Upstream, Downstream and formulation development can often be the rate-limiting step in the early introduction of biopharmaceuticals into clinical trials. For instance, Dengue is the most important mosquito-borne viral disease affecting humans. Half of the world population lives in areas at risk for dengue, resulting in an estimated 390 million infections per year globally. Currently no antiviral agents are approved for treating dengue and recent vaccine trials have fallen short of expectations. The leading vaccine candidate recently demonstrated limited efficacy, estimated to be between 30%- 60%, with limited to no significant protection against DENV-2. Recently a non-immunodominant, but functionally relevant, epitope in domain III of the E protein has been identified, and subsequently an engineered antibody, Ab513 is being developed that exhibits high-affinity binding to, and broadly neutralizes, multiple genotypes within all four serotypes (Refer Ram Sasisekharan et al Cell 162, 1-12, July 30, 2015; Samir Bhatt et al, Nature, 2013 April 25; 496 7446: 504-507). Thus if we consider global medical demand for a Dengue monoclonal antibody, recent estimates indicate that up to 390 million dengue infections occur every year globally with >90 million presenting with disease making DENV a major global threat. If we assume that 30% of the 16 million diagnosed dengue cases go to hospital then about 5 million will require said Dengue monoclonal antibody, which implies that "purified antibody" above 4 gm/L becomes a prerequisite to meet global demand of such a life saving antibody. Further, Dengue disease burden is high in developing countries where availability of electrical power and refrigeration are often inadequate and therefore antibody stability across temperature excursions assumes greater relevance for these regions.

Indeed, if it was possible to have a platform process that could be employed for manufacturing and formulating all monoclonal antibody (mAb) candidates it would greatly reduce the time and resources needed for process development. This can have a significant impact on the number of clinical candidates that can be introduced into clinical trials. Also, processes developed for early stage clinical trials, including those developed using a platform, may be non-optimal with respect to process economics, yield, pool volumes, throughput and may not be suitable for producing the quantities required for late stage or commercial campaigns. Another important consideration is the speed of process development given that process development needs to

occur prior to introduction of a therapeutic candidate into clinical trials. (Refer Abhinav A. Shukla et al Journal of Chromatography B, 848 (2007) 28-39).

Typically mammalian cell culture media is based on commercially available media formulations, including, for example, DMEM or Ham's F12. Often media formulations are not sufficiently enriched to support increases in both cell growth and biologic protein expression. There remains a need for improved cell culture media, supplements, and cell culture methods for improved protein production. Increases in cell culture antibody titers to >2 g/L have been reported earlier. Refer F. Wurm, Nat. Biotechnol. 22 (2004) 1393. Further, in perfusion reactors, cells can reach much higher cell densities than in conventional batch or fed-batch reactors. (Refer Sven Sommerfeld et al Chemical Engineering and Processing 44 (2005) 1123-1137). However perfusion based processes are complex, costly and may also result in sterility issues and undesired heterogeneity in glycosylation pattern. Addition of animal-component-free hydrolysates (Bacto TC Yeastolate, Phytone Peptone) to chemically defined media is a common approach to increase cell density, culture viability and productivity in a timely manner. Hydrolysates are protein digests composed of amino acids, small peptides, carbohydrates, vitamins and minerals that provide nutrient supplements to the media. Non-animal derived hydrolysates from soy, wheat and yeast are used commonly in cell culture media and feeds to improve antibody titer (Refer US9284371). However, because of its composition complexity, lot-to-lot variations, undesirable attribute of making culture viscous, Yeast extract and hydrolysates can be a significant source of medium variability. Due to the complexity of antibody products that include isoforms and micro heterogeneities, the performance of the cell culture process can have significant effects on product quality and potency, especially with respect to glycosylation, post-transcriptional modifications and impurity profiles.

At higher concentrations, proteins, particularly antibodies often exhibit characteristic problems including aggregation, precipitation, gelation, lowered stability, and/or increased viscosity.

Antibodies are recognized as possessing characteristics that tend to form aggregates and particulates in solution as they undergo degradation or aggregation or denaturation or chemical modifications resulting in the loss of biological activity during the manufacturing process and / or during storage with time. Antibody aggregates could be formed during cell culture expression, downstream purification, formulation and on storage. Cell culture harvest usually contains the highest level of aggregate in the process (Refer Deqiang Yu Journal of Chromatography A, 1457 (2016) 66-75). Degradation pathways for proteins can involve chemical instability (e.g., any process which involves modification of the protein by bond

formation or cleavage resulting in a new chemical entity) or physical instability (e.g., 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). Further, proteins also are sensitive to, for example, pH, ionic strength, thermal stress, shear and interfacial stresses, all of which can lead to aggregation and result in instability. For a protein to remain biologically active, a formulation must therefore preserve intact the conformational integrity of at least a core sequence of the protein's amino acids while at the same time protecting the protein's multiple functional groups from degradation.

A major problem caused by the aggregate formation is that during the administration the formulation may block syringes or pumps and rendering it unsafe to patients. Such protein modifications can also make them immunogenic resulting in the generation of anti-drug antibodies by the patient which can reduce the drug availability during subsequent injections or worse induce an autoimmune reaction. A major aim in the development of antibody formulations is to maintain protein solubility, stability and bioactivity.

Early suggestions about how to solve the problems of instability of protein therapeutics formulations included the lyophilization of the drug product, followed by reconstitution immediately or shortly prior to administration. However, Lyophilized formulations of antibodies have a number of limitations, including a prolonged process for lyophilization and resulting high cost for manufacturing. In addition, a lyophilized formulation has to be reconstituted aseptically and accurately by healthcare practitioners prior to administering to patients. The reconstitution step itself requires certain specific procedures, i.e. (1) a sterile diluent (i.e., water for intravenous administration and 5% dextrose in water for intramuscular administration) is added to the vial containing lyophilized antibody, slowly and aseptically, and the vial must be swirled very gently for 30 seconds to avoid foaming; (2) the reconstituted antibody may need to stand at room temperature for a minimum of 20 minutes until the solution clarifies; and (3) the reconstituted preparation must be administered within six (6) hours after the reconstitution. Such reconstitution procedure is cumbersome and the time limitation after the reconstitution can cause a great inconvenience in administering the formulation to patients, leading to significant waste, if not reconstituted properly, or if the reconstituted dose 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 therapeutics, i.e. antibodies should be long-term stable, contain a safe and effective amount of the pharmaceutical compound.

Removal of aggregates is more difficult than removal of process related impurities due to the biophysical similarities between the aggregate and monomer, the multiple sources and types of aggregate, and less understanding of aggregation mechanism.

One of the more recent challenges encountered during formulation development of high concentration monoclonal antibody dosage forms is the formation of proteinaceous subvisible and visible particulates during manufacturing and long-term storage. The level of proteinaceous and non-proteinaceous particulates in IgG formulations is an increasingly important part of purification and formulation development. (Refer Klaus Wuchner et al Journal of Pharmaceutical Sciences, vol. 99, no. 8, august 2010). Further the liquid formulation should be stable across different temperatures viz temperatures 2-8° C, 25° C, 40° C , and 55° C.

Many antibody preparations intended for human use require stabilizers to prevent denaturation, aggregation and other alternations to the proteins prior to the use of the preparation. Previously reported antibody Liquid antibody formulations (Lucentis, Avastin) had mannitol, trehalose as stabilizers. (Refer Susumu Uchiyama et al Biochimica Biophysica Acta 1844 (2014) 2041-2052; US20160137727; WO2009120684; US8568720). However trehalose is costly and not feasible from large scale process economics.

Also, the IV administration of antibody is usually given as an infusion rather than a bolus, and thus requires dilution of mAb formulation, including excipients into appropriate fluids suitable for IV administration. The resulting dilution of excipients, especially surfactants, which may decrease below the concentration required for prevention of aggregation during agitation, thereby resulting in generation of aggregates and subvisible particles following gentle agitation after dilution into PVC and PO IV bags containing 0.9% saline.

Hydrophobic interaction chromatography, ceramic hydroxyapatite and cation exchange resins have all been used for aggregate removal but none are ideal. Majority of previously reported antibody purification processes have heavily relied upon use of Hydrophobic interaction chromatography in combination with Protein A chromatography, Anion exchange chromatography, Cation exchange chromatography as a three or four step process (Refer WO2010141039 , WO 2014/207763, WO2013066707, WO2015099165, WO2014102814, WO2015038888, WO2004087761). However, Hydrophobic interaction chromatography resins require large amounts of salts that are expensive, show low binding capacity, can be difficult to dispose of, and may not be compatible with the materials of construction of buffer and product holding tanks. Furthermore, the density difference between the buffers used for a HIC step can cause bed stability problems. Ceramic hydroxyapatite can also be used for the separation of aggregate from monomer, but the ceramic resin can be very difficult to unpack without

damaging the resin. Therefore, storing the resin outside the column for re-use in a subsequent manufacturing campaign may not be possible (Refer Suzanne Aldington Journal of Chromatography B, 848 (2007) 64-78).

Three-step combinations of cation-exchange, anion-exchange flow through, hydrophobic interaction chromatography and mixed mode cation-exchange chromatography were found to deliver adequate clearance of host cell protein contaminants for a CHO derived monoclonal antibody. However, such purification schemes by-and-large have not caught on in commercial downstream operations due to the need to design the purification sequence separately for each mAb.

Thus, there is an urgent unmet need for an efficient platform process for antibody manufacturing and formulation that meets multiple criterion including robustness, reliability and scalability, in particular a platform that provides i) antibody titer of atleast 2 gm/L; ii) minimum aggregation/particulate formation across cell culture, purification and formulation processes; iii) improved purification showing optimal percentage recovery, high monomer content and minimum impurity levels; and iv) high concentration antibody formulation showing low viscosity.devoid of aggregation and sub-visible particles; thereby showing long-term stability.

Summary of the invention

Applicant has surprisingly found

a) A feed composition and feeding strategy that takes into consideration nutrient consumption, by-product accumulation and the balance between promoting growth versus volumetric productivity wherein in particular, mammalian cell culture process parameters like use of particular basal medium, use of concentrated basal medium as feed solution, use of different feed solutions along-with a definite feeding strategy, maintaining lower concentrations of lactate and ammonia, are found to enhance cell growth, cell longevity and protein expression; thereby resulting in an increased antibody titer.

b) Specific salt concentration as part of buffer during Protein A affinity and Cation exchange steps that minimizes aggregation; thereby achieving a monomer content of greater than 99% with a recovery of greater than 80%.

c) Particle free liquid antibody formulations comprising of sucrose in combination with Histidine, Arginine, Polysorbate-80, Sodium chloride that impart higher potency and stability, reduces viscosity of highly concentrated antibody solutions at 2-8°C for at least 9months, at 25° C for atleast 1 month, at 40° C for atleast 42 days, at 55° C for atleast 2 days as compared to formulation devoid of sucrose.

List of Figures:

1. Figure 1 : Flow chart - Downstream processing for purification of monoclonal antibody

2. Figure 2 : Flow chart - Formulation process for monoclonal antibody

Description of the Invention

Therapeutic proteins of the present invention include, but are not limited to antigen binding protein, humanized antibody, chimeric antibody, human antibody, bi-specific antibody, multivalent antibody, multi-specific antibody, antigen binding protein fragments, polyclonal, monoclonal, diabodies, nanobodies, monovalent, hetero-conjugate, multi-specific, autoantibodies, single chain antibodies, Fab fragments, F(ab)'2, fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, epitope-binding fragments and CDR-containing fragments or combination thereof.

In an embodiment of the present invention, the therapeutic protein is an antigen binding protein or immunoglobulin; more preferably is an IgG and most preferably is an lgG1 molecule. In first aspect of the present embodiment, immunoglobulin/antibody is a human lgG1 (G1m3 allotype) with a human kappa light chain specific to the Dengue virus epitope in domain III of the E protein. In second aspect of the present embodiment, the antibody is a fully human lgG1 monoclonal antibody specific to the rabies virus surface G glycoprotein. In third aspect of the present embodiment, the therapeutic protein can be selected from the group comprising 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 modification of 4E11A,adatacept, abciximab, adalimumab, aflibercept, alefacept, alemtuzumab.trastuzumab, basiliximab, bevacizumab, belatacept, bectumomab, certolizumab, cetuximab, daclizumab, eculizumab, efalizumab, entanercept, gemtuzumab, ibritumomab, infliximab, muromonab-CD3, omalizumab, palivizumab; panitumumab, pertuzumab, ranibizumab, rilonacept, rituximab, tositumomab, trastuzumab, zanolimab, nivolumab, pembrolizumab ,hA20, AME-I33, IMC-3G3, zalutumumab, nimmotuzumab, matuzumab, ch*)A,

visilizumab, HuZAF, volocixmab, ING, MLN2201, daclizumab, HCD 122, CDP860, PR0542, C 14, oregovomab, edrecolomab, etaracizumab, atezolizumab Jplimumab.mogamulizumab, lintuzumab, HulDIO, Lym-1, efalizumab, ICM3, galiximab, eculizumab obinutuzumab.pexelizumab, LDP-oi, huA33, WX-G250, sibrotuzumab, ofatumumab, Chimeric KW-2871, hu3S193, huLK26; bivatuzumab.raxibacumab, chl4.18, 3F8, BC8, huHMFGI, MORAb-003, MORAb-004, MORAb-009, denosumab, PRO-140, 1 D09C3, huMikbeta-1, NI-0401, NI-501, cantuzumab, HuN901 8H9, chTNT-1 / B, bavituximab, huJ591, HeFi-l, Pentacea, abagovomab, tositumomab, ustekinumab, Gmail 105AD7, 61, GMA321. eculizumab, obinutuzumab.pexelizumab, LDP-OI, huA33, WX-G250, sibrotuzumab, ofatumumab, Chimeric KW-2871, hu3S193, huLK26; bivatuzumab.raxibacumab, chl4.18, 3F8, BC8, huHMFGI, Morab-003, Morab-004, Morab-009, denosumab, PRO-140, 1 D09C3, huMikbeta-1, NI-0401, NI-501, cantuzumab, HuN901 , 8H9, chTNT-1 / B, bavituximab, huJ591, HEFI-l, Pentacea, abagovomab, tositumomab, ustekinumab, 105AD7, gmai 61, GMA321. eculizumab, obinutuzumab.pexelizumab, LDP-OI, huA33, WX-G250, sibrotuzumab, ofatumumab, Chimeric KW-2871, hu3S193, huLK26; bivatuzumab.raxibacumab, chl4.18, 3F8, BC8, huHMFGI, Morab-003, Morab-004, Morab-009, denosumab, PRO-140, 1 D09C3, huMikbeta-1, NI-0401, NI-501, cantuzumab, HuN901 , 8H9, chTNT-1 / B, bavituximab, huJ591, HEFI-l, Pentacea, abagovomab, tositumomab, ustekinumab, 105AD7, gmai 61, GMA321.

In other aspect of this embodiment, therapeutic protein is an antibody having binding affinity towards epitopes present on Dengue virus, Rabies virus, RSV, MPV, Influenza virus, Zika virs, 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 pappiloma virus, polyoma virus, metapneumovirus, coronavirus, VSV and VEE.

In another aspect of this embodiment, isoelectric point (pi) of said antigen binding protein is 7.5 - 8.5, more preferably about 7.8 to about 8.2, most preferably 8.12.

In particular, the antigen binding protein is a therapeutic, prophylactic or diagnostic antibody as described in WO2014025546, WO2015122995, WO2015123362, WO2006084006, WO2017027805 and WO2017165736, the contents of which are incorporated herein by reference in its entirety. More preferably, therapeutic protein is an antibody having 80% similarity to that VIS513 (Seq ID 1 or Seq ID 2). In other preferred aspect of the present

embodiment, therapeutic protein is an antibody having more than 80% similarity to that of rabies monoclonal antibody (Seq ID 3 and Seq ID 4).

It is very well understood that any host may be used for the expression of therapeutic protein in the methods described herein. The cells may be wild or genetically engineered to contain a recombinant nucleic acid sequence, e.g. a gene, which encodes a polypeptide of interest (e.g., an antibody).

In 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, GS-CHO, CHO DUX-B11 , CHO-K1 , BSC-1 , NSO myeloma cells, CV-1 in Origin carrying SV40 (COS) cells, COS-1, COS-7, P3X3Ag8.653, C127, 293 EBNA, MSR 293, Colo25, U937, SP2 cells, L cell, human embryonic kidney (HEK 293) cells, baby hamster kidney (BHK 21) cells, African green monkey kidney VERO-76 cells, HELA cells, VERO, BHK, MDCK, WI38 cells, NIH-3T3, W138, BT483, Hs578T, HTB2, BT20, T47D, NSO (a murine myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7030, HsS78Bst cells, PER.C6, SP2/0-Ag14, a myeloma cell line, a hybridoma cell line, human lung cells (W138), Retinal cells, human hepatoma line (Hep G2), and hybridoma cells.

In other aspect of the second embodiment, animal or mammalian host cells includes but not limited to Chinese hamster ovary cells (CHO) such as 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 L. A. Chasin, 1980 Proc. Natl. Acad. Sci., 77: 4216-4220. L. H. Graf, and L. A. 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, 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)), dihydrofolate reductase negative CHO cells (CHO/-DHFR, Urlaub and Chasin, 1980, Proc. Natl. Acad. Sci. 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 CRL-1651); human embryonic kidney cells (e.g., 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 (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 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.

In first aspect of the second embodiment, 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.

In third embodiment of the present invention, the cells are cultivated in a batch, fed batch or continuous mode; more particularly in a fed batch mode. It is very 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 fed batch mode providing enhanced cell growth, cell longevity and increased protein expression i.e. provides a harvest yield of atleast 2 gm/L, preferably in the range of 3 gm/L to about 6 gm/L.

In first aspect of the third embodiment, cell culture is conducted in a flask, a bioreactor, a tank bioreactor, a bag bioreactor or a disposable bioreactor. Preferably said bioreactor is selected from the group of stirred tank bioreactor, a bubble column bioreactor, an air lift bioreactor, a fluidized bed bioreactor or a packed bed bioreactor; and the said bioreactor has a volume selected from 1 L, 2L, 3L, 5L, 10L, 20L, 100L, 200L, 250L, 350L, 500 L, 1000L, 1500L, 3000L, 5000L, 10000L , 20000L and 30,000 liters.

In second aspect of the third embodiment, the present cell culture media and methods may 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% , most preferably about 40% to 60% as measured over a course of a fortnight. The time period of the fed batch method can be about 12 to 20 days; about 15 to 20 days or about 15 to 18 days.

In' fourth embodiment of the present invention, cell culture medium is selected from the group comprising one or more of 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 vento 200 (Merck Millipore); Cell vento 220 (Merck Millipore); Actipro (Hyclone); and combination thereof. Preferably the cell culture medium is selected from Cell Vento 220 (Merck), ACTIPRO (HyClone/GE), or Gibco™ Dynamis™ Medium (Thermo Fisher).

The cell culture medium is further supplemented with glucose and other feed solutions so as to increase cell growth, cell longevity, protein expression and yield. It is very well understood in the art that the feed solutions may be supplemented in rapid bolus or gradual drip manner.

The supplementation of feed solution in cell culture medium with a feeding strategy comprising of:

Initial feeding with Feed solution A, at 0.05% to 0.5% reactor volume, preferably at 0.1% to 0.2% reactor volume, from day 4;

Feeding with Feed solution A, at 0.1% to 0.5% reactor volume on day 6, 8, 10,11 and 13; Feed solution B at less than 8% reactor volume, from day 2 to day 14, on alternate day or in continuous manner;

Feeding with Feed solution C at less than 8% reactor volume for atleast 2 consecutive days beginning from day 2 or day 3, having an intermittent gap of 2 consecutive days, till day 12th or day 14th or day 15th or day 16th or day 18th.

Feeding with Feed solution D at less than 0.5% reactor volume for atleast 2 consecutive days beginning from day 4 or day 5, on alternate day or in continuous manner or having an intermittent gap of 2 consecutive days, till day 12th or day 14th or day 15th or day 16th or day 18th. Optionally feeding atleast one feed solution selected from EfficientFeed™ A, EfficientFeed™ B, EfficientFeed™ C, and Dow corning Antifoam C.

In a preferred aspect of fourth embodiment, said Feed solution A, Feed solution B, Feed solution C, Feed solution D is selected one or more from group comprising of Glucose, Cell Boost™ 5 Supplement (Hyclone), EX-CELL 293 (Sigma Aldrich), Cell Boost 7a and 7b supplements (Hyclone), 3X Actipro (Hyclone/GE), Cell Vento 220 (1X medium), EX-CELL® Advanced™ CHO Feed 1 , EfficientFeed™ A, EfficientFeed™ B, and EfficientFeed™ C, and combination thereof.

In a most preferred aspect of the fourth embodiment, said Feed solution A is Cell Boost™ 5 Supplement (Hyclone); Feed solution B is EX-CELL 293 (Sigma Aldrich), Feed solution C is Cell Boost 7a supplements (Hyclone), Feed solution D is Cell Boost 7b supplements (Hyclone). Further, the cell culture medium is supplemented with 10% "3X Actipro" (Hyclone) on 3rd day and 8% Cell Vento 220 (1X medium) on 7th day of cell culturing. It is very well understood that all the feedings addition can vary by ± 1% and ± 1 day by a person skilled in the art.

Another aspect of the fourth embodiment includes cell culture conditions employed for increasing the cell growth and longevity and protein expression. The following cell culture conditions employed during the process includes but not limited to:

pH of cell culture medium is in the range of 6.5 and 7.5;

Osmolality of the culture medium is in the range of 250 - 500 mOsm/kg; more preferably 400 - 500 mOsm/kg.

Dissolved oxygen is in the range of 10 - 60%; preferably 20-40%; more preferably 30%. Cell culture temperature is in the range of 30°C to 38°C; first temperature preferably 36 - 37 °C and optionally second temperature preferably 30 - 35 °C.

Glucose concentration is maintained below 7%; preferably between 4% and 5%.

Harvesting the cell culture when viability is decreased to 80 %;

Wherein the cell culture conditions are maintained in a manner that secondary metabolites such as Lactate concentration is not more than 5g/L; and Ammonia concentration is not more than 5 mMol/L

In fifth embodiment of the present invention, the said therapeutic protein obtained from the cell culture harvest is subjected to a purification process comprising of following steps i) affinity chromatography, ii) Viral inactivation, iii) ion exchange chromatography, and iv) filtration; wherein the overall process recovery is more than 70% and the final purified therapeutic protein has a purity/monomer content of atleast 90%, preferably more than 98%. Other impurities including residual cell DNA, residual cell protein, and residual protein A in final purified therapeutic protein is less than 1%.

In general aspect of the fifth embodiment, the inventors of this invention have succeeded in dealing with problem of aggregation of therapeutic protein during downstream processing of the said protein, by using i) Salt in a affinity chromatography wash step and ii) Linear gradient of salt solution for elution in ion exchange chromatography step. In preferred aspect of the said embodiment, the salt concentration of the buffers used in purification is in the range of 30 mM - 500 mM, more preferably the salt concentration of the buffers used in purification is in the range of 50 mM - 300 mM.

In first aspect of the fifth embodiment, affinity chromatography selected from the group comprising one or more of Protein A chromatography, Protein G chromatography, Protein L chromatography, and combination thereof; preferably the affinity chromatography used is Protein A chromatography.

We Claim

1. The method of manufacturing pharmaceutical antigen binding protein with high yield and minimum aggregation, comprising:
a) culturing in large scale mammalian cells that express antigen binding protein in a cell culture production medium, wherein the process effectively maintains the cell count and results in yield of atleast 2 gm/L;
b) purification of antigen binding protein from harvested supernatant obtained in step (a), wherein the process results in recovery of atleast 80%, purity of atleast 99%;
c) stable formulation, wherein Osmolality is in range of 300 - 400 mOsm/Kg and viscosity is less than 2.5 mPa-S.
2. The method of claim 1 comprising culturing in large scale mammalian cells that express antibody in a cell culture production medium; wherein the cell culture process includes use of basal medium, use of concentrated basal medium as feed solution, use of feed solutions along-with a definite feeding strategy, results in enhanced cell growth, maintaining lower concentrations of lactate and ammonia, effectively maintaining the cell count thereby increasing cell longevity and high yield.
3. The method of claim 2, wherein the cell culture medium comprises of atleast one medium selected from the group comprising of Cell Vento 220 (Merck), ACTIPRO (HyClone/GE), Gibco™ Dynamis™ Medium (Thermo Fisher).
4. The method of claim 1 - 3, wherein the cell culture medium is supplemented with one or more other nutrients, atleast once during the process.
5. The method of claim 1 and 4, wherein the cell culture production medium is supplemented on a schedule comprising supplementation that is continuous, daily, every other day, every two days and combination thereof.
6. The method of claim 4, wherein the cell culture production medium is supplemented with feed solution comprising of atleast one medium selected from the group comprising of Glucose, Cell Boost™ 5 Supplement (Hyclone), EEX-CELL 293 (Sigma Aldrich), Cell Boost 7a and 7b supplements (Hyclone), 3X Actipro medium, Cell Vento 220 (3X medium), EX-CELL® Advanced™ CHO Feed 1, EfficientFeed™ A, EfficientFeed™ B, and EfficientFeed™ C, and combination thereof.
7. The method of claim 1 ; wherein the cell count is in the range of 10 x 106 - 20 x 10e cells/ml.
8. The method of claim 1, wherein the cell culture medium has Osmolality in range of 250 - 500mOsm/Kg; pH in range of 6.5 - 7.5; dissolved oxygen is maintained in range of 10 - 60%; Cell culture temperature is in the range of 30°C to 38°C; first temperature preferably 36 - 37 °C and optionally second temperature preferably 30 - 35 °C; Glucose concentration is maintained below 7%; preferably between 4% and 5% ; harvesting the cell culture when viability is decreased to 80 %; wherein the cell culture conditions are maintained in a manner that secondary metabolites such as Lactate concentration is not more than 5g/L; and Ammonia concentration is not more than 5 mMol/L
9. The method of claim 8, wherein the Osmolality of the fermentation medium is 400 - 500 mOsm/kg.
10. The method of claim 1, wherein the dissolved oxygen of the fermentation medium is maintained in the range of 20 - 40%.
11. The method of claim 1, wherein antigen binding protein is selected from the group comprising of humanized antibody, chimeric antibody, human antibody, bispecific antibody, multivalent antibody, multi-specific antibody.antigen binding protein fragments, polyclonal, monoclonal, diabodies, nanobodies, monovalent, bispecific, heteroconjugate, multispecific, autoantibodies, single chain antibodies, Fab fragments, F(ab)'2, fragments, fragments produced by a Fab expression library, anti- idiotypic (anti-Id) antibodies, epitope-binding fragments and CDR-containing fragments and combination thereof.
12. The method of claim 1, wherein 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, NS0 myeloma cells, CV-1 in Origin carrying SV40 (COS) cells, COS-1, COS-7, P3X3Ag8.653, SP2 cells, human embryonic kidney (HEK 293) cells, baby hamster kidney (BHK 21) cells, African green monkey kidney VERO-76 cells, HELA cells, human lung cells (W138), Retinal cells, and human hepatoma line (Hep G2). VERO, BHK, DCK, WI38 cells, NIH-3T3, W138, BT483, Hs578T, HTB2, BT20, T47D, NS0 (a murine myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7030, HsS78Bst cells, PER.C6, SP2/0-Ag14, a myeloma cell line, a hybridoma cell line, human lung cells (W138), Retinal cells, human hepatoma 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 L. A. Chasin, 980 Proc. Natl. Acad. Sci., 77: 4216-4220. L. H. Graf, and L A. Chasin 1982, Molec. Cell. Biol., 2: 93- 96), CHO— K1 Tet-On cell line (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)), dihydrofolate reductase negative CHO cells (CHO/-DHFR, Urlaub and Chasin, 1980, Proc. Natl. Acad. Sci. USA, 77:4216), and dpi 2. CHO cells (U.S. Pat. No. 5,721,121); monkey kidney CV1 cells transformed by SV40 (COS cells, COS-7, ATCC CRL-1651); human embryonic kidney cells (e.g., 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 (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 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 and hybridoma cells
13. The method of claim 1, wherein the cell line is CHO-K1 SV GS-KO.
14. The method of claim 1 , wherein the cell line is GS - CHO.
15. The method of claim 1, the cells are cultivated in a batch, fed batch , continuous mode, perfusion mode; more particularly in a fed batch mode.
16. The method of claim 1, wherein the salt concentration of the buffers used in purification is in the range of 30 mM - 500 mM.
17. The method of claim 16, wherein the salt concentration of the buffers used in purification is in the range of 50 mM - 300 mM.
18. The method of claim 1, wherein the purification steps comprise of Protein A affinity chromatography, cation exchange chromatography and anion exchange chromatography.
19. The method of claim 1, wherein the purification steps comprises of affinity chromatography, Low pH viral inactivation, cation exchange chromatography, anion exchange chromatography, nanofiltration,
Tangential flow fi Itrati o n/U Itrafi Itrati on ; in a sequential manner.
20. The method of claim 1 , wherein the purification steps comprises of affinity chromatography, Low pH viral inactivation, anion exchange chromatography, cation exchange chromatography, nanofiltration, Tangential flow filtration/Ultrafiltration; in a sequential manner.
21. The method of claim 1, wherein the affinity chromatography matrix is selected from Protein A, Protein G and Protein L, preferably Protein A.
22. The method of claim 1 , further comprising additional chromatography step selected from the group comprising one or more of Hydrophobic interaction chromatography, Hydrophobic charge induction chromatography, Ceramic hydroxyapatite chromatography, Multimodal chromatography (Capto MMC and Capto Adhere), Membrane chromatography (Q membranes including Intercept™ (Millipore), Mustang® (Pall Corporation) and Sartobind™ (Sartorius)).

23. The method of claim 21, wherein the protein A chromatography comprises of one or more resins selected from the group comprising 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; Preferrably the Protein A affinity chromatography resin is MabSelect SuRe™. Eshmuno A, Kancap A or Poros MabCapture.
24. The method of claim 21, wherein the protein A chromatography comprises of
a) Equilibration buffer: 20 mM Phosphate Buffer; 100 - 150 mM NaCI; 0.05% Polysorbate 80; pH 7.0 ± 0.2.
b) Loading : Clarified Harvest
c) Wash I Buffer: 20 mM Phosphate buffer; 100 - 150 mM NaCI, more particularly 150mM;
0.05% Polysorbate 80; pH 7.0 ± 0.2.
d) Wash II Buffer: 20 mM Phosphate buffer; 250 mM - 1 M NaCI, more particularly 1M; 0.05% Polysorbate 80; pH 7.0 ± 0.2.
e) Wash III Buffer: 10 mM Phosphate buffer; 100 - 150 mM NaCI, more particularly 125mM; 0.05% Polysorbate 80; pH 7.0 ± 0.2.
f) Elution Buffer: 20mM Citrate buffer; pH 3.0 ± 0.2; and optionally 0.025 % (w/v) Polysorbate 80.
g) CIP Buffer: 0.1 M NaOH.
h) Residence time: 4.00 - 8.00 minutes
i) Column used: XK26
j) Linear flow rate is 10 - 500 cm/hr, more particularly 100-150 cm/hr.
25. The method of claim 19 and 20, wherein viral inactivation of the eluate from protein A chromatography is accomplished by holding the eluate at pH 3.3 - 3.5 for a period of 50 - 100 minutes.
26. The method of claim 19 and 20, wherein the cation exchange chromatography comprises of resin selected from the group comprising one or more of sulfonate based group (e.g., 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 (e.g., FRACTOGEL® SE, from EMD, POROS® S-10 and S-20 from Applied Biosystems); a sulphopropyl based group (e.g., TSK Gel SP 5PW and SP-5PW- HR from Tosoh, POROS® HS-20, HS 50, and POROS® XS from Life Technologies); a sulfoisobutyl based group (e.g., FRACTOGEL® EMD S03 " from EMD); a sulfoxy ethyl based group (e.g., SE52, SE53 and Express-Ion S from Whatman), a carboxymethyl based group (e.g., 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); sulfonic and carboxylic acid based groups (e.g., BAKERBOIMD® Carboxy-Sulfon from J.T. Baker); a carboxylic acid based group (e.g., WP CBX from J.T Baker, DOWEX® MAC-3 from Dow Liquid Separations, AMBERLITE® Weak Cation Exchangers, DOWEX® Weak Cation Exchanger, and DIAION® Weak Cation Exchangers from Sigma- Aldrich and FRACTOGEL® EMD COO- from EMD); a sulfonic acid based group (e.g., Hydrocell SP from Biochrom Labs Inc., DOWEX® Fine Mesh Strong Acid Cation Resin from Dow Liquid Separations, UNOsphere S, WP Sulfonic from J.T. Baker, SARTOBIND® S membrane from Sartorius, AMBERLITE® Strong Cation Exchangers, DOWEX® Strong Cation and DIAION® Strong Cation Exchanger from Sigma-Aldrich); and a orthophosphate based group (e.g., PI 1 from Whatman). Preferred Cation-exchange chromatography resin for this invention is Fractogel® EMD SOD" , Fractogel® EMD SE Hicap (Merck), CMM HyperCel™ (Pall Corporation), Capto S ImpAct(GE).
27. The method of claim 19 and 20, wherein the cation exchange chromatography comprises of
a) Pre-equilibration buffer: 200 mM Citrate buffer; pH 6.0 ± 0.2
b) Equilibration buffer: 10 mM Citrate buffer; 0.025 % (w/v) Polysorbate 80; pH 6.0 ± 0.2 c) Wash Buffer A: 10 mM Citrate buffer; pH 6.0 ± 0.2
d) Wash buffer B: 20 mM Citrate buffer; 300 - 500 mM NaCI; pH 6.0 ± 0.2
e) CIP buffer: 0.5M NaOH
f) Residence time: 4.00 - 7.00 minutes
g) Column used: XK26
28. The method of claim 19 and 20, the anion exchange chromatography comprises of 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 from Sartorius, MonoQ, MiniQ, Source 15Q and 30Q, Q, DEAE and ANX SEPHAROSE® Fast Flow, Q SEPHAROSE(GE), Q SEPHAROSE® High Performance, QAE SEPHADEX® and FAST Q SEPHAROSE® (GE Healthcare) ,WP PEI, WP
DEAM, WP QUAT from J.T. Baker, Hydrocell DEAE and Hydrocell QA from Biochrom Labs Inc., U Osphere Q, MACRO-PREP® DEAE and MACRO-PREP® High Q from 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, DOWEX® Fine Mesh Strong Base Type I and Type II Anion Resins and DOWEX® MONOSPHER E 77, weak base anion 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, AMBERLITE® weak strong anion exchangers type I and II, DOWEX® weak and strong anion exchangers type I and II, DIAION® weak and strong anion exchangers 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 Anion-exchange chromatography resin is Sartobind Q (Sartorius).
29. The method of claim 19 and 20, wherein the anion exchange chromatography comprises of
a) Cleaning buffer: 0.5M NaOH
b) Pre-equilibration buffer: 200 mM Citrate buffer; pH 6.0 ± 0.2
c) Equilibration buffer: 20 mM Citrate buffer; pH 6.0 ± 0.2; and optionally 0.025% Polysorbate 80
d) Storage buffer: 0.1 M NaOH
e) Linear Flow rate is 10 - 500 cm/hr, more particularly 100-150 cm/hr
f) Column used: XK26
30. The method of claim 19 and 20, the anion exchange chromatography is "flow through and wash mode" or "bind and elute mode".
31. The method of claim 19 and 20, wherein the removal of viral particles is accomplished by nanofiltration using virus retentive filter selected from the group comprising one or more 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 50N or BioEx from Asahi.
32. The method of claim 19 and 20, wherein the antigen binding protein is concentrated using Tangential Flow Filtration (TFF).
33. The method of claim 32, wherein the TFF is carried out using 30 kDa membrane, selected from the group comprising one or more of Centramate T series PES membrane (Pall Corporation), Hydrosart (Sartorius), and Pelicon 3 (Merck), preferably using Centramate T series PES membrane (Pall Corporation).
34. The method of claim 32, wherein the Tangential Flow Filtration process comprises of
a) Diafilteration using diafilteratron buffer: 25 mM Histidine buffer; 75 mM Arginine buffer; 50 - 150 mM NaCI; pH 6.50 ± 0.5.
b) Cleaning buffer: 0.5M NaOH
c) Storage buffer: 0.1 M NaOH
d) Equilibration using 5 - 10 X membrane volume
e) Concentration and Diafiltration using 10 - 20 diafiltration volume
f) WFI wash using 3 - 5 membrane volume
g) cleaning using 0.5 - 1.0 M NaOH
h) Storage 0.1 M NaOH
35. The method of claim 1 , wherein the purified therapeutic protein preparation contain no greater than 2% aggregates, preferably less than 1% aggregates.

36. The method of claim 1, wherein the stable antigen binding protein formulation comprises of atleast one antigen binding protein, atleast one Stabilizer, atleast one Buffer, atleast one Tonicity agent , and atleast one surfactant.

37. The method of claim 36, wherein stabilizer is a carbohydrate selected from the group comprising one or more of sucrose, sorbitol, trehalose, mannitol, dextran, inositol, glucose, fructose, lactose, xylose, mannose, maltose, or Raffinose; more preferably stabilizer is sucrose.
38. The method of claim 37, wherein the stable antigen binding protein formulation comprises of <2.5% sucrose, more preferably <1% sucrose, and most preferably 0.5% sucrose.
39. The method of claim 36, wherein the buffering agent is selected from group comprising of one or more of Histidine, Glycine, Sodium Citrate, Sodium Phosphate, Arginine, Citric Acid, HEPES, Potassium Acetate, Potassium Citrate, Potassium Phosphate, Sodium Acetate, Sodium Bicarbonate, Tris Base, and Tris-HCI.

40. The method of claim 39, wherein the buffering agent is Histidine or Arginine or combination thereof.
41. The method of claim 36 and 39, wherein the buffering agent is Histidine.
42. The method of claim 41, wherein the concentration of Histidine is in the range of 10 - 50 mM; preferably 25 mM.
43. The method of claim 36 and 39, wherein the buffering agent is Arginine.
44. The method of claim 43, wherein the concentration of Arginine is in the range of 10 - 150 mM; preferably 75 mM.
45. The method of claim 36, wherein the tonicity agent is selected from the group comprising one or more of sodium chloride, dextrose, glycerin, mannitol, and potassium chloride.
46. The method of claim 45, wherein the tonicity agent is sodium chloride.
47. The method of claim 45, wherein the concentration of sodium chloride is in the range of 50 - 250 mM; preferably 100 - 145 mM.
48. The method of claim 36, wherein the surfactant Is selected from the group comprising of one or more of polysorbates (e.g. polysorbate-20 or polysorbate-80); poloxamers (e.g. poloxamer 188); Triton; sodium dodecyl sulfate (SDS); sodium laurel sulfate; sodium octyl 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 (e.g. lauroamidopropyl); myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or disodium methyl oleyl-taurate; and the MONAQUAT® series (Mona Industries, Inc., Paterson, N.J.), polyethyl glycol, polypropyl glycol, and copolymers of ethylene and propylene glycol (e.g. Pluronics, PF68 etc); preferably the surfactant is polysorbate 80.
49. The method of claim 48, wherein the concentration of Polysorbate 80 is in the range of 0.002 - 0.2% (w/v) ; preferably 0.02%( w/v).
50. The method of claim 1 and 36 - 48, wherein the stable antigen binding protein formulation comprises of about 1 mg/ml to about 100 mg/ml of antigen binding protein.
51. The method of claim 50, wherein the stable antigen binding protein formulation comprises of about 1 mg/ml to about 50 mg/ml of antigen binding protein.
52. The method according to any of the preceding claims, wherein antigen binding protein formulation comprises of not more than 3% aggregation, minimum amount of sub-visible particles and improved potency.
53. The method of claim 1, wherein the concentration of the antigen binding protein monomer is greater than 99%; residual CHO DNA is not more 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 of antigen binding protein, more particularly not more than 10 ng/mg of 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 more than 0.1 EU/mg of protein, viral clearance LRV for MuLV is atleast 20 fold and for MMV is atleast 10 fold.
54. A pharmaceutical composition prepared according to any of the preceding claims.
55. A pharmaceutical formulation comprising of antigen binding protein, a buffering agent, a tonicity agent, a surfactant and a stabilizing agent.
56. A pharmaceutical formulation of claim 55, wherein stabilizer is a carbohydrate selected from the group comprising one or more of sucrose, sorbitol, trehalose, mannitol, dextran, inositol, glucose, fructose, lactose, xylose, mannose, maltose, or Raffinose; more preferably stabilizer is sucrose.

57. A pharmaceutical formulation of claim 56, wherein the stable antigen binding protein formulation comprises of <2.5% sucrose w/v, more preferably <1% sucrose w/v.
58. A pharmaceutical formulation of claim 55, wherein the buffering agent is selected from group comprising of one or more of Histidine, Glycine, Sodium Citrate, Sodium Phosphate, Arginine, Citric
Acid, HEPES, Potassium Acetate, Potassium Citrate, Potassium Phosphate, Sodium Acetate, Sodium Bicarbonate, Tris Base, and Tris-HCI.
59. A pharmaceutical formulation of claim 58, wherein the buffering agent is Histidine or Arginine or combination thereof.
60. A pharmaceutical formulation of claim 55, wherein the buffering agent is Histidine.
61. A pharmaceutical formulation of claim 60, wherein the concentration of Histidine is in the range of 10
- 50 mM; preferably 25 mM.
62. A pharmaceutical formulation of claim 55, wherein the buffering agent is Arginine.
63. A pharmaceutical formulation of claim 62, wherein the concentration of Arginine is in the range of 10
- 150 mM; preferably 75 mM.
64. A pharmaceutical formulation of claim 55, wherein the tonicity agent is selected from the group comprising one or more of sodium chloride, dextrose, glycerin, mannitol, and potassium chloride.
65. A pharmaceutical formulation of claim 64, wherein the tonicity agent is sodium chloride.
66. A pharmaceutical formulation of claim 65, wherein the concentration of sodium chloride is in the range of 50 - 250 mM; preferably 100 - 145 mM.
67. A pharmaceutical formulation of claim 55, wherein the surfactant is selected from the group comprising of one or more of polysorbates (e.g. polysorbate-20 or polysorbate-80); poloxamers (e.g. poloxamer 188); Triton; sodium dodecyl sulfate (SDS); sodium laurel sulfate; sodium octyl 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 (e.g. lauroamidopropyl); myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or disodium methyl oleyl-taurate; and the MONAQUAT® series (Mona Industries, Inc., Paterson, N.J.), polyethyl glycol, polypropyl glycol, and copolymers of ethylene and propylene glycol (e.g. Pluronics, PF68 etc); preferably the surfactant is polysorbate 80.
68. A pharmaceutical formulation of claim 67, wherein the concentration of Polysorbate 80 is in the range of 0.002 - 0.2% (w/v) ; preferably 0.02% (w/v).
69 A pharmaceutical formulation comprising of
a) 1 - 100 mg/ml of atleast one antigen binding protein;
b) 20 - 40 mM of Histidine;
c) 50 - 100 mM of Arginine;
d) 0.002 - 0.02% Polysorbate 80 (w/v);
e) 50 - 150 mM NaCl;
f) not more than 2.5% Sucrose w/v; wherein pH of the formulation Is 6.5 ± 0.5.
wherein the Osmolality of the formulation is 300 - 450 mOsmol/kg and viscosity less than 2.5 mPa-S and said formulation is stable at 2-8 deg C for atleast 9 months, at 25 deg C for atleast 1 month , at 40 deg C for atleast 40 days, at 50 deg C for atleast 2 days
70. A pharmaceutical formulation comprising of 2 - 80 mg/ml of atleast one antigen binding protein; 25 mM of Histidine; 75 mM of Arginine; 101 mM NaCI; 0.02% Polysorbate 80 (w/v); and 0.5% Sucrose w/v; wherein pH of the formulation is 6.5 ± 0.5.
71. A pharmaceutical formulation according to any of the preceding claims, wherein isoelectric point (pi) of said antigen binding protein is 7.5 - 8.5, more preferably 8.12.
72. A pharmaceutical formulation according to any of the preceding claims, wherein the Osmolality of the formulation is about 380 mOsmol/kg.
73. A pharmaceutical formulation according to any of the preceding claims, wherein said antigen binding protein is an humanized antibody, chimeric antibody, human antibody, bi-specific antibody, multivalent antibody, multi-specific antibody, antigen binding protein fragments, polyclonal antibody, monoclonal antibody, diabodies, nanobodies, monovalent, hetero-conjugate, multi-specific, autoantibodies, single chain antibodies, Fab fragments, F(ab)'2, fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, epitope-binding fragments and CDR-containing fragments or combination thereof.
74. A pharmaceutical formulation according to claim 73, wherein said antigen binding protein is a monoclonal antibody.
75. A pharmaceutical formulation according to claim 73, wherein said monoclonal binds to a dengue virus.
76. A pharmaceutical formulation according to claim 73, wherein said monoclonal antibody binds to a rabies virus.
77. A pharmaceutical formulation comprising of 2 - 80 mg/ml of Dengue monoclonal antibody; 25 mM of Histidine; 75 mM of Arginine; 101 mM NaCI; 0.02% Polysorbate 80 (w/v); and 0.5% Sucrose w/v; wherein pH of the formulation is 6.5 ± 0.5 Osmolality 380 mOsm/Kg, viscosity less than 2.5 mPa-S.
78. A pharmaceutical formulation comprising of 25 mg/ml of Dengue monoclonal antibody; 25 mM of Histidine; 75 mM of Arginine; 101 mM NaCI; 0.02% Polysorbate 80 (w/v); and 0.5% Sucrose w/v; wherein pH of the formulation is 6.5 ± 0.5 Osmolality 380 mOsm/Kg, viscosity less than 2.5 mPa-S.
79. A pharmaceutical formulation comprising of 50 mg/ml of Dengue monoclonal antibody; 25 mM of Histidine; 75 mM of Arginine; 101 mM NaCI; 0.02% Polysorbate 80 (w/v); and 0.5% Sucrose w/v; wherein pH of the formulation is 6.5 ± 0.5 Osmolality 380 mOsm/Kg, viscosity less than 2.5 mPa-S. 80. A pharmaceutical formulation comprising of 2 - 80 mg/ml of Rabies monoclonal antibody; 25 mM of Histidine; 75 mM of Arginine; 101 mM NaCI; 0.02% Polysorbate 80 (w/v); and 0.5% Sucrose w/v; wherein pH of the formulation is 6.5 ± 0.5 Osmolality 380 mOsm/Kg, viscosity less than 2.5 mPa-S.
81. A pharmaceutical formulation comprising of 25 mg/ml of Rabies monoclonal antibody; 25 mM of Histidine; 75 mM of Arginine; 101 mM NaCI; 0.02% Polysorbate 80 (w/v); and 0.5% Sucrose w/v; wherein pH of the formulation is 6.5 ± 0.5 Osmolality 380 mOsm/Kg, viscosity less than 2.5 mPa-S.
82. A pharmaceutical formulation comprising of 50 mg/ml of Rabies monoclonal antibody; 25 mM of Histidine; 75mM of Arginine; 101 mM NaCI; 0.02% Polysorbate 80 (w/v); and 0.5% Sucrose w/v; wherein pH of the formulation is 6.5 ± 0.5 Osmolality 380 mOsm/Kg, viscosity less than 2.5 mPa-S.
83. A pharmaceutical formulation according to any of the preceding claims, wherein the formulation is a liquid formulation.
84. A pharmaceutical formulation according to any of the preceding claims, wherein the formulation is a lyophillized formulation.
85. A pharmaceutical composition as claimed in claim 70, wherein the antigen binding protein is an antibody having binding affinity towards epitopes present on Dengue virus, Rabies virus, RSV, MPV, Influenza virus, Zika virs, West Nile virus, Yellow fever virus, chikungunya virus, HSV, CMV, MERS, 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 pappiloma virus, polyoma virus, metapneumovirus, coronavirus, Ebola virus, VSV and VEE.
86. A pharmaceutical composition as claimed in claim 70, wherein the antigen binding protein is selected from the group comprising of one or more of CTP19 , CR57 , CR4098, RVFab8, MabJA, MabJB-1, Mab S7.17C7, 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 modification of 4E11A,adatacept, abciximab, adalimumab, aflibercept, alefacept, alemtuzumab.trastuzumab, basiliximab, bevacizumab, belatacept, bectumomab, certolizumab, cetuximab, daclizumab, eculizumab, efalizumab, entanercept, gemtuzumab, ibritumomab, infliximab, muromonab-CD3, omalizumab, palivizumab; panitumumab, pertuzumab, ranibizumab, rilonacept, rituximab, tositumomab, trastuzumab, zanolimab, nivolumab, pembrolizumab ,hA20, AME-I33, IMC-3G3, zalutumumab, nimmotuzumab, matuzumab, ch*)A, KSB- 102, MR1-1, SC100, SC101, SC103, muromonab-CD3, OKT4A, ibritumomab, gemtuzumab, motavizumab, infliximab, pegfilgrastin, CDP-571, etanercept, ABX-CBL , ABX-IL8, ABX-MAI pemtumomab, Therex, AS 1405, natalizumab, HuBC-l, I DEC- 131, VLA-I; CAT- 152; J695, CAT- 192,
CAT-213, BR3-Fc, LymphoStat-B, TRAIL-RlmAb , bevacizumab, omalizumab, efalizumab, MLN-02, HuMax-IL 15, HuMax-lnflam, HuMax-Cancer, HuMax-Lymphoma, HuMax-TAC, clenoliximab, lumi!iximab, BEC2, IMC-ICI 1, DCIOI, labetuzumab, arcitumomab, epratuzumab, tacatuzumab, Cetuximab, MyelomaCide, LkoCide, ProstaCide, ipilimumab, MDX-060, 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, MO RIOI, Mori 02, MOR201, visilizumab, HuZAF, volocixmab, Ing-I, MLN2201, daclizumab, HCD 122, CDP860, PR0542, C 14, oregovomab, edrecolomab, etaracizumab, atezolizumab, iplimumab, mogamulizumab, lintuzumab, HulDIO, Lym-1, efalizumab, ICM3, galiximab, eculizumab, obinutuzumab.pexelizumab, LDP-oi, huA33, WX-G250, sibrotuzumab, ofatumumab, a non-Chimeric 2871, hu3S193, huLK26; bivatuzumab.raxibacumab, chl4.18, 3F8, BC8, huHMFGI, MORAb-003, MORAb-004, MORAb-009, denosumab, Protestant-140, 1D09C3, Hu Mikbeta-1, N 1-0401, 1-501 N, cantuzumab, HuN901, 8H9, chTNT-
1 / B, bavituximab, huJ591, EFI-l, Pentacea, abagovomab, tositumomab, USTEKINUMAB, 105AD7, GMAI 61, GMA321.
87. A pharmaceutical formulation according to any of the preceding claims, wherein 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. A container comprising the formulation of any of the preceding claims, wherein the container is selected from a bottle, a vial, an ampule.an IV bag, a wearable injector, a bolus injector, a syringe, a pen, a pump, a multidose needle syringe, a multidose pen, a injector, a syrette, an autoinjector, a pre-filled syringe, or a combination thereof.
89. A container comprising the formulation of claim 88, wherein at least one primary packaging component comprises a container closure selected from polypropylene (PP), polyethylene terephthalate (PETG), high-density polyethylene (HDPE), polyethylene terephthalate (PET), polypentafluorostyrene (PFS), polycarbonate, polyvinyl chloride (PVC), polycyclopentane (CZ.RTM.), cyclic olefin copolymer (COC), polyolefin, and combinations or copolymers thereof.