 5,721,121); monkey kidney CV1 cells transformed by SV40 (COS cells, COS-7, ATCC CRL1651); human embryonic kidney cells (for example, 293 cells or 293 cells subcloned for growth in suspension culture, Graham et al., 1977, J. Gen. Virol., 36:59); baby hamster kidney cells (BHK, ATCC CCL-10); CAP cell, AGE1.HN cell, monkey kidney cells (CV 1, ATCC CCL-70); African green monkey kidney cells (VERO-76, ATCC CRL-1587; VERO, ATCC CCL-81); mouse Sertoli cells (ΤΜ4, Mather, 1980, Biol. Reprod., 23: 243-251); human cervical carcinoma cells (HELA, ATCC
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CCL-2); canine kidney cells (MDCK, ATCC CCL-34); human lung cells (W138, ATCC CCL-75); human hepatoma cells (HEP-G2, HB 8065); mouse mammary tumor cells (MMT 060562, ATCC CCL-51); Buffalo rat liver cells (BRL 3A, ATCC CRL-1442); TR1 cells (Mather, 1982, Ann. NY Acad. Sci., 383: 44-68); MCR 5 cells; and FS4 cells.
[0029] In a first aspect of the second modality, the cell line used for the expression of therapeutic proteins is Chinese hamster ovary cells; more particularly, the cell line is CHOK1SV GS-KO or GS-CHO.
[0030] In a third embodiment of the present invention, cells are cultured in batch mode, fed batch or continuous mode; more particularly in a batch feed mode. It is well understood that a person skilled in the art can modulate a process described in this invention according to available facilities and individual needs. More particularly, the cell culture process is carried out in batch feeding mode providing improved cell growth, cell longevity and increased protein expression ie providing a yield of at least 2 g / L, preferably in the range from 3 g / L to about 6 g / L.
[0031] In a first aspect of the third modality, cell culture is performed in a flask, in a bioreactor, a tank bioreactor, a bag bioreactor or a disposable bioreactor. Preferably, said bioreactor is selected from the group of agitated tank bioreactor, bubble column bioreactor, pneumatically agitated "Airlift" bioreactor, fluidized bed bioreactor or fixed bed bioreactor; and said bioreactor has a volume selected between 1L, 2L, 3L, 5L, 10L, 20L, 100L, 200L, 250L, 350L, 500L, 1000L, 1500L, 3000L, 5000L, 10000L, 20000L and 30,000 liters.
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15/60 [0032] In a second aspect of the third embodiment, the present cell culture media and methods can be used to increase antibody yield by about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 180% or 200%, more preferably, about 40% to 60%, as measured over a fortnight. The batch feeding time can be about 12 to 20 days; about 15 to 20 days or about 15 to 18 days.
[0033] In a fourth embodiment of the present invention, the cell culture medium is selected from the group comprising one or more among CD CHO, CD OptiCHO ™, CD FortiCHO ™ (Life Technologies); Ex-Cell ™ CD CHO (Sigma Aldrich); ProCHO ™ 5 (Lonza); BalanCD ™ CHO Growth A (Irvine Scientific); CDM4Mab (Hyclone); Cellvento ™ CHO-100 (EMD Millipore); Cell wind 200 (Merck Millipore); Cell wind 220 (Merck Millipore); Actipro (Hyclone); and combination of them. Preferably, the cell culture medium is selected from Cell Vento 220 (Merck), ACTIPRO (HyClone / GE) or Gibco ™ Dynamis ™ Medium (Thermo Fisher).
[0034] The cell culture medium is further supplemented with glucose and other food solutions, in order to increase cell growth, cell longevity, and protein expression and yield. It is well understood in the art that the feeding solutions can be supplemented in a fast bolus or in a gradual drip.
[0035] The supplementation of feed solution in cell culture medium with a feeding strategy comprises: [0036] Initial feeding with feed solution A, at 0.05% to 0.5% of the reactor volume, preferably 0.1% to 0.2% of the reactor volume, from day 4;
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16/60 [0037] Feeding with feeding solution A, at 0.1% to 0.5% of the reactor volume on days 6, 8, 10, 11 and 13;
[0038] Feed solution B with less than 8% of the reactor volume, from day 2 to day 14, on alternate days or continuously;
[0039] Feeding with feed solution C with less than 8% of the reactor volume for at least 2 consecutive days from day 2 or day 3, with an intermittent interval of 2 consecutive days until the 12th or 14th day or 15-day or 16-day or 18-day.
[0040] Feeding with food solution D at less than 0.5% of the reactor volume for at least 2 consecutive days from day 4 or day 5, on alternate days or continuously or with an intermittent interval of 2 days consecutive until 12 ² day or 14 ² day or 15 ² day or 16-day or 18-day. Optionally, feed with at least one feed solution selected from EfficientFeed ™ A, EfficientFeed ™ B, EfficientFeed ™ C and Dow Corning Antifoam C.
[0041] In a preferred aspect of the fourth embodiment, said feed solution A, feed solution B, feed solution C, feed solution D, is selected, one or more, from the group comprising Glucose, Cell Boost ™ supplement 5 (Hyclone), EX-CELL 293 (Sigma Aldrich), Cell Boost supplements 7a and 7b (Hyclone), 3X Actipro (Hyclone / GE), Cell Vento 220 (1X medium), EXCELL® Advanced ™ CHO Feed 1, EfficientFeed ™ A, EfficientFeed ™ B and EfficientFeed ™ C, and their combinations.
[0042] In a more preferred aspect of the fourth embodiment, said feeding solution A is the supplement Cell Boost ™ 5 (Hyclone); feed solution B is EX-CELL 293 (Sigma Aldrich), feed solution C is Cell Boost 7a supplement (Hyclone), feed solution D is Cell Boost 7b supplement (Hyclone). Furthermore, the cell culture medium is supplemented with 10% 3X Actipro (Hyclone) at 3 ² day and 8% Cell Wind 220 (half
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1X) ² day 7 in cell culture. It is well understood that any feed addition can vary by ± 1% and ± 1 day by a person skilled in the art.
[0043] Another aspect of the fourth modality includes cell culture conditions used to increase cell growth and longevity and protein expression. The following cell culture conditions employed during the process include, but are not limited to:
[0044] The pH of the cell culture medium is in the range of 6.5 to 7.5;
[0045] The osmolality of the culture medium is in the range of 250 500 mOsm / kg; more preferably 400 - 500 mOsm / kg.
[0046] The dissolved oxygen is in the range of 10 to 60%; preferably 20-40%; more preferably 30%.
[0047] The temperature of the cell culture is in the range of 30 ° C to 38 ° C; the first temperature, preferably, 36-37 ° C and, optionally, the second temperature, preferably, 30-35 ° C.
[0048] The glucose concentration is kept below 7%; preferably between 4% and 5%.
[0049] Harvest the cell culture when the viability is reduced to 80%;
[0050] In which the cell culture conditions are maintained in such a way that the secondary metabolites, such as the lactate concentration does not exceed 5 g / L; and the ammonia concentration does not exceed 5 mMol / L [0051] In the fifth embodiment of the present invention, said therapeutic protein obtained from the cell culture harvest is subjected to a purification process comprising the following steps: i) affinity chromatography , ii) viral inactivation, iii) ion exchange chromatography and iv) filtration; where the overall recovery of the process is
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18/60 greater than 70% and the final purified therapeutic protein has a purity / monomer content of at least 90%, preferably greater than 98%. Other impurities, including residual cell I DNA, residual cell protein and residual protein A in the final purified therapeutic protein are less than 1%.
[0052] In the general aspect of the fifth modality, the inventors of this invention were able to deal with the problem of therapeutic protein aggregation during the downstream processing of said protein, using i) salt in a washing step by affinity chromatography and ii) linear gradient of salt solution for elution in ion exchange chromatography step. In a preferred aspect of said embodiment, the salt concentration of the buffers used in the purification is in the range of 30 mM - 500 mM, more preferably the salt concentration of the buffers used in the purification is in the range of 50 mM - 300 mM.
[0053] In a first aspect of the fifth modality, affinity chromatography selected in the group comprising one or more between protein A chromatography, protein G chromatography, protein L chromatography and combination thereof; preferably, the affinity chromatography used is Protein A chromatography.
[0054] In a second aspect of the fifth modality, the resin used for Protein A chromatography is selected from the group comprising one or more of Eshmuno A, KanCapATM, MabSelect SuRe ™, MabSelect SuRe LX, MabSelect Xtra, rProtein A Sepharose Fast Flow, Poros® MabCapture A, Protein A JWT203 Amsphere ™, ProSep HC, ProSep Ultra and ProSep Ultra Plus. Preferably, the Protein A affinity chromatography resin is MabSelect SuRe ™, Eshmuno A, Kancap A or Poros MabCapture; more preferably, the Protein A affinity chromatography resin is MabPetição 870190053787, of 12/06/2019, pg. 129/197
19/60
Select SuRe ™.
[0055] In a third aspect of the fifth modality, the wash buffer used for Protein A chromatography is selected from the group comprising one or more of:
10-30 mM phosphate buffer, preferably 20 mM phosphate buffer; 100 - 150 mM NaCl, preferably 150 mM NaCl; polysorbate 80 0.05%; pH 7.0 ± 0.2.
10-30 mM phosphate buffer, preferably 20 mM phosphate buffer; 250 mM NaCl - 1M, preferably 1M NaCl; polysorbate 80 0.05%; pH 7.0 ± 0.2.
1-30 mM phosphate buffer, preferably 10 mM phosphate buffer; 100 mM NaCl - 150 mM, preferably 125 mM NaCl; polysorbate 80 0.05%; pH 7.0 ± 0.2.
[0056] In a fourth aspect of the fifth embodiment, the elution buffer used for protein A chromatography comprises 10 - 30 mM citrate buffer; pH 3.0 ± 0.5; and optionally 0.01 - 0.05% (w / v) of polysorbate 80; preferably the elution buffer consists of 20 mM citrate buffer; pH 3.0 ± 0.2; and optionally 0.025% (w / v) of polysorbate 80.
[0057] In a fifth aspect of the fifth modality, the eluate obtained from the affinity chromatography step is subjected to viral reduction and inactivation. It is well understood in the art that the reduction and viral inactivation of the eluate can be carried out by a method selected individually or in combination in the group comprising pH treatment, detergent treatment, heat treatment and filtration for virus reduction. In a preferred aspect of this modality, viral inactivation is carried out by subjecting the eluate to low pH, i.e. 3.3-3.5 for 50 to 100 minutes. In addition, the eluate was neutralized by pH by subjecting it to neutralization buffer, that is, 1M Tris / Citrate buffer, pH 7.0 ± 0.2. It is very well understood in
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20/60 art that any other compatible buffer can be used alternatively for effective neutralization of the eluate by pH.
[0058] In the sixth aspect of the fifth modality, the inactivated viral eluate is subjected to ion exchange chromatography. According to one aspect of this modality, ion chromatography is cation exchange chromatography or anion exchange chromatography or its combination; and chromatography can be carried out in the bind and elute mode or in the flow mode. In the preferred aspect of this modality, cation exchange chromatography and anion exchange chromatography are performed in any sequential order. Another aspect of the fifth embodiment is that said chromatography resin is optionally a multimodal resin such as Capto MMC resin (GE Healthcare).
[0059] In the seventh aspect of the fifth modality, the inactivated viral eluate is subjected to cation exchange chromatography. In a preferred aspect of this embodiment, chromatographic parameters including chromatographic resin and buffering conditions are selected so that the positively charged therapeutic protein binds to the chromatography resin while negatively charged molecules enter the flow, other therapeutic proteins are eluted using a salt gradient. In a preferred aspect of this modality, the cation exchange chromatography resin is selected from the group comprising one or more of: sulfonate-based group (eg MonoS, MiniS, Source 15S and 30S, SP SEPHAROSE® Fast Flow, SP SEPHAROSE® High Performance from GE Healthcare, TOYOPEARL® SP-650S and SP-650M from Tosoh, MACRO-PREP® High S from BioRad, Ceramic HyperD S, TRISACRYL® M and LS SP and Spherodex LS SP from Pall Technologies); a sulfoethyl-based group (eg, FRACTOGEL® SE, from EMD, POROS® ΒΙΟ and S-20 from Applied Biosystems); a sulfopropyl-based group (eg TSK Gel SP 5PW and SP-5PW-HR from Tosoh, POROS®
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21/60
HS-20, HS 50 and POROS® XS from Life Technologies); a sulfoisobutyl-based group (for example, EMD SOs 'FRACTOGEL® EMD'); a sulfoxyethyl-based group (eg What52's SE52, SE53 and Express-Ion S), a carboxymethyl-based group (eg, GE SEPHAROSE® Fast Flow by GE Healthcare, Hydrocell CM by Biochrom Labs Inc., MACRO-PREP ® CM from BioRad, Ceramic HyperD CM, TRISACRYL® M CM, TRISACRYL® LS CM, from Pall Technologies, Matrex CELLUFINE® C500 and C200 from Millipore, CM52, CM32, CM23 and Express-Ion C from Whatman, TOYOPEARL® CM-650S , CM-650M and CM-650C by Tosoh), groups based on sulfonic and carboxylic acid (for example, BAKERBOND® Carboxy-Sulfon by JT Baker), a group based on carboxylic acid (for example, WP CBX by JT Baker, DOWEX ® MAC-3 by Dow Liquid Separations, AMBERLITE® weak cation exchanger resins, DOWEX® weak cation exchanger, and EMION DIAION® weak cation exchanger by SigmaAldrich and EMD COO 'FRACTOGEL® EMD); a sulfonic acid-based group (for example, Hydrocell SP from Biochrom Labs Inc., fine mesh strong acid acid resin (DOC) Resin) DOWEX® from Dow Liquid Separations, UNOsphere S, WP Sulfonic from JT Baker, SARTorius' SARTOBIND® S membrane, AMBERLITE® strong cation exchange resins, DOWEX® strong cation exchange resin and SION-Aldrich DIAION® strong cation exchange resin); and an orthophosphate-based group (for example, Whatman's PI 1). In the most preferred aspect of this modality, the resin used for cation exchange chromatography is Fractogel® EMD SO3 ·, Fractogel® EMD SE Hicap (Merck), CMM HyperCel ™ (Pall Corporation), Capto S ImpAct. In another aspect of the fifth embodiment, process parameters for cation exchange chromatography include, but are not limited to, pre-equilibrium buffer [200 mM citrate buffer; pH 6.0 ± 0.2]; equilibration buffer [10 mM citrate buffer; Polis
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22/60 sorbate 80 (0.025% (w / v)); pH 6.0 ± 0.2]; low pH for neutralization; wash buffer A [10 mM citrate buffer; pH 6.0 ± 0.2]; wash buffer B [20 mM citrate buffer; 300 - 500 mM NaCI; pH 6.0 ± 0.2]; CIP buffer [0.5 MJ NaOH; residence time [4.00 - 7.00 minutes]; column used [XK26], [0060] In the eighth aspect of the fifth modality, the inactivated viral eluate is subjected to anion exchange chromatography. In a preferred aspect of this embodiment, chromatographic parameters including chromatographic resin and buffering conditions are selected in such a way that all negatively charged impurities bind to the membrane while the therapeutic protein elutes in a flow through the membrane. In a preferred aspect of this modality, the anion exchange chromatography resin is selected from the group comprising one or more of: DEAE-cellulose, POROS® PI 20, PI 50, HQ 10, HQ 20, HQ 50, D 50 from Applied Biosystems, SARTOBIND® Q by Sartorius, MonoQ, MiniQ, Source 15Q and 30Q, Q, DEAE and ANX SEPHAROSE® Fast Flow, Q SEPHAROSE, Q SEPHAROSE® High Performance, QAE SEPHADEX® and FAST Q SEPHAROSE® (GE Healthcare), WP PEI, WP DEAM, WP QUAT by JT Baker, Hydrocell DEAE and Hydrocell QA by Biochrom Labs Inc., U Osphere Q, MACRO-PREP® DEAE and MACRO-PREP® High Q by Biorad, Ceramic HyperD Q, ceramic HyperD DEAE, TRISACRYL ® M and LS DEAE, Spherodex LS DEAE, QMA SPHEROSIL® LS, QMA SPHEROSIL® M and MUSTANG® Q from Pall Technologies, strong base type I and type II anionic resins with fine mesh DOWEX® and DOWEX® MONOSPHER E 77, resin anionic weak base from Dow Liquid Separations, INTERCEPT® Q membrane, Matrex CELLUFINE® A200, A500, Q500, and Q800, from Millipore, FRACTOGEL® EMD TMAE, FRA CTOGEL® EMD DEAE and FRACTOGEL® EMD DMAE from EMD, weak and strong AMBERLITE® type I and II anion exchange resins, anion exchange resins
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23/60 weak and strong DOWEX® type I and II, weak and strong anion exchange resins DIAION® type I and II, DUOLITE® from Sigma-Aldrich, TSK gel Q and DEAE 5PW and 5PW-HR, TOYOPEARL® SuperQ-650S , 650M and 650C, QAE-550C and 650S, DEAE-650M and 650C from Tosoh, QA52, DE23, DE32, DE51, DE52, DE53, Express-Ion D and Express-Ion Q from Whatman; more preferably the anion exchange chromatography resin is selected from Sartobind Q (Sartorius), Eshmuno Q (Merck), MUSTANG® Q (Pall Corporation) and Poros X (Thermo). In another aspect of the fifth embodiment, process parameters for anion exchange chromatography include, but are not limited to, cleaning buffer [0.5 M NaOH]; pre-equilibration buffer [200 mM citrate buffer; pH 6.0 ± 0.2]; equilibration buffer [20 mM citrate buffer; pH 6.0 ± 0.2; and optionally Polysorbate 80 0.025%]; storage buffer [0.1 M NaOH]; Linear flow [10 - 500 cm / h, more particularly 100-150 cm / h]; column used [XK26], [0061] The purification process of the aforementioned modalities may further comprise at least one additional chromatography step selected from the group comprising one or more between hydrophilic interaction chromatography, hydrophobic charge induction chromatography, ceramic chromatography hydroxyapatite, multimodal chromatography (Capto MMC and Capto Adhere), membrane chromatography (Q membranes including Intercept ™ (Millipore), Mustang® (Pall Corporation) and Sartobind ™ (Sartorius)).
[0062] In the ninth aspect of the fifth embodiment, virus particles were removed using a 20 nm filter. The filter used to remove viral particles includes, but is not limited to, a virus retaining filter selected from the group of Viresolve PRO (Merck), Planova 20N (Asahi Kasei), Bio EXL PALL PEGASUS PRIME, PEGASUS SV4 (Pall Life Sciences) and Virosart (Sartorius), Virosart CPV filter from Sartorius, Virosolve from Millipore, Ultipor DV20 or DV50 from Pall, Planova 20N and
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24/60
50N or BioEx from Asahi. It is well understood in the art that any other virus-capable filter can be used at this stage; preferably, the filter used for removing viral particles is selected from Viresolve PRO (Merck), BioEx PALL PEGASUS PRIME, PEGASUS SV4 (Pall Life Sciences), and Virosart (Sartorius).
[0063] In the tenth aspect of the fifth embodiment, the therapeutic protein is concentrated to a desired concentration and the buffer is exchanged in the buffer formulation. The plug is exchanged in a tangential flow filtering system or in an ultrafiltration system. The other tangential flow filtration parameters comprise one or more diafiltration selected using diafiltration buffer [25 mM histidine buffer; 75 mM arginine buffer; 50 - 150 mM NaCI; pH 6.50 ± 0.5]; cleaning buffer [0.5 M NaOH]; storage buffer [0.1 M NaOH]; equilibrium using 5 - 10 X membrane volumes; concentration and diafiltration using 10-20 volumes of diafiltration; WFI washing using 3 - 5 volumes of membrane; cleaning using 0.5 - 1.0 M NaOH; storage [NaOH 0.1 M], In one of the preferred aspects of this modality, tangential flow filtration is performed using a 30 kDa MWCO membrane selected from the group comprising one or more PES membranes of the Centramate T series (Pall Corporation), Hydrosart ( Sartorius) and Pelicon 3 (Merck).
[0064] In the sixth embodiment of the present invention, said purified therapeutic protein is formulated with pharmaceutical excipients, where the osmolality of the formulation is in the range of 300 mOsm / Kg to 500 mOsm / Kg and the viscosity of the formulation is less than 2, 5 mPa-S.
[0065] In a first aspect of the sixth embodiment, the therapeutic protein formulation comprises at least one antigen-binding protein, at least one stabilizer, at least one agent
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25/60 buffering, at least one tonicity agent, and at least one surfactant. Optionally, the formulation includes a preservative. [0066] In a second aspect of the sixth modality, the stabilizer is a carbohydrate. The stabilizer is selected from the group comprising one or more of sucrose, sorbitol, trehalose, mannitol, dextran, inositol, glucose, fructose, lactose, xylose, mannose, maltose, raffinose and a combination thereof; more preferably the stabilizer is sucrose. In yet another aspect of this embodiment, the stabilizer includes sucrose in a concentration of about 0.1% to about 2.5% w / v, preferably <1% sucrose w / v.
[0067] In a third aspect of the sixth modality, the buffering agent is selected from the group consisting of one or more among histidine, arginine, glycine, sodium citrate, sodium phosphate, citric acid, HEPES, potassium acetate, potassium citrate , potassium phosphate, sodium acetate, sodium bicarbonate, Tris base or Tris-HCI, and combinations thereof. Preferably, the buffering agent provides a pH of about 5.5 to 7.5, about 6.0 to 7.0, about 6.3 to about 6.8 or about 6.5.
[0068] In a fourth aspect of the sixth modality, the buffering agent is histidine. In a preferred aspect of this embodiment, the buffering agent comprises histidine in a concentration of about 5 mM to about 150 mM, about 10 mM to about 50 mM, about 20 mM to about 40 mM. In the most preferred aspect of this embodiment, the buffering agent includes histidine at a concentration of about 25 mM.
[0069] In a fifth aspect of the sixth modality, the buffering agent is arginine. In a preferred aspect of this embodiment, the buffering agent comprises arginine in a concentration of about 5 mM to about 200 mM, about 50 mM to about 150 mM, about 50 mM to about 100 mM. In the most preferred aspect
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26/60 of this embodiment, the buffering agent includes arginine at a concentration of about 70 to 80 mM.
[0070] In a sixth aspect of the sixth modality, the tonicity agent is selected from the group consisting of one or more among sodium chloride, dextrose, glycerin, mannitol and potassium chloride. In a preferred aspect of this embodiment, the tonicity agent consists of sodium chloride and is present in a concentration of about 10 mM to about 500 mM; preferably at a concentration of about 50 mM to about 250 mM; more preferably at a concentration of about 100 - 145 mM. In the seventh aspect of the sixth modality, the surfactant is present in a concentration of about 0.001 to about 0.2% (w / v); and is selected from the group consisting of one or more polysorbates (for example polysorbate20 or polysorbate-80); poloxamers (for example, poloxamer 188); Triton; sodium dodecyl sulfate (SDS); Sodium lauryl sulfate; octyl sodium glycoside; lauryl-, myristyl-, linoleyl- or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl- or stearyl-sarcosine; linoleyl-, myristyl- or cetyl-betaine; lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, palmidopropyl- or isostearamidopropyl-betaine (for example, lauroamidopropyl); myristamidopropyl-, palmidopropylor isostearamidopropyl-dimethylamine; sodium methyl-cocoyl, or disodium methyloloyl-taurate; and the MONAQUAT® series (Mona Industries, Inc., Paterson, N.J.), polyethylene glycol, polypropyl glycol and copolymers of ethylene and propylene glycol (e.g., Pluronics, PF68, etc.). In a preferred aspect of this embodiment, the surfactant consists of Polysorbate 80 and is present in a concentration of about 0.001% to about 0.2% w / v; preferably at a concentration of about 0.002% to about 0.02%; about 0.005% to about 0.02%, most preferably at a concentration of about 0.02%.
[0071] In the eighth aspect of the sixth modality, the formulation
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27/60 consists of a therapeutic protein at a concentration of about 1 mg / L to about 150 mg / L, about 1 mg / L to about 50 mg / L, about 20 mg / L to about 40 mg / L. Preferably, the formulation consists of a therapeutic protein at a concentration of about 1 mg / L to about 50 mg / L.
[0072] In the ninth aspect of the sixth modality, the formulation also includes preservative, the preservative being able to be selected from the group consisting of benzyl alcohol, m-cresol and phenol.
[0073] In the seventh embodiment of the present invention, the therapeutic protein formulation comprises at least one therapeutic protein, sucrose, arginine, histidine, sodium chloride, polysorbate 80. Preferably the therapeutic protein formulation comprises from about 1 mg / ml to about 50 mg / ml of therapeutic protein; about 20 mM to about 25 mM histidine; from about 50 mM to about 100 mM arginine; from about 0.002% to about 0.02% of polysorbate 80 (w / v); from about 50 mM to about 150 mM NaCI; and <2.5% sucrose w / v. The pH of the formulation is in the range of 6.0 to about 7.0 and the osmolality of the formulation is in the range of 300 mOsm / Kg to about 450 mOsm / Kg.
[0074] In one of the preferred aspects of the seventh embodiment, a pharmaceutical formulation comprises 2-80 mg / ml of Dengue monoclonal antibody; 25 mM histidine; 75 mM arginine; 101 mM NaCI; 0.02% polysorbate 80 (w / v); and 0.5% sucrose w / v; where the pH of the formulation is 6.5 ± 0.5 osmolality of 380 mOsm / Kg, viscosity less than 2.5 mPa-S.
[0075] In one of the preferred aspects of the seventh modality, a pharmaceutical formulation comprises 25 mg / ml of Dengue monoclonal antibody; 25 mM histidine; 75 mM arginine; 101 mM NaCI; 0.02% polysorbate 80 (w / v); and 0.5% sucrose w / v; where the pH of the formulation is 6.5 ± 0.5, osmolality of 380 mOsm / Kg, viscoside
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28/60 of less than 2.5 mPa-S.
[0076] In one of the preferred aspects of the seventh modality, a pharmaceutical formulation comprises 50 mg / ml of Dengue monoclonal antibody; 25 mM histidine; 75 mM arginine; 101 mM NaCI; 0.02% polysorbate 80 (w / v); and 0.5% sucrose; where the pH of the formulation is 6.5 ± 0.5, osmolality of 380 mOsm / Kg, viscosity less than 2.5 mPa-S.
[0077] In one of the preferred aspects of the seventh embodiment, a pharmaceutical formulation comprises 2-80 mg / ml of rabies monoclonal antibody; 25 mM histidine; 75 mM arginine; 101 mM NaCI; 0.02% polysorbate 80 (w / v); and 0.5% sucrose w / v; where the pH of the formulation is 6.5 ± 0.5, osmolality of 380 mOsm / Kg, viscosity less than 2.5 mPa-S.
[0078] In one of the preferred aspects of the seventh embodiment, a pharmaceutical formulation comprises 25 mg / ml of rabies monoclonal antibody; 25 mM histidine; 75 mM arginine; 101 mM NaCI; 0.02% polysorbate 80 (w / v); and 0.5% sucrose w / v; where the pH of the formulation is 6.5 ± 0.5, osmolality of 380 mOsm / Kg, viscosity less than 2.5 mPa-S.
[0079] A pharmaceutical formulation comprises 50 mg / ml of rabies monoclonal antibody; 25 mM histidine; 75 mM arginine; 101 mM NaCI; 0.02% polysorbate 80 (w / v); and 0.5% sucrose; where the pH of the formulation is 6.5 ± 0.5, osmolality of 380 mOsm / Kg, viscosity less than 2.5 mPa-S.
[0080] According to another aspect of the seventh embodiment, said pharmaceutical antibody formulation can be a lyophilized formulation.
[0081] In the eighth embodiment of the present invention, the affinity and potency of the therapeutic protein is measured by one or more of: ELISA or flow cytometry. In a preferred aspect of the eighth modality,
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29/60 the method based on indirect ELISA is used to quantify the binding of the therapeutic protein to the specific antigen. In a preferred aspect of this modality, the Dengue mAb formulation is tested against all dengue virus serotypes and the amount of Dengue mAb is determined. The potency of the therapeutic protein is reported as% of activity relative to the reference standard. It is well understood that any other similar method can be used to demonstrate the potency and affinity of the therapeutic protein.
[0082] In the ninth embodiment of the present invention, the spot reduction neutralization test (PRNT / FRNT) or a related test is performed to assess the neutralization of viral activity by therapeutic protein. In a preferred aspect of this modality, the Dengue mAb formulation is tested against all dengue virus serotypes and EC50 values are calculated for neutralization of dengue viruses. It is well understood that any other similar method can be used to demonstrate the neutralizing activity of the therapeutic protein.
[0083] In the tenth embodiment of the present invention, HPLC-based size exclusion chromatography is used to assess the presence of aggregates in the therapeutic protein formulation. In a preferred aspect of this modality, the Phenomenex BioSec-S 3000 column is used to demonstrate the percentage of aggregate and monomer in the formulation of Dengue mAb. It is well understood that any other similar method can be used to assess the presence of aggregates in the therapeutic protein formulation.
[0084] In the eleventh embodiment of the present invention, the formulation can be stored in a suitable container. The container can be selected from: a bottle, a bottle, a glass bottle, an IV bag, a puff-filled shape / container
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30/60 sealing, a reusable injector, a bolus injector, a syringe, a pen, a pump, a multidose needle syringe, a multidose pen, an injector, a syrette, an autoinjector, a pre-filled syringe or a combination of these .
[0085] At least one primary packaging component comprises a container closure system selected from: polypropylene (PP), polyethylene terephthalate (PETG), high density polyethylene (HDPE), polyethylene terephthalate (PET), polypentafluoro styrene (PFS), polycarbonate, poly (vinyl chloride) (PVC), polyolefin, polycyclopentane (CZ.RTM), cyclic olefinic copolymer (COC), and combinations or copolymers thereof.
[0086] The formulations of anti-dengue antibody or anti-rabies antibody disclosed herein may be used (alone or in combination with other agents or therapeutic modalities) to treat, prevent and or diagnose dengue or rabies viruses. For example, combination therapy may include an anti-dengue antibody molecule co-formulated with, and / or administered with, one or more additional therapeutic agents, for example, antiviral agents (including other anti-dengue antibodies), vaccines (including dengue virus vaccines) , or agents that improve an immune response. In other embodiments, the antibody molecules are administered in combination with other therapeutic treatment modalities, such as intravenous hydration, fever-reducing agents (such as acetaminophen) or blood transfusion. Such combination therapies can advantageously use lower dosages of the administered therapeutic agents, thus avoiding possible toxicities or complications associated with the various monotherapies.
Examples:
[0087] Example 1: Upstream process for cell culture and therapeutic protein expression, that is, monoclonal antibody from
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Dengue (VIS513)
Protocol:
• Cultivation of cells on a 10 L scale was carried out in a batch feeding mode using the parameters mentioned below during the upstream / fermentation process.
• Dengue monoclonal antibody was expressed in a CHO-K1 SV GS-KO cell line obtained from Visterra Inc. USA.
• The cell culture medium used for cell growth and therapeutic protein expression, that is, dengue monoclonal antibody was ΊΧ Cellvento ™ CHO-220 Liquid Medium ”.
• Feed solution A, feed solution B, feed solution C, feed solution D selected from the group comprising glucose, Cell Boost ™ 5 supplement (Hyclone), EX-CELL 293 (Sigma Aldrich), Cell Boost supplements 7a and 7b (Hyclone), 3X Actipro (Hyclone), Cell Vento 220 (3 X medium), “EX-CELL® Advanced ™ CHO Feed 1” were used as supplementary feed.
• pH of the fermentation medium was maintained at 6.7 to 7.5.
• The osmolality of the fermentation medium was maintained at <490 mOsm / Kg.
• Dissolved oxygen from the fermentation medium was maintained at about 20% to about 40%.
• The temperature of the fermentation medium was maintained at 36.5 ± 0.5 ° C.
• The culture was harvested after the cell count dropped to 60%.
[0088] Feed supplementation was done by gradual drip, according to Table 1:
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Table 1
Day feeding strategy protocol Power A Power B Power C Power D Basal medium 1 (3X Actipro (Hyclone)) Basal medium2 (Cellvento ™ CHO-220 (3X)) 0 1 24% 3 10%4 0.2%4% 0.4% 56% 4% 0.4% 6 0.2% 4% 4% 0.4% 76% 8% 8 0.2%4% 0.4% 94% 4% 4.0% 10 0.1% 2% 4% 0.4% 11 0.2% 2% 2% 0.2% 124% 13 0.1% 2% 2% 0.2% 143% 153% 16
Results and conclusion:
[0089] The viable colony count and the yield obtained during the fermentation process were as follows:
Table 2: Counting of viable colonies and yield obtained during the fermentation process
Day Batch 1Batch 2 Viable colony count Title g / l Viable colony count Title g / l 0 0.80.811 1.51.72 2.83.453 4.35.94 6.57.45 8.411.86 10.1 0.54 15.5 0.63 7 12.7 0.77 18.5 0.91 8 14.5 1.18 19 1.29 9 17 1.62 18.5 1.76 10 17.25 2.15 18.8 2.42 11 17.25 2.64 18 2.85 12 16.8 3.0518 3.39 13 15.3 3.617.2 4.0 14 15 3.9917 4.32 15 14.7 4.4314.9 4.68 16 14.5 4.5613.5 4.86
[0090] The Applicant has found that using the cell culture process comprising basal medium, concentrated basal medium as the feed solution, use of feed solutions together with a defined feeding strategy, increased cell growth, lower concentrations of lactate and ammonia, effectively maintaining cell count and increasing
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33/60 of cell longevity and high yield. Yield greater than 4 g L was obtained in the fermentation process. The harvest obtained was also submitted to purification / downstream processing.
Example 2:
[0091] The cell culture obtained in example 1 was harvested and then subjected to a protocol for purification of dengue monoclonal antibody (VIS513) as shown in Figure 1.
The detailed process used was as follows:
Protein-A Affinity Chromatography:
[0092] In this step, the target monoclonal antibody was separated from the components of the medium in the collected supernatant. The clarified supernatant was passed through the chromatography column and then eluted using compatible elution buffers.
Materials used:
Resin (Matrix): Mab Select Sure / Eshmuno A (Protein-A Affinity)
Residence Time: 4.0-8.0 minutes
Column used: XK 26
Equilibration Buffer: 20 mM Phosphate Buffer + 150 mM NaCI + 80 0.05% polysorbate (w / v), pH 7.0 ± 0.2.
Wash Buffer I: 20 mM Phosphate Buffer + 150 mM NaCl + 0.05% polysorbate 80 (w / v), pH 7.0 ± 0.2.
Wash Buffer II: 20 mM Phosphate Buffer + 1 M NaCl + 0.05% polysorbate 80 (w / v), pH 7.0 ± 0.2.
Wash Buffer III: 10 mM Phosphate Buffer + 125 mM NaCl + 0.025% polysorbate 80 (w / v), pH 6.0 ± 0.2.
Elution buffer: 20 mM citrate buffer + 0.025% (w / v) polysorbate 80, pH 3.0 ± 0.2.
CIP buffer: 0.1 M NaOH [0093] Process parameters used:
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Table 3:
Step No. Process step Column volume Linear flow (cm / h) 1 balance 5 <300 2 Load (ml) Actual (as current) <300 3 Wash I 2 <300 4 Wash II 4 <300 5 Wash III 4 <300 6 Elution 5 <300 7 Cleaning 3 <300 8 Storage 2 <300
1. Viral inactivation at low pH
i. Protein A affinity chromatography eluate was subjected to low pH, that is, 3.5 ± 0.1 for 60 ± 10 minutes to inactivate the viral particles.
ii. After maintaining the low pH, the eluate was neutralized using neutralization buffer, that is, 1 M Tris / Citrate buffer with pH 7.0 ± 0.2.
iii. The conductivity of the neutralized eluate was adjusted using WFI with polysorbate 80 0.025% (w / v)
2. Cation exchange chromatography [0094] Positively charged molecules bound to the column while negatively charged molecules enter the flow. Antibody molecules bound to the column are eluted using a salt gradient.
Materials used:
Resin used: Fractogel SOs' / Fractogel SE Hicap (Merck)
Length of stay: 4.00 to 7.00 minutes
Column used: XK 26
Pre-Balance: 200 mM citrate buffer pH 6.0 ± 0.2.
Balance: 10 mM citrate buffer + 0.025% polysorbate 80 (w / v), pH 6.0 ± 0.2.
Load: kept at low pH; neutralized (low pH hold neutralized). Wash buffer A: 10 mM citrate buffer, pH 6.0 ± 0.2.
Wash buffer B: 20 mM citrate buffer + 300 mM NaCI, pH 6.0
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35/60 ± 0.2.
CIP Buffer: 0.5 M NaOH
Storage Buffer: 0.1 M NaOH
Process Parameters:
Table 4:
Step No. Process step CV Linear flow (cm / h) 1 Pre-balance 2 <300 2 balance 5 <300 3 Charge Actual (as current) <300 4 Lavaqem 5 <300 5 Elution 0-60% B (Gradient) 15 <300 6 100% B 2 <300 7 CIP 3 <300
[0095] Fraction collection during the gradient
Table 5:
Stage Fraction name Collection criteria (UV 280) mAU 1 Fraction 01 Up to 300 2 Fraction 02 300 - 700 3 Fraction 03 700 to baseline 4 Fraction 04 (100% B) Baseline to baseline
Anion Exchange Chromatography:
[0096] All negatively charged impurities are bound to the membrane while the antibody enters the flow. Materials used:
Membrane / Resin used: Sartobind Q single Sep mini (Sartorius) / Eshmuno Q
Loading volume: 150mg / ml_-1000 mg / ml
Column used: XK 26
Cleaning Buffer: 0.5 M NaOH
Pre-equilibration buffer: 200 mM citrate buffer, pH 6.0 ± 0.2.
Equilibration Buffer: 20 mM citrate buffer, pH 6.0 ± 0.2; and optionally from PS-80 0.025% w / v pH 6.0 ± 0.2
Storage Buffer: 0.1 M NaOH
Process Parameters:
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Table 6:
Step No. Process step Column volume (CV) Linear flow (cm / h) 1 Cleaning 10 <300 2 Pre-balance 10 <300 3 balance 20 <300 4 Charge Actual (as current) <300 5 Lavaqem post carqa 20 <300 6 Cleaning 10 <300
3. Nano-filtration:
[0097] Nanofilter of 20 nm, that is, Viresolve PRO (Merck) was used to remove any virus particles from the therapeutic protein.
4. Tangential flow filtration / ultra-flow filtration [0098] The antibody was concentrated to the desired concentration and subjected to buffer exchange in one of the three buffers in the formulation. Used material:
Formulation Buffer:
Buffer 1: 25 mM histidine buffer + 75 mM arginine buffer + 75 mM NaCl, pH 6.50 ± 0.25;
Buffer 2: 25 mM histidine, 75 mM arginine, 101 mM NaCl;
Bufffer 3: 25mM histidine, 75mM arginine, 75mM NaCI - 101mM, polysorbate-80 0.002% w / v
Cleaning Buffer: 0.5 M NaOH
Storage Buffer: 0.1 M NaOH
Membrane used: PALL Centramate T Series, PES MWCO membrane: 30 kDa
Process Parameters:
Table 7
Step No. Process step description Note 1 Cleaning 0.5 M NaOH 30 min recirculation 2 WFI Cleaning WFI until the conductivity is below 1.3 pS / cm - 3 balance 400 ml4 Concentration and diafiltration -10-12 DV pass5 WFI Washing with 1000 ml WFI6 Cleaning 0.5 M NaOH 30 min recirculation
Sterile filtration
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37/60 [0099] Stabilizer was added to the antibody solution and sterile filtered through a 0.2 μ filter.
Results:
[00100] Staged recovery of the various stages used in the purification process.
Table 8:
Step No phase Recovery (%) Purity (%) 1 Protein A affinity chromatography 98 99.3 2 Viral inactivation by low pH 98 - 3 Cation exchange chromatography 90 99.52 4 Anion exchange chromatography 95 99.46 5 Nanofiltration 100 - 6 TFF / UFF 98 - 7 Sterile formulation and filtration 100 -
[00101] The overall recovery process was found to be ~ 80% and the total purity was> 99%.
Table 9: Impurity data
Test Criterion accepted Batch 1 Batch 2 HP-SEC purity (% monomer) Monomer must be> 90.0%Monomer retention time should be comparable to the reference standard 99.74 99.11 Residual CHO DNA (pg / mg mAb) <2 pg / mg IgG 0.005 0.004 Residual protein A (ng / mg mAb) <10.00 ng / mg IgG 1.05 0.82 Residual CHO protein (ng / mg mAb) <100.00 ng / mg IgG 1.74 3.80 Endotoxin (EU / mg protein) <0.1 EU / mg protein <0.05 <0.05
Table 10: Batch Recovery & Purity
Step No Batch No. Recovery (%) Purity (%) 1 Batch 1 85 99.21 2 Batch 2 87 99.26
Example 3:
[00102] The purified Dengue monoclonal antibody (VIS513) was formulated as follows:
[00103] Excipients ie arginine, histidine, NaCI, sucrose and polysorbate-80 were added and mixed carefully using a magnetic stirrer at 50-60 rpm to form a mixture of
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38/60 excipients. This mixture was then added to the Dengue mAb TFF harvest gradually with a stirring rate of 50-60 RPM. The pH was checked (pH 6.5) and, if necessary, adjusted by histidine-arginine buffer. The final formulation was filtered through a 0.2 μΜ filter filling the final container.
[00104] The concentration of each component in the final formulation was as follows:
Table 11
Ingredient Formulation 1 Formulation 2 Formulation 3 Dengue mAb (VIS 513) 10 mg / ml 25 mg / ml 50 mg / ml Histidine 25 mM 25 mM 25 mM Arginine 75 mM 75 mM 75 mM Sodium chloride 101 mM 101 mM 101 mM Sucrose 0.5% w / v 0.5% w / v 0.5% w / v Polysorbate-80 0.02% w / v 0.02% w / v 0.02% w / v PH 6.5 ± 0.5 6.5 ± 0.5 6.5 ± 0.5 Osmolality 380 mOsm / kg 380 mOsm / kg 380 mOsm / kg
[00105] These formulations have been tested for purity, stability, effectiveness and potency for 9 months.
Example 4:
[00106] The effect of the presence of sucrose in Dengue VIS513 antibody formulation was studied to test potency through ELISA assay on DV1 EDIII protein. Formulation studies were carried out for temperatures of 2 - 8 ° C, 25 ° C and 40 ° C.
Results:
1. Dengue VIS513 antibody formulation without sucrose
Table 12:
Ingredient (QTY) Power (%) compared to reference standard stored at 2-8 ° C2 to 8 ° C RT (25 ° C) 40 ° C15 days 30 days 15 days 30 days 25 mM L-histidine 78.8 73.2 79.8 53.8 75 mM L-arginine 75 mM sodium chloride Polysorbate 80 0.02% w / v
2. Dengue VIS513 antibody formulation with sucrose
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Table 13:
Ingredient (QTY) Power (%) compared to reference standard stored at 2-8 ° C2 to 8 ° C RT (25 ° C) 40 ° C15 days 30 days 15 days 30 days 25 mM L-histidine 90.5 86.3 94.6 81.6 75 mM L-arginine 101 mM sodium chloride Polysorbate 80 0.02% w / v Sucrose 0.5% w / v
Composition of the reference standard formulation
Ingredient (QTY) L-histidine 25 mM L-arginine 75 mM Sodium chloride 101 mM Polysorbate 80 0.02% w / v Sucrose 0.5% w / v
Conclusion: The addition of 0.5% w / v improves stability compared to the corresponding sampling point without sucrose.
Example 5:
[00107] The VIS513 antibody formulation was stored at 40 ° C for 20 days and the posterior potency of VIS513 was assessed by the ELISA test. The effect of increasing sucrose content was studied in the VIS513 antibody formulation at 40 ° C, in which the sucrose concentration of 0.1.0.2 and 0.5% was evaluated.
Results:
Table 14:
Ingredient (QTY) Power (%) compared to reference standard stored at 2-8 ° C 25 mM L-histidine 53.8 75 mM L-arginine 101 mM sodium chloride Polysorbate 80 0.02% w / v
Table 15:
Ingredient (QTY) Power (%) compared to reference standard stored at 2-8 ° C 25 mM L-histidine 70.8 75 mM L-arginine 101 mM sodium chloride Polysorbate 80 0.02% w / v Sucrose 0.1% w / v
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Table 16:
Ingredient (QTY) Power (%) compared to reference standard stored at 2-8 ° C 25 mM L-histidine 65.7 75 mM L-arginine 101 mM sodium chloride Polysorbate 80 0.02% w / v Sucrose 0.2% w / v
Table 17:
Ingredient (QTY) Power (%) compared to reference standard stored at 2-8 ° C 25 mM L-histidine 81.6 75 mM L-arginine 101 mM sodium chloride Polysorbate 80 0.02% w / v Sucrose 0.5% w / v
Composition of the standard reference formulation:
Ingredient (QTY) L-histidine 25 mM L-arginine 75 mM Sodium chloride 101 mM Polysorbate 80 0.02% w / v Sucrose 0.5% w / v
Conclusion: The greatest stability was observed in the formulation containing 0.5% sucrose.
Example 6:
[00108] Analytical test for purity, stability, efficacy and potency of the Dengue mAb formulation (VIS513) with storage at • 2-8 ° C for a period of 0 months, 3 months, 6 months, months, 12 months and 18 months;
• 25 ° C for a period of 0 days and 30 days;
• 40 ° C for a period of 0 days, 7 days, 14 days, 28 days, 35 days and 42 days.
6.1: The potency of the VIS513 antibody formulation was tested by indirect ELISA.
[00109] The indirect ELISA-based method was used when
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41/60 to bind Dengue mAb (VIS513) to the DVII antigen EDIII protein. EDIII protein was immobilized on the plate. Unbound antigen was washed away. In the next step, standard and test samples were added, which bound to the antigen. To determine the amount of bound Dv-mAb, mouse anti-human IgG Fc-HRP, specific for Dv-Mab (human immunoglobulin Fc fragment), was used to recognize the presence of Dv-Mab. The test was developed with the TMB Microwell Peroxidase Substrate System, which quantifies the extent of binding by the amount of color formed at 450 nm. The data analysis program generated a connection curve for each sample using a curve fitting model with 4 parameters and compared the connection curve of the test sample to the standard curve by calculating the relative power. The power of a test sample is reported as% of activity in relation to a reference standard (relative power x 100).
Results:
Table 18: Dengue mAb potency (VIS513) (%) by indirect ELISA for formulation stored at 2 - 8 ° C.
0 day 3 months 6 months 9 months 12 months 18 months Batch 1 74.90 79.30 89.30 84.9 88.5 93.90 Batch 2 74.30 80.05 82.20 86.7 79.00 94.70 Batch 3 91 98.30 125 99.70 88.80 118.40 Batch 4 97.5 102.2 92.6 84 83.80 96.70
Table 19: Dengue mAb potency (VIS513) (%) by indirect ELISA for formulation stored at 25 ° C.
0 day 30 days Batch 1 74.90 79.30 Batch 2 74.30 87.8
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Table 20: Dengue mAb potency (VIS513) (%) by indirect ELISA for formulation stored at 40 ° C.
0 day 14 days 21 days 28 days 35 days 42 days Batch 1 70.3 82.80 96.20 71.90 89.70 90.80
[00110] The dengue mAb formulation (VIS513) did not show any time-dependent loss of binding affinity.
6.2: PRNT assay to determine EC50 [00111] The assay means premixing antibodies serially diluted with virus to allow antibody binding, neutralization and then transfer of the mixture to a Vero cell monolayer, overlapping with a viscous medium , incubation (~ 3-7 days, depending on the virus serotype) to allow limited virus replication and spread, followed by plaque detection. Neutralization was captured by the reduction of plaque formation. Robust detection was achieved with immunostaining methods, using mouse anti-dengue antibody 4G2 and goat anti-mouse antibody labeled with HRP with peroxidase substrate.
[00112] Samples of dengue mAb formulation (VIS513) were tested against all four dengue virus serotypes, namely DV1, DV2, DV3 and DV4. The EC50 value was calculated for the neutralization of Dengue viruses. The EC50 value represents the effective concentration for 50% effective neutralization of dengue viruses, the EC50 value being calculated from the number of plaques present in the virus control wells and the number of plaques in the wells in which mAb-virus samples incubated have been added. Results:
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Table 21: EC50 value of Dengue mAb (VIS513) (ng / ml) per PRNT test for formulation stored at 2 - 8 ° C.
Batch # Dengue virus serotypes 0 day 3 months 6 months 9 months 12 months 18 months Batch 1 DV1 47.15 49.74 23.88 24.1242.58 DV2 5.9 8.03 3.58 3.3113.03 DV3 14.38 14.7111 5.58 4.5718.14 DV4 30.29 50.75 23.96 23.35117597 Batch 2 DV1 21.03 21.01 16.12 29.4162.94 DV2 3.26 5.69 4.15 2.878.59 DV3 13.19 24.14 6.53 7.0527.83 DV4 22.46 22.44 16.61 19.56155207 Batch 3 DV1 32.77 13.74 27.76 34.2260.48 DV2 4.34 7.37 2.94 4.2616.80 DV3 15.43 4.96 6.76 7.7414.21 DV4 30.49 16.36 39.71 31.75141205 Batch 4 DV1 22.81 24.67 23.34 46.7223.48 DV2 3.16 3.25 1.25 6.3523.49 DV3 11.42 7.35 4.65 5.34128.6 DV4 21.39 24.58 19.56 22.3115814
Table 22: EC50 value of Dengue mAb (VIS513) (ng / ml) per PRNT test for formulation stored at 25 ° C.
Batch # Dengue virus serotypes 0 day 30 days Batch 1 DV1 47.15 29.82 DV2 5.9 5.6 DV3 14.38 9.63 DV4 30.29 35.28 Batch 2 DV1 21.03 28.34 DV2 3.26 5.51 DV3 13.19 9.25 DV4 22.46 30.69
Table 23: EC50 value of Dengue mAb (VIS513) (ng / ml) per PRNT test for formulation stored at 40 ° C.
Batch # DV2 serotype of the dengue virus Control test Batch 1 0 day 6.66 - 7 days 6.37 26.89 14 days 6.18 36.12
[00113] The dengue mAb formulation (VIS513) did not show any time-dependent loss of virus neutralization efficacy at 2-8 ° C & 25 ° C. VIS513 formulation even when kept at 40 ° C, does not lose its ability to neutralize the dengue virus.
6.3: Analysis of aggregation and purity [00114] A size exclusion chromatography based on
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HPLC (HPLC-SEC) was used to evaluate the aggregates in the crude and final formulation of DV mAb. In this method, a phenomenex BioSec-S 3000 column was used to demonstrate the aggregates and the percentage of Dengue mAb monomer (VIS513) by injecting 50 pg of total antibody and running at a flow rate of 1 ml / minute for 35 minutes. Phosphate buffered saline (PBS), pH 6.5, was used as the mobile phase.
[00115] Results: SEC-HPLC (acceptance range is not less than (not less than -NLT) 90%)
Table 24: SEC-HPLC analysis of formulation stored at 2 - 8 ° C
0 day 3 months 6 months 9 months 12 months 18 months Batch 1 % Monomers 99.21 98.60 99.14 98.55 98.25 98.14 % Aggregates 0.78 1.39 0.85 1.44 1.74 1.85 Batch 2 % Monomers 99.26 98.81 98.89 98.53 98.36 98.18 % Aggregates 0.73 1.18 0.86 1.45 1.63 1.81 Batch 3 % Monomers 99.25 99.35 98.99 98.81 98.78 98.22 % Aggregates 0.74 0.64 1.00 1.18 1.18 1.77 Batch 4 % Monomers 99.25 99.12 98.69 98.93 98.42 93.48 % Aggregates 0.74 0.82 1.30 1.06 1.42 1.95
Protein Concentration Analysis
Table 24A: Protein Concentration (mg / mL) Analysis by UV measurement of the formulation stored at 2 - 8 ° C
0 day 3 months 6 months 9 months 12 months 18 months Batch 1 25.94 25.24 25.13 24.71 25.37 22.71 Batch 2 25.80 25.20 25.75 24.59 26.63 23.72 Batch 3 24.72 24.39 24.75 25.67 24.02 24.79 Batch 4 26.04 26.23 26.27 26.92 26.63 25.40
Analysis of Subvisible Particles of formulation stored at 2 - 8 ° C [00116] The particulate material in the injections consists of undissolved mobile particles, other than gas bubbles, involuntarily present in the solutions. Dengue mAb particulate material (VI513) formulated in bulk was analyzed over time. For parenterals with a volume of less than 100 mL, the test is approved when per container, less than 6000 particles of size equal to or greater than 10μ are observed, or less than 600 particles of size equal to or greater than 25 μ.
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Table 24B:
Particle size Sampling Point (No. of particles / mL)Initial 3 months 6 months 12 months > 10 μ 1 3 5 9 > 25 μ 0 0 3 1
[00117] The bulk formulation has stability, since only a marginal increase in particulate matter was observed. Table 25: SEC-HPLC analysis of formulation stored at 25 ° C
0 day 30 days Batch 1 99.21 97.63 Batch 2 99.26 97.58
Table 26: SEC-HPLC analysis of formulation stored at 40 ° C
0 day 7 days 14 days 21 days 29 days 35 days 42 days Batch 1 99.21 99.30 99.50 97.74 97.30 98.70 95.30
[00118] Dengue mAb formulation (VIS513) did not show any significant time-dependent aggregation; and the purity / monomer content was found to be> 98%.
Protein Concentration Analysis
Table 26A: Protein Concentration (mg / mL) Analysis by UV measurement of the formulation stored at 40 ° C
0 day 3 days 7 days 15 days Batch 1 27.53 26.09 25.46 26.58
[00119] The variation in the measurement of protein concentration was found within the variability of the test analysis by UV measurement.
[00120] Studies of pH and isoelectric focusing (protein separation based on its isoelectric point) on the formulation of Dengue mAb (VIS513) stored at 40 ° C.
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Table 26B: pH measurement of Dengue mAb (VIS513) stored at 40 ° C.
0 day 3 days 7 days 15 days Batch 1 6.4 6.38 6.38 6.33
Table 26C: Identification by SDS PAGE (Reducer / Non-Reducer) & Isoelectric Focusing of Dengue mAb (VIS513) stored at
40 ° C.
test 0 day 3 days 7 days 15 days SDS PAGE (Reducer) Single diffused band corresponding in position and intensity with reference standard AT AT Single diffused band corresponding in position and intensity with reference standard SDS PAGE (Non-Reducer) Single diffused band corresponding in position and intensity with reference standard AT AT Single diffused band corresponding in position and intensity with reference standard Isoelectric focusing Std. PI = 7.8 ± 0.3 Compliant with internal reference standard (SR) AT AT Compliant with internal reference standard (SR)
Example 7:
Effect of surfactant concentrations in Formulations:
[00121] The effect of the surfactant concentration was evaluated by analysis of subvisible particles. Formulations varying Polysorbate-80 concentrations were prepared and analyzed for subvisible particles.
Table 27
Formulation with 80 cone polysorbate. (% w / v)0.0016% 0.002% 0.005% Particle size > 2μ 916 211 20 > 5 μ 55 48 5 > 10 μ 6 16 2 > 25 μ 1 1 0
Conclusion:
[00122] In the formulation containing 0.005% w / v polysorbate-80 minimal subvisible particles were observed. Depending on the dose, if the formulation requires dilution, the concentration of polysorbate 80 was
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Example 8: Study to determine the minimum concentration of the stabilizers used [00123] The required minimum buffer force (10-30mM) has been referred to the available literature. In order to discover the minimal arginine (used as a solubilizing agent and viscosity reducing agent), the buffer of the mAb sample was changed to normal saline and a solution of arginine stock (300 mM) was gradually added. The aggregation of the solution was monitored by OD @ 350 nm. The saline solution with 75 mM Arginine provided the lowest OD, resulting in the completion of 75 mM arginine.
Example 8
Viscosity studies of the dengue antibody formulation (VIS513) [00124] The viscosity of the mAb DV samples was measured on a microchip-based viscometer, Model: microVISCTM (Manufacture: RheoSense, CA USA) according to the procedure mentioned in the instrument manual.
Table 28
Sample Measuring temperature Viscosity MPa.s Day 0 5 ° C 2.04 25 ° C 1,164 Day 90 stored at 2 - 8 ° C 25 ° C 1,173 Day 180 stored at 2 - 8 ° C 25 ° C 1,167 Day 180 stored at 2 - 8 ° C 25 ° C 1,166 Day 180 stored at 2 - 8 ° C 25 ° C 1,174 Day 365 stored at 2 - 8 ° C 25 ° C 1,175 Day 30 stored at 25 ° C 25 ° C 1,137 Day 60 stored at 25 ° C 25 ° C 1,127 Day 90 stored at 25 ° C 25 ° C 1,122
Conclusion:
[00125] No time-dependent increase in viscosity was observed in the mAb formulation stored at 2-8 ° C for 90 days, as well as in a sample maintained at 25 ° C for 1 month; this is main
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48/60 mainly due to the excipient - 75 mM Arginine. It was found that the viscosity of our formulation was 1.1 to 1.2 mPa-S / cP, which is lower than that of other commercial formulations that have viscosity between 11-50 mPa-S / cP The viscosity of the aggregate sample does not increase even after storage for 1 year at 2-8 ° C or for 3 months at 25 ° C.
Example 9: Viral Spiking studies in the purification process of Dengue mAb (VIS513) [00126] Virus validation was performed for the actual manufacturing process, to test the effectiveness of virus removal by virus filtration in the manufacturing process monoclonal antibody.
[00127] Murine Leukemia Virus (MuLV) and Mouse Minute Virus (MMV / MVM) were used as model organisms. The inventors of this invention compared the ability of their invention purification process to that of the general and well-established method of purifying monoclonal antibodies.
[00128] The general and well-established method of purifying monoclonal antibodies composed of Protein A Affinity Chromatography (GE Resin); Low pH treatment; Sartobind Q chromatography (Anion Exchange Membrane, Sartorius, single use); Chromatography of Fenila Sartobind (Membrane Chromatography, Sartorius, single use); Viresolve Pro filtration (Nanofiltration, Merck).
Table 29:
Table 29Logw viral reduction factor (LRV) MuLV MMV THE General / standard method > 12.64 ± 0.60 7.02 ± 0.69 B Invention Method (SIIPL) > 18.3 ± 0.60 14.72 ± 0.70
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Table 29A: Details of the Methods used in row A of table 29.
General / standard method of the Dengue Monoclonal Antibody Purification Process Stage Logio Viral Reduction Factor MuLV MMv Protein A Affinity Chromatography (GE Resin) 2.75 ± 0.10 1.52 ± 0.46 Chromatography Q Sartobind (Anion exchange membrane, Sartorius, single use) 1.15 ± 0.43 2.19 ± 0.44 Sartobind phenyl chromatography (Membrane chromatography, Sartorius, single use) 2.09 ± 0.17 Not tested Viresolve Pro Filtration (Nanofiltration, Merck) > 3.76 ± 0.25 3.31 ± 0.26 Cumulative Logio Reduction Factor > 12.64 ± 0.60 7.02 ± 0.69
Table 29B: Details of the Methods used in row B of table 29.
SIIPL’s Inventive Method of Dengue Monoclonal Antibody Purification Process Stage Logio Viral Reduction Factor MuLV MMv Protein A Affinity Chromatography (Merck) 2.27 ± 0.17 2.35 ± 0.45 Low pH treatment 4.14 ± 0.26 Not applicable Cation exchange chromatography (Merck) 2.31 ± 0.31 2.73 ± 0.44 Anion exchange chromatography (Merck) 4.38 ± 0.36 2.96 ± 0.44 Viresolve Pro Filtration (Nanofiltration, Merck) > 5.20 ± 0.28 6.68 ± 0.90 Cumulative Logio Reduction Factor 18.3 ± 0.60 14.72 ± 0.70
NOTE: * MMV is a non-enveloped virus and highly resistant to acidic pH. Therefore, the low pH treatment stage is not assessed.
In the calculation of retrovirus particles per dose according to the general method and the SIIPL method. According to ICH Q5A, less than one particle per million doses is expected.
Table 29C: Comparison of the Methods used in lines A and B of table 29.
description General / standard method Invention Method (SIIPL) Maximum charge of RVLPs / mL in unprocessed mass determined by electron microscopy (Visterra Data) 8.5x106 (A) 8.5x106 (A) $ Total volume of unprocessed bulks entering the Purification Process (L) 770.85 (B) 300 (B) Product yield for batch (g) 720.32 (C) 625 (C) Proposed maximum single dose 75mg / kg (Visterra), 25mg / kg (SIIPL) average patient 80KG 6 (D) 2 * (D) Volume of unprocessed bulk needed to manufacture a single dose of product (L) 6.42089 (D) HC / B} 0.960(D) ^ {C / B} Volume of unprocessed bulk needed to make a single dose of productJmL) 6420.89 (E) 960.0 (E)
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Virus reduction factor determined by the spiking study > 12.64log10 18.3 Yog10 Reduction factor antilog > 4.37X10 ¹² (F) 1.99X10 ¹⁸ (F) Estimated virus particles per dose ΑΧ E 8.5X10® Χ6420.89 4.37X10 ¹² 8.5X10® Χ961.5 1.99X10 ¹⁸ F 1,25X10- = ² (G) 4,1X10- = ⁹ (G) Therefore, 1 virus is expected as a particle for each maximum dose (1 / G) 8.0X10 ¹ serving 2.4X10 ⁸ Conclusion 1 viral particle would be present in 80 doses of the product. 1 viral particle would be present in 240 million doses of the product.
Results:
- The SIIPL inventive purification process is highly efficient in viral clearance, the total LRV (MuLV) achieved was 18.3, that is, at least 18 log ™, a reduction factor, whereas with the General / Standard method it was 12.64 ; the total LRV (MMV) achieved was at least 14 log 10, a reduction factor, while with the General / Standard method it was 7.02;
- A viral particle would be present in 240 million doses of the product using the inventive SIIPL purification process (dose of 2.0 grams);
- A viral particle would be present in 80 doses of the product using the general / standard method purification process. [00130] The dengue monoclonal antibody purified by the inventive SIIPL purification process can be used in clinical trials in humans without any viral risk.
[00131] The SIIPL purification process was highly efficient in viral clearance, the total achieved LRV is in accordance with ICH guidelines. (standard process - LRV 12.64 as an inventive process (SIIPL) - LRV 23.74). The dengue antibody purified using our inventive process has been shown to be suitable for human clinical trials without any viral risk.
Example 10: Upstream process for cell culture and therapeutic protein expression, ie rabies monoclonal antibody (Rabies).
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Protocol:
[00132] Cultivation of cells on a 2 L scale was carried out in a batch feeding mode using the parameters mentioned below during the upstream / fermentation process.
• Rabies monoclonal antibody was expressed in GS-CHO cell line ”.
• The cell culture medium used for cell growth and therapeutic protein expression, that is, rabies monoclonal antibody was ΊΧ Cellvento ™ CHO-220 Liquid Medium "or" Actipro1X (Hyclone) ".
• Feed solution A, feed solution B, feed solution C, feed solution D selected from the group comprising glucose, Cell Boost ™ 5 supplement (Hyclone), EX-CELL 293 (Sigma Aldrich), Cell Boost supplements 7a and 7b (Hyclone), 3X Actipro (Hyclone), Cell Vento 220 (1 X medium), EX-CELL® Advanced ™ CHO Feed were used as supplementary feed.
• The pH of the fermentation medium was maintained at 6.5 to 7.5.
• The osmolality of the fermentation medium was maintained between 270 and 450 mOsm / Kg.
• Dissolved oxygen from the fermentation medium was maintained at about 20% to about 60%.
• The temperature of the fermentation medium was maintained at 36.5 ± 1 ° C.
[00133] Feed supplementation was done by gradual drip, according to the following table:
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Table 30:
Day Power A Power B Power C Power D Basal Medium 1 (3 X Actipro (Hyclone)) Basal Medium 2 (Cellvento ™ CHO-220 (1X)) 0 1 24% 3 0.2%4 4% 0.4% 10% 8% 56% 4% 0.4% 6 0.2% 4% 4% 0.4% 76% 8 0.2%4% 0.4% 94% 4% 4.0% 10 0.1% 2% 4% 0.4% 11 0.2% 2% 2% 0.2% 124% 13 0.1% 2% 2% 0.2% 143% 153% 16
Note: All feeding solutions can vary by ± 1% and by ± 1 day.
[00134] Cell culture was harvested after a drop in OD of up to 60% Results and conclusion:
[00135] The yield of 3 - 5 g / L was obtained from the fermentation process. The harvest obtained was also submitted to purification / downstream processing.
Example 11:
[00136] The cell culture obtained according to example 9 was harvested and then submitted to a protocol for purification of rabies monoclonal antibody as shown in Figure 1.
[00137] The detailed process used was as follows:
[00138] Protein-A Affinity Chromatography:
[00139] In this step, the target monoclonal antibody was separated from the components of the medium in the collected supernatant. The clarified supernatant was passed through the chromatography column and then eluted using compatible elution buffers.
Materials used:
Resin (Matrix): Mab Select Sure / Eshmuno A (Protein-A Affinity)
Residence Time: 4.0-8.0 minutes
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Column used: XK 26
Equilibration Buffer: 20 mM Phosphate Buffer + 150 mM NaCI + 80 0.05% polysorbate (w / v), pH 7.0 ± 0.2.
Wash Buffer I: 20 mM Phosphate Buffer + 150 mM NaCl + 0.05% polysorbate 80 (w / v), pH 7.0 ± 0.2.
Wash Buffer II: 20 mM Phosphate Buffer + 1 M NaCl + 0.05% polysorbate 80 (w / v), pH 7.0 ± 0.2.
Wash Buffer III: 10 mM Phosphate Buffer + 125 mM NaCl + 0.025% polysorbate 80 (w / v), pH 6.0 ± 0.2.
Elution buffer: 20 mM citrate buffer + 0.025% (w / v) polysorbate 80, pH 3.0 ± 0.2.
CIP Buffer: 0.1 M NaOH
Process parameters used:
Table 31
Step No. Process step Column volume Linear flow (cm / h) 1 balance 5 <300 2 Load (ml) Actual (as current) <300 3 Wash I 2 <300 4 Wash II 4 <300 5 Wash III 4 <300 6 Elution 5 <300 7 Cleaning 3 <300 8 Storage 2 <300
1. Viral inactivation at low pH
i. Protein A affinity chromatography eluate was subjected to low pH, that is, 3.5 ± 0.1 for 60 ± 10 minutes to inactivate the viral particles.
ii. After maintaining the low pH, the eluate was neutralized using neutralization buffer, that is, 1 M Tris / Citrate buffer with pH 7.0 ± 0.2. Subsequently, neutralized solution was filtered using 0.8 / 0.45 μ or 0.8 / 0.2 μ.
iii. The conductivity of the neutralized eluate was adjusted using WFI with polysorbate 80 0.025% (w / v)
2. Cation exchange chromatography
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54/60 [00140] Positively charged molecules bound to the column while negatively charged molecules enter the flow. Antibody molecules bound to the column are eluted using a salt gradient. Materials used:
Resin used: Fractogel SOs' / Fractogel SE Hicap (Merck)
Length of stay: 4.00 to 7.00 minutes
Column used: XK 26
Pre-Balance: 200 mM citrate buffer pH 6.0 ± 0.2.
Balance: 10 mM citrate buffer + 0.025% polysorbate 80 (w / v), pH 6.0 ± 0.2.
Load: Maintenance of low pH followed by neutralization Wash buffer A: 10 mM citrate buffer, pH 6.0 ± 0.2.
Wash buffer B: 20 mM citrate buffer + 300 mM NaCI, pH 6.0 ± 0.2.
CIP Buffer: 0.5 M NaOH
Storage Buffer: 20% ethanol + 150mM NaCI
Table 32: Process Parameters:
Step No Process step CV Linear flow cm / h 1 Pre-balance 2 <300 2 balance 5 <300 3 Charge Actual (as current) <300 4 Washing 5 <300 5 Elution 0 - 60% B (gradient) 15 <300 6 100% B 2 <300 7 CIP 3 <300
Table 33: Fraction collection during 0 gradient
Step No Fraction name Collection criteria (UV280) mAU 1 Fraction 01 Up to 300 2 Fraction 02 300-700 3 Fraction 03 700 to baseline 4 Fraction 04 (100% B) Baseline to baseline
Anion Exchange Chromatography:
[00141] All negatively charged impurities are bound to the membrane while the antibody enters the flow.
Materials used:
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Membrane I Resin used: Sartobind Q single Sep mini (Sartorius) I Eshmuno Q
Loading volume: 150mg / ml-1000 mg / ml
Column used: XK 26
Cleaning Buffer: 0.5 Na NaOH
Pre-equilibration buffer: 200 mM citrate buffer, pH 6.0 ± 0.2.
Equilibration Buffer: 20 mM citrate buffer, pH 6.0 ± 0.2; and optionally PS-80 0.025% w / v pH 6.0 ± 0.2
Storage Buffer: 20% ethanol + 150mM NaCI or 0.1 M NaOH
Table 34: Process Parameters:
Step No Process step CV Linear flow cm / h 1 Cleaning 10 <300 2 Pre-Balance 10 <300 3 balance 20 <300 4 Charge Real <300 5 After load washing 20 <300 6 Cleaning 10 <300
Filtration:
[00142] 20 nm nanofilter, that is, Viresolve PRO (Merck) was used to remove any virus particles from the therapeutic protein. 4. Tank flow filtration / ultrafiltration [00143] The antibody was concentrated to the desired concentration and subjected to buffer exchange in one of the three buffers in the formulation. Used material:
Formulation Buffer:
Buffer 1: 25 mM histidine buffer + 75 mM arginine buffer + 75 mM NaCl, pH 6.50 ± 0.25;
Buffer 2: 25 mM histidine, 75 mM arginine, 101 mM NaCI;
Bufffer 3: 25mM histidine, 75mM arginine, 75mM NaCI - 101mM, polysorbate-80 0.002% w / v
Cleaning Buffer: 0.5 M NaOH
Storage Buffer: 20% ethanol + 150mM NaCI or NaOH
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0.1 Μ
Membrane used: PALL Centramate T Series, PES membrane
MWCO: 30 kDa
Table 35: Process Parameters:
Step No. Process step description Note 1 Cleaning 0.5 M NaOH 30 min recirculation 2 WFI Cleaning WFI until the conductivity is below 1.3 pS / cm - 3 balance 400 ml - 4 Concentration and diafiltration Passage of ~ 10-12 DV - 5 WFI Washing with 1000 ml WFI - 6 Cleaning 0.5 M NaOH 30 In decirculation
Sterile filtration [00144] Stabilizer was added to the antibody solution and sterile filtered through a 0.2 μ filter.
Results:
Table 36: Staged recovery of the various stages used in the purification process.
Step No phase Recovery (%) * 1 Protein A affinity chromatography 94 2 Viral inactivation by low pH 98 3 Cation exchange chromatography 94 4 Anion exchange chromatography 95 5 Nanofiltration 100 6 TFF / UFF 98 7 Sterile formulation and filtration 100
[00145] It was found that the overall recovery process was>
80%
Table 37: Impurity data
Test Criterion accepted Batch 1 Batch 2 Concentration by UV280 (mg / ml) - 26.19 24.39 Purity by SEC-HPLC (% monomer) Monomer must be> 90.0% Monomer retention time must be comparable to the reference standard 100 100 SDS PAGE NR (kD) - 156.0 156.0 SDS PAGE R (kD) - 50.4 / 27.7 50.7 / 27.9 Residual CHO DNA (pg / mg mAb) <2 pg / mg IgG 0.34 0.17 Residual protein A (ng / mg mAb) <10.00 ng / mg IgG <10 0.82 Residual CHO protein (ng / mg mAb) <100.00 ng / mg IgG 5.50 7.94 Bacterial endotoxin (EU / mg protein) <0.1 EU / mg protein <0.35 <0.5
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Table 38: Batch & Purity Recovery
Step No Batch No Recovery% Purity (%) 1 Batch 1 82 99.4 2 Batch 2 80 99.6
[00146] The overall purity of rabies mAb after purification was found to be> 99% and the overall recovery> 80%.
Example 12:
[00147] The rabies monoclonal antibody was formulated as in the flowchart of Figure 2.
[00148] Excipients i.e. arginine, histidine, NaCl, sucrose and polysorbate-80 were added and mixed carefully using a magnetic stirrer at 50-60 rpm to form a mixture of excipients. This mixture was then added to the Dengue mAb TFF harvest gradually with a stirring rate of 50-60 rpm. The pH was checked (pH 6.5) and, if necessary, adjusted by histidine-arginine buffer. The final formulation was filtered through a 0.2 μΜ filter filling the final container.
[00149] The concentration of each component in the final formulation was as follows:
Table 39:
Ingredient Formulation 1 Formulation 2 Formulation 3 Rabies mAb 10 mg / ml 25 mg / ml 50 mg / ml Histidine 25 mM 25 mM 25 mM Arginine 75 mM 75 mM 75 mM Sodium chloride 101 mM 101 mM 101 mM Sucrose 0.5% w / v 0.5% w / v 0.5% w / v Polysorbate-80 0.02% w / v 0.02% w / v 0.02% w / v PH 6.5 + 0.5 6.5 + 0.5 6.5 + 0.5 Osmolality 386 mOsm / kg 386 mOsm / kg 386 mOsm / kg
[00150] These formulations were tested for purity, stability, effectiveness and potency for 9 months.
Example 13: Analytical test for purity and stability of rabies mAb formulation with storage at 2-8, 25 and 40 ° C for a period of 0 months, 1 month, 3 months & 6 months.
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13.1 Purity and aggregation analysis [00151] An HPLC-based size exclusion chromatography (HPLC-SEC) was used to evaluate aggregates in the crude and final formulation of DV mAb. In this method, a phenomenex BioSec-S 3000 column was used to demonstrate the aggregates and the percentage of rabies mAb monomer by injecting ~ 50 pg of total antibody and running at a flow rate of 1 ml / minute for 35 minutes. Phosphate buffered saline solution (Phosphate buffered Saline PBS), pH 6.5 was used as the mobile phase.
Results:
Table 40: SE-HPLC results (%)
Temp. 0 day 1 month 3 months 6 months 2 - 8 ° C 100 99.70 99.40 99.60 25 ° C 100 99.60 - - 40 ° C 100 99.20 (7 days) - -
[00152] Rabies mAb formulation did not show any time-dependent aggregation; and the purity / monomer content was found to be> 99%.
13.2 Batch 1 SDS Page Analysis - Test sample at 28 ° C
Table 41:
0 day 1 M 3 M 6M Reducing SDS PAGE (kD)Molecular weight of heavy and light chains should be within ± 10% of the molecular weight of the reference standard. Total% of light and heavy chain must be> 95%.Total%: 100 Test sample at 2 - 8 ° C Heavy chain 50.8 49.8 49.3 49.2 Light Chain 25.6 25.7 25.6 26.4 Reference standard Heavy chain 50 50 50 50 Light chain 25 25 25 25Non-reducing SDS PAGE (kD)RS (reference standard) - reference standardMolecular weight of the largest band must be within ± 10% of the molecular weight of the reference standard. (LOL) Test sample at 2-8 ° C 151.0 156.7 157.2 157.6 Reference standard 150 150 150 150
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Test sample at 25 ° C
0 day 30 days Reducing SDS PAGE(kD)Molecular weight of heavy and light chains should be within ± 10% of the molecular weight of the reference standard. Total% of heavy and light chain must be> 95%.Total%: 100 Test sample at 25 ° C Heavy chain 50.8 47.5 Light chain 25.6 25.5 Reference standard Heavy chain 50 50 Light chain 25 25Non-reducing SDS PAGE(kD)RS = reference standardMolecular weight of the largest band must be within ± 10% of the molecular weight of the reference standard (RS) Test sample at 25 ° C 151.0 151.6 Reference standard 150 150
[00153] Conclusion: The rabies mAb formulation did not show any significant time-dependent changes in aggregation and change in molecular weight. Thus, the formulation is considered stable at 2 - 8 ° C, 25 ° C and 40 ° C.
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Sequence_ST25. Txt
Seq ID 1: VIS513 amino acid heavy chain (VH) variable sequence (dengue monoclonal antibody) QVQLVQSGAEVKKP GASVKVSCKAGFNIKDVYMSWVRQAPEQGLEWMGRIDPENGDTKYD 60
PKLQGRVTMTADTSTNTAYMELRSLRSDDTAVYYCARGWEGFAYW GQGTLVTVSSASTKG
120
PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVH TFPAVLQSSGLYSL
180
SSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPC PAPELLGGPSVFL
240
FPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRV
300
VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY TLPPSREEMTKNQ
360
VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNV
420
FSCSVMHEALHNHYTQKS LS PGK
445
Seq ID 2: Variable light chain (VL) amino acid sequence of VIS513 (dengue monoclonal antibody)
DIVMTQSPASLAVSLGERATISCRASENVDKYGNSFMHWYQQKPGQ PPKLLIYRASELQW
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GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQRSNEVPWTFGQGT KLEIKRTVAAPSVF
120
IFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESV
TEQDSKDSTYSLS
180
STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
218 seq ID 3: Sil RmAb (RABI) heavy chain amino acid sequence (17c7)
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMHWVRQAPGKGL EWVAWSYDGRTKDY
ADSVKGRFTISRDNSKNTLYLQMNSLRTEDTAVYFCARERFSGAYFD YWGQGTLVTVSSA
120
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT
SGVHTFPAVLQSSG
180
LYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHT
CPPCPAPELLGGP
240
SVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVH
NAKTKPREEQYNS
300
TYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP QVYTLPPSREEM
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQ
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420
QGNVFSCSVMHEALHNHYTQKS LS LS PGK
449
Seq ID 4: Sil RMAb (RAB1) (17C7) light chain amino acid sequence
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRL
LIYDASNRATGIPA
RFSGSGSGTDFTLTISSLEPEDFAVYSCQQRNNWPPTFGGGTKVE
IKRTVAAPSVSVFIF
120
PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES
VTEQDSKDSTYSLSST
180
LTLSKADYEKHKVYACEVTHQGLSS PVTKS FNRGEC
219
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权利要求:
Claims (89)
[1]
1. Method of manufacturing pharmaceutical antigen-binding protein with high yield and minimal aggregation, characterized by the fact that it comprises:
a) large-scale culture of mammalian cells expressing antigen-binding protein in a cell culture production medium, in which the process effectively maintains the cell count and results in a yield of at least 2 g / L;
b) purification of the antigen-binding protein from the collected supernatant obtained in step (a), in which the process results in recovery of at least 80% and purity of at least 99%;
c) stable formulation, in which the osmolality is in the range of 300 - 400 mOsm / kg and the viscosity is less than 2.5 mPa-S.
[2]
A method according to claim 1, consisting of large-scale culture of mammalian cells expressing antibodies in a cell culture production medium; characterized by the fact that the cell culture process includes the use of basal medium, use of concentrated basal medium as a feeding solution, use of feeding solutions together with a defined feeding strategy, results in improved cell growth, maintaining lower concentrations of lactate and ammonia, effectively maintaining the cell count, thus increasing the longevity of the cells and obtaining high yield.
[3]
3. Method according to claim 2, characterized in that the cell culture medium includes at least one medium selected from the group consisting of Cell Vento 220 (Merck), ACTIPRO (HyClone / GE), Gibco ™ Dynamis ™ Medium (Thermo Fisher).
[4]
Method according to any one of claims 1 to 3, characterized in that the cell culture medium is
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2/22 supplemented with one or more other nutrients, at least once during the process.
[5]
5. Method according to claims 1 and 4, characterized by the fact that the cell culture production medium is supplemented in a scheme comprising supplementation that is continuous, daily, on alternate days, every two days and a combination thereof .
[6]
6. Method according to claim 4, characterized in that the cell culture production medium is supplemented with a feed solution comprising at least one medium selected from the group comprising Glucose, Cell Boost ™ 5 (Hyclone), EEX supplement -CELL 293 (Sigma Aldrich), Cell Boost supplements 7a and 7b (Hyclone), 3X Actipro medium, Cell Vento 220 (3X medium), EX-CELL® Advanced ™ CHO Feed 1, EfficientFeed ™ A, EfficientFeed ™ B, and EfficientFeed ™ C, and combinations thereof.
[7]
A method according to claim 1; characterized by the fact that the cell count is in the range of 10 x 10 ⁶ to 20 x 10 ⁶ cells / ml.
[8]
8. Method according to claim 1, characterized by the fact that the cell culture medium has osmolality in the range of 250 - 500 mOsm / Kg; pH in the range of 6.5 - 7.5; dissolved oxygen is maintained in the range of 10 to 60%; the temperature of the cell culture is in the range of 30 ° C to 38 ° C; the first temperature is preferably 36-37 ° C and, optionally, the second temperature is preferably 30-35 ° C; the glucose concentration is kept below 7%; preferably between 4% and 5%; the harvest of the cell culture is done when the viability is reduced to 80%; the cell culture conditions are maintained in such a way that secondary metabolites, such as lactate concentration, do not exceed 5 g / L; and the ammonia concentration does not exceed 5 mMol / L.
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[9]
9. Method according to claim 8, characterized by the fact that the osmolality of the fermentation medium is 400 500 mOsm / kg.
[10]
10. Method according to claim 1, characterized by the fact that the dissolved oxygen in the fermentation medium is maintained in the range of 20 - 40%.
[11]
11. Method according to claim 1, characterized in that the antigen binding protein is selected from the group consisting of humanized antibody, chimeric antibody, human antibody, bispecific antibody, multivalent antibody, multispecific antibody, binding protein fragments antigen, polyclonal, monoclonal, diabody, nanobody, monovalent, bispecific, heteroconjugate, multispecific, autoantibody, single chain antibodies, Fab fragments, F (ab) '2 fragments, fragments produced by a Fab expression library, antiidiotypic antibodies ( anti-ld), epitope-binding fragments and fragments containing CDR and their combinations.
[12]
12. Method according to claim 1, characterized by the fact that the cell line is selected from the group consisting of Chinese hamster ovary (CHO) cells, GS - CHO, CHOK1SV GS-KO, CHO DUX-B11, CHO-K1, BSC-1, myeloma cells NS0, CV-1 of Origin carrying SV40 cells (COS), COS-1, COS-7, P3X3Ag8.653, SP2 cells, embryonic human kidney cells (HEK 293), baby hamster kidney cells (BHK 21), VERO-76 African green monkey kidney cells, HELA cells, human lung cells (W138), retinal cells, and human hepatoma cell line (Hep G2). VERO, BHK, MDCK, WI38, NIH3T3, W138, BT483, Hs578T, HTB2, BT20, T47D, NS0 cells (a murine myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7030, HsS78Bst cells,
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PER.C6, SP2 / 0-Ag14, a myeloma cell line, a hybridoma cell line, human lung cells (W138), retinal cells, human hepatoma cell line (Hep G2), CHO— K1 ( ATCC CCL-61), DG44 (Chasin et al., 1986, Som. Cell Molec. Genet., 12: 555-556; and Kolkekar et al., 1997, Biochem., 36: 10901-10909), SH87 cellCHO- DXB11 (G. Urlaub and LA Chasin, 1980 Proc. Natl. Acad. Sci., 77: 4216-4220. LH Graf, and L A. Chasin 1982, Molec. Cell. Biol., 2: 93-96), lineage of CHO cells— K1 Tet-On (Clontech), CHO designated ECACC 85050302 (CA R, Salisbury, Wiltshire, UK), CHO clone 13 (GEIMG, Genova, IT), CHO clone B (GEIMG, Genova, IT), CHO - K1 / SF designated ECACC 93061607 (CAMR, Salisbury, Wiltshire, UK), RR— CHOK1 designated ECACC 92052129 (CAMR, Salisbury, Wiltshire, UK), CHOKIsv (Edmonds et al., Mol. Biotech. 34: 179-190 ( 2006)), CHO— S (Pichler et al., Biotechnol. Bioeng. 108: 386-94 (2011)), CHO cells negative for dihydrofolate reductase (CH O / -DHFR, Urlaub and Chasin, 1980, Proc. Natl. Acad. Know. USA, 77: 4216), and dp12.CHO cells (U.S. Patent No. 5,721,121); monkey kidney CV1 cells transformed by SV40 (COS, COS-7 cells, ATCC CRL-1651); embryonic human kidney cells (for example, 293 cells or 293 cells subcloned for growth in suspension culture, Graham et al., 1977, J. Gen. Virol., 36:59); baby hamster kidney cells (BHK, ATCC CCL-10); CAP cell, AGE1.HN cell, monkey kidney cells (CV1, ATCC CCL-70); African green monkey kidney cells (VERO-76, ATCC CRL-1587; VERO, ATCC CCL-81); mouse Sertoli cells (TM4, Mather, 1980, Biol. Reprod., 23: 243-251); human cervical carcinoma cells (HELA, ATCC CCL-2); canine kidney cells (MDCK, ATCC CCL-34); human lung cells (W138, ATCC CCL-75); human hepatoma cells (HEP-G2, HB 8065); mouse mammary tumor cells (MMT 060562, ATCC
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CCL-51); Buffalo rat liver cells (buffalo rat liver cells) (BRL 3A, ATCC CRL-1442); TR1 cells (Mather, 1982, Ann. NY Acad. Sci., 383: 44-68); MCR 5 cells; and FS4 cells, and hybridoma cells.
[13]
13. Method according to claim 1, characterized by the fact that the cell line is CHO-K1 SV GS-KO.
[14]
14. Method according to claim 1, characterized by the fact that the cell line is GS - CHO.
[15]
15. Method according to claim 1, characterized by the fact that the cells are cultured in batch, with batch feeding, in continuous mode, in perfusion mode; more particularly in batch feeding mode.
[16]
16. Method according to claim 1, characterized by the fact that the salt concentration of the buffers used in the purification is in the range of 30 mM - 500 mM.
[17]
17. Method according to claim 16, characterized by the fact that the salt concentration of the buffers used in the purification is in the range of 50 mM - 300 mM.
[18]
18. Method according to claim 1, characterized by the fact that the purification steps consist of protein A affinity chromatography, cation exchange chromatography and anion exchange chromatography.
[19]
19. Method according to claim 1, characterized by the fact that the purification steps consist of affinity chromatography, low pH viral inactivation, cation exchange chromatography, anion exchange chromatography, nanofiltration, tangential flow / ultrafiltration filtration; sequentially.
[20]
20. Method according to claim 1, characterized by the fact that the purification steps consist of affinity chromatography, low pH viral inactivation, anion exchange chromatography, cation exchange chromatography, nanofiltration, filtration of
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6/22 tangential flow / ultrafiltration; sequentially.
[21]
21. Method according to claim 1, characterized by the fact that the affinity chromatography matrix is selected from Protein A, Protein G and Protein L, preferably Protein A.
[22]
22. Method according to claim 1, characterized in that it includes an additional chromatography step selected from the group comprising one or more hydrophobic interaction chromatography, hydrophobic charge induction chromatography, hydroxyapatite ceramic chromatography, multimodal chromatography (Capto MMC and Capto Adhere), membrane chromatography (Q membranes including Intercept ™ (Millipore), Mustang® (Pall Corporation) and Sartobind ™ (Sartorius)).
[23]
23. Method according to claim 21, characterized by the fact that protein A chromatography consists of one or more resins selected from the group consisting of Eshmuno A, KanCapA ™, MabSelect SuRe ™, MabSelect SuRe LX, MabSelect Xtra, rProtein A Sepharose Fast Flow, Poros® MabCapture A, Amsphere ™ Protein A JWT203, ProSep HC, ProSep Ultra, and ProSep Ultra Plus; preferably the protein A affinity chromatography resin is MabSelect SuRe ™. Eshmuno A, Kancap A or Poros MabCapture.
[24]
24. Method according to claim 21, characterized by the fact that protein A chromatography consists of
a) Balance buffer: 20 mM phosphate buffer; 100 - 150 mM NaCI; 0.05% polysorbate 80; pH 7.0 ± 0.2.
b) Load: clarified harvest
c) Wash buffer I: 20 mM phosphate buffer; 100 - 150 mM NaCI, more particularly 150 mM; 0.05% polysorbate 80; pH 7.0 ± 0.2.
d) Wash buffer II: 20 mM phosphate buffer; 250 mM NaCI - 1M, more particularly 1M; 0.05% polysorbate 80; pH
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7.0 ± 0.2.
e) Wash buffer III: 10 mM phosphate buffer; 100 - 150 mM NaCI, more particularly 125 mM; 0.05% polysorbate 80; pH 7.0 ± 0.2.
f) Elution buffer: 20 mM citrate buffer; pH 3.0 ± 0.2; and optionally 0.025% (w / v) of polysorbate 80;
g) CIP buffer: 0.1 M NaOH.
h) Residence time: 4.00 - 8.00 minutes
i) Column used: XK26
j) Linear flow is 10 - 500 cm / h, more particularly 100-150 cm / h.
[25]
25. Method according to claims 19 and 20, characterized by the fact that the viral inactivation of the eluate with protein A chromatography is carried out keeping the eluate at pH 3.3 - 3.5 for a period of 50 - 100 minutes.
[26]
26. Method according to claims 19 and 20, characterized by the fact that cation exchange chromatography consists of resin selected from the group comprising one or more of a group based on sulfonate (for example, MonoS, Minis, Source 15S and 30S , SP SEPHAROSE® Fast Flow, SP SEPHAROSE® High Performance by GE Healthcare, TOYOPEARL® SP-650S and SP-650M by Tosoh, MACRO-PREP® High S by BioRad, Ceramic HyperD S, TRISACRYL® M and LS SP and Spherodex LS Pall Technologies SP); a sulfoethyl-based group (for example, FRACTOGEL® SE, from EMD, POROS® S-10 and S-20 from Applied Biosystems); a sulfopropyl-based group (for example, TSK Gel SP 5PW and SP-5PW-HR from Tosoh, POROS® HS-20, HS 50, and POROS® XS from Life Technologies); a group based on sulfoisobutyl (for example, FRACTOGEL® EMD SO3 · from EMD); a sulfoxyethyl based group (e.g. Whatman SE52, SE53 and Express-Ion S), a carboxy based group
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8/22 methyl (for example, CM SEPHAROSE® Fast Flow from GE Healthcare, Hydrocell CM from Biochrom Labs Inc., MACRO-PREP® CM from BioRad, Ceramic HyperD CM, TRISACRYL® M CM, TRISACRYL® LS CM, from Pall Technologies , Matrex CELLUFINE® C500 and C200 from Millipore, CM52, CM32, CM23 and Express-Ion C from Whatman, TOYOPEARL® CM-650S, CM-650M and CM-650C from Tosoh); groups based on sulfonic and carboxylic acid (for example, BAKERBOIMD® CarboxySulfon by J.T. Baker); a group based on carboxylic acid (for example, WP CBX from JT Baker, DOWEX® MAC-3 from Dow Liquid Separations, AMBERLITE® weak cation exchange resins, DOWEX® weak cation exchange resin, and DIAION® weak cation exchange resins from Sigma-Aldrich and FRACTOGEL® EMD COO- from EMD); a sulfonic acid-based group (for example, Hydrocell SP from Biochrom Labs Inc., DOWEX® fine-meshed strong acid cation resin from Dow Liquid Separations, UNOsphere S, JT Baker Sulfonic WP, SARTOBIND® S membrane from Sartorius, resins AMBERLITE® strong cation exchangers, DOWEX® strong cation exchanger resins and DIAION® strong cation exchanger resins from Sigma-Aldrich); and an orthophosphate-based group (for example, Whatman's PI 1). Preferred cation exchange chromatography resins for this invention are Fractogel® EMD SOs', Fractogel® EMD SE Hicap (Merck), CMM HyperCel ™ (Pall Corporation), Capto S ImpAct (GE).
[27]
27. Method according to claims 19 and 20, characterized by the fact that cation exchange chromatography comprises
a) Pre-balance buffer: 200 mM citrate buffer; pH 6.0 ± 0.2
b) Equilibration Buffer: 10 mM citrate buffer; 0.025% (w / v) of polysorbate 80; pH 6.0 ± 0.2
c) Wash Buffer A: 10 mM citrate buffer; pH 6.0
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9/22 ± 0.2
d) wash buffer B: 20 mM citrate buffer; 500 mM NaCI; pH 6.0 ± 0.2
e) CIP buffer: 0.5 M NaOH
f) Residence time: 4.00 - 7.00 minutes
g) Column used: XK26
[28]
28. Method according to claims 19 and 20, characterized by the fact that anion exchange chromatography consists of resin selected from the group comprising one or more of DEAE-cellulose, POROS® PI 20, PI 50, HQ 10, HQ 20, HQ 50, D 50 from Applied Biosystems, SARTOBIND® Q from Sartorius, MonoQ, MiniQ, Source 15Q and 30Q, Q, DEAE and ANX SEPHAROSE® Fast Flow, Q SEPHAROSE, Q SEPHAROSE® High Performance, QAE SEPHADEX® and FAST Q SEPHAROSE® (GE Healthcare), WP PEI, WP DEAM, WP QUAT by JT Baker, Hydrocell DEAE and Hydrocell QA by Biochrom Labs Inc., U Osphere Q, MACRO-PREP® DEAE and MACROPREP® High Q by Biorad, Ceramic HyperD Q, ceramic HyperD DEAE, TRISACRYL® M and LS DEAE, Spherodex LS DEAE, QMA SPHEROSIL® LS, QMA SPHEROSIL® M and MUSTANG® Q from Pall Technologies, strong base type I and type II anionic resins with fine mesh DOWEX® and DOWEX® MONOSPHER E 77, weak anionic resin from Dow Liquid Separations, INTERCEPT® Q membrane, Matrex CELLUFINE® A200, A500, Q500 , and Q800, from Millipore, FRACTOGEL® EMD TMAE, FRACTOGEL® EMD DEAE and FRACTOGEL® EMD DMAE from EMD, weak and strong AMBERLITE® type I and II anion exchange resins, weak and strong DOWEX® type I and II anion resins II, weak and strong anion exchange resins DIAION® type I and II, DUOLITE® from Sigma-Aldrich, TSK gel Q and DEAE 5PW and 5PW-HR, TOYOPEARL® SuperQ-650S, 650M and 650C, QAE550C and 650S, DEAE- 650M and 650C from Tosoh, QA52, DE23, DE32,
Petition 870190053787, of 6/12/2019, p. 183/197
10/22
DE51, DE52, DE53, Express-Ion D and Express-Ion Q by Whatman; more preferably the anion exchange chromatography resin is Sartobind Q (Sartorius).
[29]
29. Method according to claims 19 and 20, characterized by the fact that anion exchange chromatography includes
a) Cleaning buffer: 0.5M NaOH
b) Pre-balance buffer: 200 mM citrate buffer; pH 6.0 ± 0.2
c) equilibrium buffer: 20 mM citrate buffer; pH 6.0 ± 0.2; and optionally 0.025% polysorbate 80
d) Storage buffer: 0.1 M NaOH
e) The linear flow rate is 10 - 500 cm / h, more particularly 100-150 cm / h
f) Column used: XK26
[30]
30. Method according to claims 19 and 20, characterized by the fact that the anion exchange chromatography is performed in the “flow and wash mode” or in the “connect and elute mode.
[31]
31. Method according to claims 19 and 20, characterized by the fact that the removal of viral particles is carried out by nanofiltration using a virus retaining filter selected from the group that includes one or more between Viresolve PRO (Merck), Planova 20N (Asahi Kasei), Bio EXL PALL PEGASUS PRIME, PEGASUS SV4 (Pall Life Sciences) and Virosart (Sartorius), Virosart CPV filter from Sartorius, Virosolve from Millipore, Ultipor DV20 or DV50 from Pall, Planova 20N and 50N or BioEx from Asahi.
[32]
32. Method according to claims 19 and 20, characterized in that the antigen-binding protein is concentrated using Tangential Flow Filtration (TFF).
[33]
33. The method of claim 32, characterized
Petition 870190053787, of 6/12/2019, p. 184/197
11/22 due to the fact that TFF is performed using a 30 kDa membrane, selected from the group comprising one or more PES membrane Centramate T series (Pall Corporation), Hydrosart (Sartorius), and Pelicon 3 (Merck), preferably using Centramate T series PES membrane (Pall Corporation).
[34]
34. Method according to claim 32, characterized by the fact that the tangential flow filtration process consists of:
a) Diafiltration using diafiltration buffer: 25 mM histidine buffer; 75 mM arginine buffer; 50-150 mM NaCI; pH 6.50 ± 0.5.
b) Cleaning buffer: 0.5 M NaOH
c) Storage buffer: 0.1 M NaOH
d) Balance using 5 -10 X membrane volume
e) Concentration and diafiltration using 10-20 volumes of diafiltration
f) WFI washing using 3 - 5 volumes of membrane
g) cleaning using 0.5 - 1.0 M NaOH
h) 0.1 M NaOH storage
[35]
35. The method of claim 1, characterized in that the purified therapeutic protein preparation contains no more than 2% aggregates, preferably less than 1% aggregates.
[36]
36. Method according to claim 1, characterized in that the antigen-binding protein formulation includes at least one antigen-binding protein, at least one stabilizer, at least one buffer, at least one tonicity agent and at least one surfactant.
[37]
37. Method according to claim 36, characterized by the fact that the stabilizer is a carbohydrate selected in
Petition 870190053787, of 6/12/2019, p. 185/197
12/22 group consisting of one or more of sucrose, sorbitol, trehalose, mannitol, dextran, inositol, glucose, fructose, lactose, xylose, mannose, maltose, or raffinose; more preferably the stabilizer is sucrose.
[38]
38. The method of claim 37, characterized in that the stable antigen binding protein formulation includes <2.5% sucrose, more preferably <1% sucrose, and most preferably 0.5% of sucrose.
[39]
39. Method according to claim 36, characterized in that the buffering agent is selected from the group comprising one or more of Histidine, Glycine, sodium citrate, sodium phosphate, arginine, citric acid, HEPES, acetate potassium, potassium citrate, potassium phosphate, sodium acetate, sodium bicarbonate, Tris Base, and Tris-HCI.
[40]
40. The method of claim 39, characterized in that the buffering agent is histidine or arginine or a combination thereof.
[41]
41. Method according to claims 36 and 39, characterized in that the buffering agent is histidine.
[42]
42. Method according to claim 41, characterized by the fact that the concentration of histidine is in the range of 10 - 50 mM; preferably 25 mM.
[43]
43. The method of claims 36 and 39, characterized in that the buffering agent is arginine.
[44]
44. Method according to claim 43, characterized by the fact that the concentration of arginine is in the range of 10 150 mM; preferably 75 mM.
[45]
45. Method according to claim 36, characterized in that the tonicity agent is selected from the group comprising one or more of sodium chloride, dextrose, glycerin, mannitol, and potassium chloride.
Petition 870190053787, of 6/12/2019, p. 186/197
13/22
[46]
46. The method of claim 45, characterized by the fact that the tonicity agent is sodium chloride.
[47]
47. Method according to claim 45, characterized by the fact that the concentration of sodium chloride is in the range of 50 - 250 mM; preferably 100-145 mM.
[48]
48. Method according to claim 36, characterized in that the surfactant is selected from the group comprising one or more among polysorbates (for example polysorbate-20 or polysorbate-80); poloxamers (for example poloxamer 188); Triton; sodium dodecyl sulfate (SDS); Sodium lauryl sulfate; octyl sodium glycoside; lauryl-, myristyl-, linoleyl- or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl- or stearyl-sarcosine; linoleyl-, myristyl- or cetylbetaine; lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, palmidopropyl- or isostearamidopropyl-betaine (for example, lauroamidopropyl); myristamidopropyl-, palmidopropyl- or isostearamidopropyl-dimethylamine; sodium methyl-cocoyl, or disodium methyl-oleyltaurate; and the MONAQUAT® series (Mona Industries, Inc., Paterson, N.J.), polyethylene glycol, polypropyl glycol and copolymers of ethylene and propylene glycol (e.g., Pluronics, PF68, etc.); preferably the surfactant is polysorbate 80.
[49]
49. Method according to claim 48, characterized by the fact that the concentration of polysorbate 80 is in the range of 0.002 - 0.2% (w / v); preferably 0.02% (w / v).
[50]
50. Method according to any of claims 1 and 36 to 48, characterized in that the formulation of stable antigen-binding protein includes from about 1 mg / ml to about 100 mg / ml of protein-binding antigen.
[51]
51. The method of claim 50, characterized in that the stable antigen-binding protein formulation includes from about 1 mg / ml to about 50 mg / ml of protein
Petition 870190053787, of 6/12/2019, p. 187/197
14/22 antigen binding
[52]
52. Method according to any one of the preceding claims, characterized in that the antigen-binding protein formulation includes no more than 3% aggregation, minimal amount of subvisible particles and improved potency.
[53]
53. Method according to claim 1, characterized in that the concentration of the antigen-binding protein monomer is greater than 99%; CHO residual DNA is not greater than 2 pg / mg of antigen binding protein, more particularly not more than 0.1 pg / mg of antigen binding protein; residual CHO protein is not more than 100 ng / mg antigen binding protein, more particularly not more than 10 ng / mg antigen binding protein; residual protein A is not more than 10 ng / mg of antigen binding protein, more particularly not more than 1.5 ng / mg of antigen binding protein; Endotoxin is not greater than 0.1 EU / mg protein, viral clearance LRV for MuLV is at least 15 Log ™ reduction factor, and for MMV it is at least 12 Logw reduction factor.
[54]
54. Pharmaceutical composition, characterized in that it is prepared as defined in any of the preceding claims.
[55]
55. Pharmaceutical formulation, characterized by the fact that it consists of antigen-binding protein, a buffering agent, a tonicity agent, a surfactant and a stabilizing agent.
[56]
56. Pharmaceutical formulation according to claim 55, characterized by the fact that the stabilizer is a carbohydrate selected from the group comprising one or more among sucrose, sorbitol, trehalose, mannitol, dextran, inositol, glucose, fructose, lactose, xylose, mannose, maltose, or raffinose; more preferably the stabilizer
Petition 870190053787, of 6/12/2019, p. 188/197
15/22 zante is sucrose.
[57]
57. The pharmaceutical formulation according to claim 56, characterized in that the stable antigen-binding protein formulation includes <2.5% sucrose w / v, more preferably <1% sucrose w / v.
[58]
58. Pharmaceutical formulation according to claim 55, characterized in that the buffering agent is selected from the group comprising one or more among histidine, glycine, sodium citrate, sodium phosphate, arginine, citric acid, HEPES, acetate potassium, potassium citrate, potassium phosphate, sodium acetate, sodium bicarbonate, Tris base and Tris-HCI.
[59]
59. The pharmaceutical formulation according to claim 58, characterized in that the buffering agent is histidine or arginine or a combination thereof.
[60]
60. Pharmaceutical formulation according to claim 55, characterized in that the buffering agent is histidine.
[61]
61. Pharmaceutical formulation according to claim 60, characterized by the fact that the concentration of histidine is in the range of 10-50 mM; preferably 25 mM.
[62]
62. Pharmaceutical formulation according to claim 55, characterized in that the buffering agent is arginine.
[63]
63. Pharmaceutical formulation according to claim 62, characterized by the fact that the concentration of arginine is in the range of 10-150 mM; preferably 75 mM.
[64]
64. Pharmaceutical formulation according to claim 55, characterized in that the tonicity agent is selected from the group comprising one or more of sodium chloride, dextrose, glycerin, mannitol, and potassium chloride.
Petition 870190053787, of 6/12/2019, p. 189/197
16/22
[65]
65. Pharmaceutical formulation according to claim 64, characterized in that the tonicity agent is sodium chloride.
[66]
66. Pharmaceutical formulation according to claim 65, characterized by the fact that the concentration of sodium chloride is in the range of 50 - 250 mM; preferably 100 - 145 mM.
[67]
67. Pharmaceutical formulation according to claim 55, characterized in that the surfactant is selected from the group comprising one or more among polysorbates (for example polysorbate-20 or polysorbate-80); poloxamers (for example, poloxamer 188); Triton; sodium dodecyl sulfate (SDS); Sodium lauryl sulfate; octyl sodium glycoside; lauryl-, myristyl-, linoleyl- or stearylsulfobetaine; lauryl-, myristyl-, linoleyl- or stearyl-sarcosine; linoleyl-, myristyl- or cetyl-betaine; lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, palmidopropyl- or isostearamidopropyl-betaine (for example, lauroamidopropyl); myristamidopropyl-, palmidopropyl- or isostearamidopropyl-dimethylamine; sodium methyl-cocoyl, or disodium methyl-oleyl-taurate; and the MONAQUAT® series (Mona Industries, Inc., Paterson, N.J.), polyethylene glycol, polypropyl glycol and copolymers of ethylene and propylene glycol (for example, Pluronics, PF68, etc.); preferably the surfactant is polysorbate 80.
[68]
68. Pharmaceutical formulation according to claim 67, characterized by the fact that the concentration of polysorbate 80 is in the range of 0.002 - 0.2% (w / v); preferably 0.02% (w / v).
[69]
69. Pharmaceutical formulation, characterized by the fact that it consists of
a) 1-100 mg / ml of at least one antigen-binding protein;
b) 20 - 40 mM histidine;
c) 50 - 100 mM arginine;
Petition 870190053787, of 6/12/2019, p. 190/197
17/22
d) 0.002 - 0.02% of polysorbate 80 (w / v);
e) 50-150 mM NaCI;
f) no more than 2.5% sucrose w / v; where the pH of the formulation is 6.5 ± 0.5, where the osmolality of the formulation is 300 - 450 mOsmol / kg and the viscosity is less than 2.5 mPa-S and the said formulation is stable at 2 -8 ° C for at least 9 months, at 25 ° C for at least 1 month, at 40 ° C for at least 40 days, at 50 ° C for at least 2 days.
[70]
70. Pharmaceutical formulation, characterized by the fact that it consists of 2 - 80 mg / ml of at least one antigen-binding protein; 25 mM histidine, 75 mM arginine; 101 mM NaCI; 0.02% polysorbate 80 (w / v); and 0.5% sucrose w / v; wherein the pH of the formulation is 6.5 ± 0.5.
[71]
71. Pharmaceutical formulation according to any of the preceding claims, characterized by the fact that the isoelectric point (pl) of said antigen-binding protein is 7.0 - 8.5.
[72]
72. Pharmaceutical formulation according to any of the preceding claims, characterized by the fact that the osmolality of the formulation is about 380 mOsmol / kg.
[73]
73. Pharmaceutical formulation according to any one of the preceding claims, characterized in that said antigen-binding protein is a humanized antibody, chimeric antibody, human antibody, bispecific antibody, multivalent antibody, multispecific antibody, binding protein fragments to antigen, polyclonal antibody, monoclonal antibody, dimeric (diabody), nanobodies, monovalent, heteroconjugate, multispecific, autoantibody, single chain antibodies, Fab fragments, F (ab) '2 fragments, fragments produced by a Fab expression library, anti-idiotypic antibodies (anti-ld), fragments of li
Petition 870190053787, of 6/12/2019, p. 191/197
18/22 epitope and fragments containing CDR or their combinations.
[74]
74. The pharmaceutical formulation according to claim 73, characterized in that said antigen-binding protein is a monoclonal antibody.
[75]
75. The pharmaceutical formulation according to claim 73, characterized in that said monoclonal antibody binds to a dengue virus.
[76]
76. The pharmaceutical formulation according to claim 73, characterized in that said monoclonal antibody binds to a rabies virus.
[77]
77. Pharmaceutical formulation, characterized by the fact that it consists of 2 - 80 mg / ml of dengue monoclonal antibody; 25 mM histidine; 75 mM arginine; 101 mM NaCI; 0.02% Polysorbate 80 (w / v); and 0.5% sucrose w / v; the pH of the formulation is 6.5 ± 0.5, osmolality of 380 mOsm / Kg, viscosity less than 2.5 mPa-S.
[78]
78. Pharmaceutical formulation, characterized by the fact that it consists of 25 mg / ml of dengue monoclonal antibody; 25 mM histidine; 75 mM arginine; 101 mM NaCI; 0.02% polysorbate 80 (w / v); and 0.5% sucrose w / v; where the pH of the formulation is 6.5 ± 0.5, osmolality of 380 mOsm / Kg, viscosity less than 2.5 mPa-S.
[79]
79. Pharmaceutical formulation, characterized by the fact that it consists of 50 mg / ml of dengue monoclonal antibody; 25 mM histidine; 75 mM arginine; 101 mM NaCI; 0.02% polysorbate 80 (w / v); and 0.5% sucrose w / v; where the pH of the formulation is 6.5 ± 0.5, osmolality of 380 mOsm / Kg, viscosity less than 2.5 mPa-S.
[80]
80. Pharmaceutical formulation, characterized by the fact that it consists of 2 - 80 mg / ml of rabies monoclonal antibody; 25 mM histidine; 75 mM arginine; 101 mM NaCI; 0.02% poly
Petition 870190053787, of 6/12/2019, p. 192/197
19/22 sorbate 80 (w / v); and 0.5% sucrose w / v; where the pH of the formulation is 6.5 ± 0.5, osmolality of 380 mOsm / Kg, viscosity less than 2.5 mPa-S.
[81]
81. Pharmaceutical formulation, characterized by the fact that it consists of 25 mg / ml of rabies monoclonal antibody; 25 mM histidine; 75 mM arginine; 101 mM NaCI; 0.02% polysorbate 80 (w / v); and 0.5% sucrose w / v; where the pH of the formulation is 6.5 ± 0.5, osmolality of 380 mOsm / Kg, viscosity less than 2.5 mPa-S.
[82]
82. Pharmaceutical formulation, characterized by the fact that it consists of 50 mg / ml of rabies monoclonal antibody; 25 mM histidine; 75 mM arginine; 101 mM NaCI; 0.02% polysorbate 80 (w / v); and 0.5% sucrose w / v; where the pH of the formulation is 6.5 ± 0.5, osmolality of 380 mOsm / Kg, viscosity less than 2.5 mPa-S.
[83]
83. Pharmaceutical formulation according to any of the preceding claims, characterized by the fact that the formulation is a liquid formulation.
[84]
84. Pharmaceutical formulation according to any of the preceding claims, characterized by the fact that the formulation is a lyophilized formulation.
[85]
85. Pharmaceutical formulation according to claim 70, characterized in that the antigen-binding protein is an antibody having an affinity for binding to epitopes present in the Dengue virus, rabies virus, RSV, MPV, influenza virus (influenza ), Zika virus, West Nile virus, yellow fever virus, chikungunya virus, HSV, CMV, MERS, Epstein-Barr virus, VaricellaZoaster virus, mumps virus, measles virus, polio virus, Rhino virus, adenovirus , hepatitis A virus, hepatitis B virus, hepatitis C virus, Norwalk virus, Togavirus, alpha virus, rubella virus,
Petition 870190053787, of 6/12/2019, p. 193/197
20/22 HIV virus, Marburg virus, Ebola virus, human papilloma virus, polyomavirus, metapneumovirus, coronavirus, VSV and VEE.
[86]
86. Pharmaceutical formulation according to claim 70, characterized in that the antigen-binding protein is selected from the group comprising one or more of CTP19, CR57, CR4098, RVFab8, MabJA, MabJB-1, Mab 57, 17C7, 2B10 , Ab513 / VIS513, N297Q-B3B9, Mab2E8, 2D22, DMScHuMab, 3CH5L1, HMB DV5, HMB DV6, HMB DV8, DB32-6, D88, F38, A48, C88, F108, B48, A68, A100, C58, C78, C68, D98, D188, C128, C98, A11, B11, R17D6, R14B3, R16C9, R14D6, R18G9, R16F7, R17G9, R16E5, antibodies derived from the 4E11A modification, adatacept, abciximab, adalimumab, afliberte, albeit , basiliximab, bevacizumab, belatacepte, bectumomab, certolizumab, cetuximab, daclizumab, eculizumab, efalizumab, entanercepte, gentuzumab, ibritumomabe, infliximabe, muzebbe, tumuzbeumab, rumizumbe, rumizumbe, rome, panivizma, , nivolumab, pembrolizumab, hA20, AME-I33, IMC-3G3 , zalutumumab, nimotuzumab, matuzumab, ch *) ^A > KSB-102, MR1-1, SC100, SC101, SC103, muromonabe-CD3, OKT4A, ibritumomab, gentuzumab, motavizumab, infliximab, pegfilgrastim, CDP-571, etaner CBL, ABX-IL8, ABX-MAI, pemtumomab, Therex, AS1405, natalizumab, HuBC-I, IDEC-131, VLA-I; CAT-152; J695, CAT-192, CAT-213, BR3-Fc, LymphoStat-B, TRAIL-RImAb, bevacizumab, omalizumab, efalizumab, MLN-02, HuMax-IL 15, HuMax-Inflam, HuMaxCancer, HuMax-Lymphoma, HuMax -TAC , clenoliximab, lumiliximab, BEC2, IMC-ICI 1, DCIOI, labetuzumab, arcitumomab, epratuzumab, tacatuzumab, cetuximab, MyelomaCide, LkoCide, ProstaCide, ipilimumab, MDX-060, MDX-070, MDX-070, MDX-070, MDX-070, MDX-070, MDX-070, MDX-070, 1103, MDX-1333, MDX-214, MDX-1100, MDX-CD4, MDX-1388, MDX-066,
Petition 870190053787, of 6/12/2019, p. 194/197
21/22
MDX-1307, HGS-TR2J, FG-3019, BMS-66513, SGN-30, SGN-40, tocilizumab, CS-1008, IDM-I, golimumab, CNTO 1275, CNTO 95, CNTO 328, mepolizumab, MORIOI, MORI 02, MOR201, visilizumab, HuZAF, volocixmab, ING-I, MLN2201, daclizumab, HCD 122, CDP860, PR0542, C 14, oregovomabe, edrecolomab, etaracizumab, atezolizumab, jplimumabe, mogamulizumabe, lint, ICM3, galiximab, eculizumab, obinutuzumab, pexelizumab, LDP-ΟΙ, huA33, WX-G250, sibrotuzumab, ofatumumab. Chimeric KW-2871, hu3S193, huLK26; bivatuzumab, raxibacumab, cl4.18, 3F8, BC8, huHMFGI, MORAb-003, MORAb-004, MORAb-009, denosumab, PRO-140, 1D09C3, huMikbeta-1, NI-0401, Nl501, cantuzumabe, HuN9 chTNT-1 / B, bavituximab, huJ591, HeFi-l, Pentacea, abagovomab, tositumomab, ustequinumab, 105AD7, GMAI 61, GMA321.
[87]
87. Pharmaceutical formulation according to any of the preceding claims, characterized in that said antigen binding protein is an anti-dengue antibody or anti-rabies antibody that can be administered alone or in combination with other agents, other prophylactic or therapeutic modalities.
[88]
88. Container containing the formulation as defined in any one of the preceding claims, characterized by the fact that the container is selected from a bottle, a flask, a glass bottle, an ampoule, an IV bag, a blow mold / container. filling-sealing, a wearable injector, a bolus injector, a syringe, a pen, a pump, a multidose needle syringe, a multidose pen, an injector, a syrette, an autoinjector, a pre-filled syringe or a combination of themselves.
[89]
89. Container containing the formulation according to claim 88, characterized in that at least one primary packaging component comprises a closure system

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法律状态:
2021-10-13| 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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IN201621044139|2016-12-23|

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PCT/IB2017/058194|WO2018116198A1|2016-12-23|2017-12-20|Improved methods for enhancing antibody productivity in mammalian cell culture and minimizing aggregation during downstream, formulation processes and stable antibody formulations obtained thereof|

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