Patent Application
20200131251
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, Jul. 30, 2015; Samir Bhatt et al, Nature, 2013 Apr. 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 U.S. Pat. No. 9,284,371). 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; U.S. Pat. No. 8,568,720). 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.
Applicant has surprisingly found
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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, auto-antibodies, 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 IgG1 molecule. In first aspect of the present embodiment, immunoglobulin/antibody is a human IgG1 (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 IgG1 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, Ab513N/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-133, IMC-3G3, zalutumumab, nimmotuzumab, matuzumab, ch*){circumflex over ( )}, 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, 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, HuMax-Cancer, 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-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, volocixmab, ING-I, MLN2201, daclizumab, HCD 122, CDP860, PRO542, C 14, oregovomab, edrecolomab, etaracizumab, atezolizumab, iplimumab, mogamulizumab, lintuzumab, HuIDIO, 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, 1D09C3, huMikbeta-1, NI-0401, NI-501, cantuzumab, HuN901, 8H9, chTNT-1/B, bavituximab, huJ591, HeFi-I, 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, W138 cells, NIH-3T3, W138, BT483, Hs578T, HTB2, BT20, T47D, NS0 (a murine myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7O3O, 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 cellICHO-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), CHOK1sv (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, 2 L, 3 L, 5 L, 10 L, 20 L, 100 L, 200 L, 250 L, 350 L, 500 L, 1000 L, 1500 L, 3000 L, 5000 L, 10000 L, 20000 L 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); ProCHOT™ 5 (Lonza); BalanCD™ CHO Growth A (Irvine Scientific); CDM4Mab (Hyclone); Celivento™ 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:
In fourth aspect of the fifth embodiment, elution buffer used for Protein A chromatography comprises of 10-30 mM Citrate buffer; pH 3.0±0.5; and optionally 0.01-0.05% (w/v) Polysorbate 80; preferably the elution buffer comprises of 20 mM Citrate buffer; pH 3.0±0.2; and optionally 0.025% (w/v) Polysorbate 80.
In fifth aspect of the fifth embodiment, eluate obtained from the affinity chromatography step is subjected to viral inactivation and reduction. It is very well understood in the art that viral inactivation and reduction of the eluate may be effected by method selected individually or in combination from the group comprising of pH treatment, detergent treatment, heat treatment, and virus reduction filtration. In preferred aspect of this embodiment, the viral inactivation is effected by subjecting the eluate to low pH i.e. 3.3-3.5 for 50-100 minutes. Further, the eluate was pH neutralized by subjecting it to neutralization buffer i.e. 1 M Tris/Citrate buffer pH 7.0±0.2. It is very well understood in the art that any other compatible buffer may be used alternatively for effective pH neutralization of the eluate.
In sixth aspect of the fifth embodiment, the viral inactivated eluate is subjected to ion exchange chromatography. According to one of the aspect of this embodiment, ion exchange chromatography is cation exchange chromatography or anion exchange chromatography or their combination; and chromatography may be carried out in “bind and elute” mode or “flow through” mode. In preferred aspect of this embodiment, cation exchange chromatography and anion exchange chromatography is carried out in any sequential order. A further aspect of the fifth embodiment is that the said chromatography resin optionally is a multi-modal resin like Capto MMC resin (GE Healthcare).
In seventh aspect of the fifth embodiment, the viral inactivated eluate is subjected to cation exchange chromatography. In preferred aspect of this embodiment, the chromatography parameters including chromatography resin and buffer conditions are selected in such a manner that the positively charged therapeutic protein binds to the chromatography resin while the negatively charged molecules comes in the flow through, further therapeutic proteins are subjected to elution using a salt gradient. In preferred aspect of this embodiment, the cation exchange chromatography resin is 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., BAKERBOND® 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). In most preferred aspect of this embodiment, 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 fifth embodiment, process parameters for cation exchange chromatography includes but not limited to Pre-equilibration buffer [200 mM Citrate buffer; pH 6.0±0.2]; Equilibration buffer [10 mM Citrate buffer; Polysorbate 80 (0.025% (w/v)); pH 6.0±0.2]; Low pH hold for neutralization; Wash Buffer A [10 mM Citrate buffer; pH 6.0±0.2]; Wash buffer B [20 mM Citrate buffer; 300-500 mM NaCl; pH 6.0±0.2]; CIP buffer [0.5M NaOH]; Residence time [4.00-7.00 minutes]; Column used [XK26].
In eighth aspect of the fifth embodiment, the viral inactivated eluate is subjected to anion exchange chromatography. In preferred aspect of this embodiment, the chromatography parameters including chromatography resin and buffer conditions are selected in such a manner that all negatively charged impurities are bound with the membrane while the therapeutic protein elutes in a flow through. In preferred aspect of this embodiment, the anion exchange chromatography resin is selected from the group comprising one or more of DEAE cellulose, POROSO 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 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 selected from Sartobind Q (Sartorius), Eshmuno Q (Merck), MUSTANG® Q (Pall Corporation) and Poros X (Thermo). In another aspect of fifth embodiment, process parameters for anion exchange chromatography includes but not limited to Cleaning buffer [0.5M 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 0.025% Polysorbate 80]; Storage buffer [0.1M NaOH]; Linear Flow rate [10-500 cm/hr, more particularly 100-150 cm/hr]; Column used [XK26].
The purification process of aforementioned embodiments can further comprise of atleast one 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)).
In ninth aspect of the fifth embodiment, virus particles were removed by using 20 nm filter. The filter used for removal of viral particles includes but not limited to virus retentive 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 50N or BioEx from Asahi. It is very well understood in the art that any other filter having retention capacity for viruses may be used in this step; preferably the filter used for removal of viral particles is selected from Viresolve PRO (Merck), Bio EXL PALL PEGASUS PRIME, PEGASUS SV4 (Pall Life Sciences), and Virosart (Sartorius).
In tenth aspect of the fifth embodiment, the therapeutic protein is concentrated to a desired concentration and buffer exchanged in formulation buffer. The buffer is exchanged in a tangential flow filtration system or an ultra flow filtration system. The other parameters of Tangential flow filtration comprises of one or more selected from Diafilteration using diafilteration buffer [25 mM Histidine buffer; 75 mM Arginine buffer; 50-150 mM NaCl; pH 6.50±0.5]; Cleaning buffer [0.5M NaOH]; Storage buffer [0.1M NaOH]; Equilibration using 5-10× membrane volume; Concentration and Diafilteration using 10-20 diafilteration volume; WFI wash using 3-5 membrane volume; cleaning using 0.5-1.0 M NaOH; Storage [0.1M NaOH]. In one of the preferred aspect of this embodiment, Tangential flow filtration is carried out using 30 kDa MWCO membrane selected from the group comprising one or more of Centramate T series PES membrane (Pall Corporation), Hydrosart (Sartorius), and Pelicon 3 (Merck).
In sixth embodiment of the present invention, the said purified therapeutic protein is formulated with pharmaceutical excipients, wherein the osmolality of the formulation is in the range of 300 mOsm/Kg to 500 mOsm/Kg and viscosity of the formulation is less than 2.5 mPa-S.
In first aspect of the sixth embodiment, therapeutic protein formulation comprises of atleast one antigen binding protein, atleast one stabilizer, atleast one buffering agent, atleast one tonicity agent, and atleast one surfactant. Optionally, formulation comprises of a preservative.
In second aspect of the sixth embodiment, stabilizer is an carbohydrate. Stabilizer is selected from the group comprising of one or more of sucrose, sorbitol, trehalose, mannitol, dextran, inositol, glucose, fructose, lactose, xylose, mannose, maltose, Raffinose and combination thereof; more preferably the stabilizer is sucrose. In yet another aspect of this embodiment, stabilizer comprises of sucrose at a concentration of about 0.1% to about 2.5% w/v, preferably <1% sucrose w/v.
In third aspect of the sixth embodiment, buffering agent is selected from the group comprising of one or more of histidine, arginine, glycine, sodium citrate, sodium phosphate, citric acid, HEPES, potassium acetate, potassium citrate, potassium phosphate, sodium acetate, sodium bicarbonate, Tris base, or Tris-HCl, and combination thereof. Preferably, 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
In fourth aspect of sixth embodiment, buffering agent is Histidine. In preferred aspect of this embodiment, the buffering agent comprises Histidine at 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 most preferred aspect of this embodiment, buffering agent comprises Histidine at a concentration of about 25 mM.
In fifth aspect of sixth embodiment, buffering agent is Arginine. In preferred aspect of this embodiment, the buffering agent comprises Arginine at 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 most preferred aspect of this embodiment, buffering agent comprises Arginine at a concentration of about 70 to 80 mM.
In sixth aspect of sixth embodiment, tonicity agent is selected from the group comprising of one or more of sodium chloride, dextrose, glycerin, mannitol, and potassium chloride. In preferred aspect of this embodiment, tonicity agent comprises of Sodium Chloride and is present at a concentration of about 10 mM to about 500 mM; preferably at concentration of about 50 mM to about 250 mM; most preferably at a concentration of about 100-145 mM.
In seventh aspect of sixth embodiment, surfactant is present at a concentration of about 0.001 to about 0.2% (w/v); and 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). In preferred aspect of this embodiment, surfactant comprises of Polysorbate 80 and is present at a concentration of about 0.001% to about 0.2% w/v; preferably at 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%.
In eighth aspect of sixth embodiment, the formulation comprises 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 comprises of a therapeutic protein at a concentration of about 1 mg/L to about 50 mg/L.
In ninth aspect of the sixth embodiment, the formulation further comprises of preservative, the preservative may be selected from the group comprising of benzyl alcohol, m-cresol, and phenol.
In seventh embodiment of the present invention, the therapeutic protein formulation comprises of atleast one therapeutic protein, sucrose, arginine, histidine, Sodium chloride, Polysorbate 80. Preferably therapeutic protein formulation comprises of about 1 mg/ml to about 50 mg/ml of therapeutic protein; about 20 mM to about mM mg/ml of Histidine; about 50 mM to about 100 mM of Arginine; about 0.002% to about 0.02% Polysorbate 80 (w/v); about 50 mM to about 150 mM NaCl; and <2.5% Sucrose w/v. The pH of the formulation is in the range of 6.0 to about 7.0 and Osmolality of the formulation is in the range of 300 mOsm/Kg to about 450 mOsm/Kg.
In one of the preferred aspect of seventh embodiment, a pharmaceutical formulation comprises of 2-80 mg/ml of Dengue monoclonal antibody; 25 mM of Histidine; 75 mM of Arginine; 101 mM NaCl; 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.
In one of the preferred aspect of seventh embodiment, a pharmaceutical formulation comprises of 25 mg/ml of Dengue monoclonal antibody; 25 mM of Histidine; 75 mM of Arginine; 101 mM NaCl; 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.
In one of the preferred aspect of seventh embodiment, a pharmaceutical formulation comprises of 50 mg/ml of Dengue monoclonal antibody; 25 mM of Histidine; 75 mM of Arginine; 101 mM NaCl; 0.02% Polysorbate 80 (w/v); and 0.5% Sucrose; wherein pH of the formulation is 6.5±0.5 Osmolality 380 mOsm/Kg, viscosity less than 2.5 mPa-S.
In one of the preferred aspect of seventh embodiment, a pharmaceutical formulation comprises of 2-80 mg/ml of Rabies monoclonal antibody; 25 mM of Histidine; 75 mM of Arginine; 101 mM NaCl; 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.
In one of the preferred aspect of seventh embodiment, a pharmaceutical formulation comprises of 25 mg/ml of Rabies monoclonal antibody; 25 mM of Histidine; 75 mM of Arginine; 101 mM NaCl; 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.
A pharmaceutical formulation comprising of 50 mg/ml of Rabies monoclonal antibody; 25 mMof Histidine; 75 mMof Arginine; 101 mM NaCl; 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.
According to another aspect of the seventh embodiment, said pharmaceutical formulation of antibody could be a lyophilized formulation.
In 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 preferred aspect of the eighth embodiment, indirect ELISA based method is used to quantify binding of therapeutic protein to the specific antigen. In preferred aspect of this embodiment, Dengue Mab formulation is tested against all serotypes of the dengue viruses and amount of Dengue mAb is determined. The potency of the therapeutic protein is reported as % activity relative to the reference standard. It is very well understood that any other similar method may be used to demonstrate the potency and affinity of the therapeutic protein.
In ninth embodiment of the present invention, focus reduction neutralization test (PRNT/FRNT) or a related test is carried out for evaluating neutralization of viral activity by therapeutic protein. In preferred aspect of this embodiment, Dengue mAb formulation is tested against all serotypes of the dengue viruses and EC50 values are calculated for neutralization of Dengue Viruses. It is very well understood that any other similar method may be used to demonstrate the neutralization activity of the therapeutic protein.
In tenth embodiment of the present invention, HPLC based size exclusion chromatography is used to assess the presence of aggregates in therapeutic protein formulation. In preferred aspect of this embodiment, Phenomenex Bio-Sec-S 3000 column is used to demonstrate the aggregate and monomer percentage of Dengue mab formulation. It is very well understood that any other similar method may be used to assess the presence of aggregates in therapeutic protein formulation.
In eleventh embodiment of the present invention, the formulation may be stored in a suitable container. The container may be selected from a bottle, a vial, a 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.
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), polyolefin, polycyclopentane (CZ®), cyclic olefin copolymer (COC), and combinations or copolymers thereof.
The anti-dengue antibody or anti-rabies antibody formulations disclosed herein can be used (alone or in combination with other agents or therapeutic modalities) to treat, prevent and or diagnose dengue or rabies virus. For example, the combination therapy can Include an anti-dengue antibody molecule co-formulated with, and/or co-administered with, one or more additional therapeutic agents, e.g., antiviral agents (Including other anti-dengue antibodies), vaccines (Including dengue virus vaccines), or agents that enhance 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 may advantageously utilize lower dosages of the administered therapeutic agents, thus avoiding possible toxicities or complications associated with the various monotheraples.
Protocol:
Cell culturing at 10 L scale was carried out in a Fed batch manner using below mentioned parameters during fermentation/upstream process.
The Dengue monoclonal antibody was expressed in cell line “CHO-K1 SV GS-KO” obtained from Visterra Inc. USA.
Feed supplementation was done in a gradual drip manner as per following Table 1:
Results & Conclusion:
Viable colony count and Yield obtained during the fermentation process was as follows:
Applicant has found that by using Cell culture process comprising of basal medium, concentrated basal medium as feed solution, use of feed solutions along-with a definite feeding strategy, enhanced cell growth, lower concentrations of lactate and ammonia can be obtained thereby effectively maintaining the cell count and increasing cell longevity and high yield. Yield of greater than 4 gm/L was obtained in fermentation process. Harvest obtained was further subjected to purification/downstream processing.
Cell culture obtained in Example 1 was harvested and later subjected to protocol for purification of the dengue (VIS513) monoclonal antibody as per
Detailed process used was as follows:
Process Parameters Used:
1. Low pH Viral Inactivation
2. Cation Exchange Chromatography
Positively charged antibody molecules bound with the column while negatively charged molecules come in the flow through. Column bound antibody molecules are eluted using salt gradient.
Materials Used:
Resin used: Fractogel SO3−/Fractogel SE Hicap (Merck)
Residence Time: 4.00-7.00 minutes
Column used: XK 26
Pre Equilibration: 200 mM Citrate buffer pH 6.0±0.2.
Equilibration: 10 mM Citrate buffer+0.025% (w/v) Polysorbate 80, pH 6.0±0.2.
Loading: 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 NaCl, pH 6.0±0.2.
CIP Buffer: 0.5 M NaOH
Storage Buffer: 0.1 M NaOH
Process Parameters:
Fraction Collection During Gradient
Anion Exchange Chromatography:
All negatively charged impurities are bound with the membrane while antibody comes in the flow through.
Materials Used:
Membrane/Resin used: Sartobind Q single Sep mini (Sartorius)/Eshmuno Q
Loading volume: 150 mg/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 0.025% PS-80 pH 6.0±0.2
Storage Buffer: 0.1 M NaOH
Process Parameters:
3. Nano-Filtration:
20 nm nanofilter i.e. Viresolve PRO (Merck), was used to remove any virus particles available in the therapeutic protein.
4. Tangential Flow Filtration/Ultra Flow Filtration:
The antibody was concentrated to desired concentration and buffer exchanged in one of the three formulation buffers.
Material Used:
Formulation Buffer:
Storage Buffer: 0.1 M NaOH
Membrane used: PALL Centramate T Series, PES membrane MWCO: 30 kDa
Process Parameters:
Sterile Filtration
Stabilizer was added to the antibody solution and sterile filtered through 0.2μ filter.
Results:
Stage wise recovery of the various steps used in the purification process.
The overall process recovery was found to be ˜80% and overall purity was found to be >99%.
Purified Dengue (VIS513) monoclonal antibody was formulated as follows:
Excipients i.e. Arginine, Histidine, NaCl, Sucrose, and polysorbate-80 were added and mixed thoroughly using a magnetic stirrer at 50-60 RPM to form a mixture of excipients. This mixture was then added into the Dengue mAb TFF harvest gradually with stirring rate 50-60 RPM. pH was checked (pH 6.5) and if required adjusted by histidine-arginine buffer. The final formulation was filtered through a 0.2 μM filter and filled into final container.
The concentration of each component in the final formulation was as follows:
These formulations were further tested for purity, stability, efficacy and potency for 9 months.
Effect of presence of Sucrose in VIS513 Dengue antibody formulation was studied for testing potency by ELISA assay on EDIII protein of DV1. The formulation studies were done for temp. 2-8° C., 25° C., and 40° C.
Results:
1. VIS513 Dengue Antibody Formulation without Sucrose
2. VIS513 Dengue Antibody Formulation with Sucrose
Reference Standard Formulation Composition:
Conclusion: Addition of 0.5% w/v improves stability as compared to the corresponding sampling point without sucrose.
VIS513 antibody formulation was stored at 40° C. for 20 days and later potency of VIS513 was evaluated by ELISA test. Effect of increasing Sucrose Strength was studied on VIS513 antibody formulation at 40° C., wherein sucrose concentration of 0.1, 0.2 and 0.5% was evaluated.
Results:
Reference Standard Formulation Composition:
Conclusion: Highest stability was observed formulation comprising 0.5% Sucrose.
Analytical test for purity, stability, efficacy and potency of Dengue (VIS513) Mab formulation with storage at
6.1: Potency of VIS513 Antibody Formulation was Tested by Indirect ELISA.
The indirect ELISA based method was used to quantify binding of Dengue Mab (VIS513) to EDIII protein of DV1 antigen. EDIII protein was immobilized to the plate. Unbound antigen was removed by washing. In the next, step standard and test samples were added, allowed to bind to the antigen. To determine the amount of bound Dv-Mab, Mouse anti-Human IgG Fc-HRP, specific to Dv-Mab (human Immunoglobulin Fc fragment), was used to recognize the presence of Dv-Mab. The assay was developed with TMB Microwell Peroxidase Substrate System which quantifies the extent of binding by amount of color formed at 450 nm. The data analysis software generated a binding curve for each sample using a four parameter curve fitting model, and compared the binding curve of the test sample to the standard curve by calculating Relative Potency. The potency of a test sample is reported as % Activity relative to reference standard (Relative Potency times 100).
Results:
6.2: PRNT Assay to Determine EC50
The assay involves premixing serially diluted antibody with virus to allow antibody binding, neutralization then transfer of mixture to a Vero cell monolayer, overlay with a viscous medium, incubation (˜3-7 days, depending on virus serotype) to allow limited virus replication and spread, followed by detection of plaques. Neutralization was captured by the reduction of plaque formation. Robust detection was achieved with immunostaining methods, using mouse 4G2 Anti-Dengue antibody and HRP-labelled goat anti-mouse antibody with Peroxidase substrate.
The Dengue (VIS513) Mab formulation samples were been tested against all four serotypes of dengue viruses i.e. DV1, DV2, DV3 and DV4. EC50 value was calculated for neutralization of Dengue viruses. EC50 value represents the 50% effective concentration required for the effective neutralization of dengue viruses and EC50 value calculated from number of plaques present in the virus control wells and number of plaques in the wells in which mab-Virus incubated samples were added.
Results:
Dengue (VIS513) mab formulation did not show any time dependent loss of virus neutralization efficacy at 2-8° C. & 25° C. VIS513 formulation even if kept at 40° C., does not lose its ability to neutralize dengue virus.
6.3: Aggregation and Purity Analysis
A HPLC-based size exclusion chromatography (HPLC-SEC) was used to assess the aggregates in the bulk and final formulation of DV Mab. In this method a phenomenex Bio-Sec-S 3000 column was used to demonstrate the aggregates and monomer percentage of Dengue (VIS513) Mab by injecting the ˜50 ug of total antibody and run at a flow rate of 1 ml/minute for 35 minutes. Phosphate buffered Saline (PBS), pH 6.5 was used as mobile phase.
Results: SEC-HPLC (Acceptance Range is NLT 90%)
Dengue (VIS513) mab formulation did not show any significant time dependent aggregation; and purity/monomer content was found to be >98%.
Effect of Surfactant Concentrations of Formulations:
Effect of Surfactant concentration was evaluated by sub-visible particle analysis. Formulations varying Polysorbate-80 strengths were prepared and analyzed for Sub visible particle analysis.
Conclusion:
In the formulation containing 0.005% w/v Polysorbate-80 minimum sub visible particles were observed. Depending on dose, if the formulation requires dilution Polysorbate 80 strength was finalized 0.02% wN with margin of 4 fold.
Minimum buffer strength required (10-30 mM) was referred from the available literature. To find out minimum Arginine (used as solubilising agent and viscosity reducing agent) Mab sample was buffer exchanged into normal saline and Arginine stock solution (300 mM) was gradually added. The aggregation of the solution was monitored by measuring OD@350 nm. The saline with 75 mM Arginine gave lowest OD hence 75 mM Arginine was finalized.
Viscosity Studies of Dengue (VIS513) Antibody Formulation
Viscosity of DV mab samples was measured on a microchip based Viscometer, Model: microVISC™ (Make: RheoSense, CA USA) as per procedure mentioned in the instrument manual.
Conclusion:
No time dependent increase in viscosity was observed in the mab formulation stored at 2-8° C. for 90 days as well at a sample kept at 25° C. for 1 month, this is primarily due to the excipient-Arginine 75 mM. Viscosity of our formulation was found to be 1.1 to 1.2 mPa-S/cP, which is lower than other marketed formulations that have viscosity between 11-50 mPa-S/cP
Virus validation was performed for actual manufacturing process, to test the effectiveness of the virus removal by virus filtration in the manufacturing process of monoclonal antibody.
Murine Leukemia Virus (MuLV) and Minute virus of mice (MMV/MVM) were used as model organisms. Inventors of this invention compared the ability of their inventive purification process with that of the general and well established method of monoclonal antibody purification.
The general and well established method of monoclonal antibody purification comprised of Protein-A Affinity Chromatography (GE Resin); Low pH Treatment; Sartobind Q Chromatography (Anion Exchange Membrane, Sartorius, single use); Sartobind Phenyl Chromatography (Membrane Chromatography, Sartorius, single use); Viresolve Pro filtration (Nanofiltration, Merck).
Results:
SIIPL purification process was highly efficient in viral clearance, total LRV achieved is as per the ICH guidelines. (Standard Process LRV 12.64 while SIIPL inventive process 23.74) Dengue antibody purified using our inventive process was found to be suitable for human clinical trials without any viral risk.
Protocol:
Cell culturing at 2 L scale was carried out in a Fed batch manner using below mentioned parameters during fermentation/upstream process.
Feed supplementation was done in a gradual drip manner as per following table:
The cell culture was harvested upon drop in OD up to 60%
Results & Conclusion:
Yield of 3-5 gm/L was obtained of the fermentation process. Harvest obtained was further subjected to purification/downstream processing.
Cell culture obtained according to example 9 was harvested and later subjected to protocol for purification of the rabies monoclonal antibody as per
Detailed process used was as follows:
Process Parameters Used:
1. Low pH Viral Inactivation
2. Cation Exchange Chromatography
Positively charged antibody molecules bound with the column while negatively charged molecules come in the flow through. Column bound antibody molecules are eluted using salt gradient.
Materials Used:
Resin used: Fractogel SO3−/Fractogel SE Hicap (Merck)
Residence Time: 4.00-7.00 minutes
Column used: XK 26
Pre Equilibration: 200 mM Citrate buffer pH 6.0±0.2.
Equilibration: 10 mM Citrate buffer+0.025% (w/v) Polysorbate 80, pH 6.0±0.2.
Loading: 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 NaCl, pH 6.0±0.2.
CIP Buffer: 0.5 M NaOH
Storage Buffer: 20% ethanol+150 mM NaCl
Anion Exchange Chromatography:
All negatively charged impurities are bound with the membrane while antibody comes in the flow through.
Materials Used:
Membrane/Resin used: Sartobind Q single Sep mini (Sartorius)/Eshmuno Q
Loading volume: 150 mg/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 0.025% PS-80 pH 6.0±0.2
Storage Buffer: 20% ethanol+150 mM NaCl or 0.1 M NaOH
3. Nano
20 nm nanofilter i.e. Viresolve PRO (Merck), was used to remove any virus particles available in the therapeutic protein.
4. Tangential Flow Filtration/Ultra Flow Filtration:
The antibody was concentrated to desired concentration and buffer exchanged in one of the three formulation buffers.
Material Used:
Formulation Buffer:
Storage Buffer: 20% ethanol+150 mM NaCl or 0.1 M NaOH
Membrane used: PALL Centramate T Series, PES membrane MWCO: 30 kDa
Sterile Filtration
Stabilizer was added to the antibody solution and sterile filtered through 0.2μ filter.
Results:
The overall process recovery was found to be >80%.
The overall purity of the rabies mab after purification was found to be >99% and overall recovery was found to be >80%.
Purified Rabies monoclonal antibody was formulated as per the flowchart given in
Excipients i.e. Arginine, Histidine, NaCl, Sucrose, and polysorbate-80 were added and mixed thoroughly using a magnetic stirrer at 50-60 RPM to form a mixture of excipients. This mixture was then added into the Dengue mAb TFF harvest gradually with stirring rate 50-60 RPM. pH was checked (pH 6.5) and if required adjusted by histidine-arginine buffer. The final formulation was filtered through a 0.2 μM filter and filled into final container.
These formulations were further tested for purity, stability, efficacy and potency for 9 months.
13.1 Aggregation and Purity Analysis
A HPLC-based size exclusion chromatography (HPLC-SEC) was used to assess the aggregates in the bulk and final formulation of DV Mab. In this method a phenomenex Bio-Sec-S 3000 column was used to demonstrate the aggregates and monomer percentage of Rabies Mab by injecting the ˜50 ug of total antibody and run at a flow rate of 1 ml/minute for 35 minutes. Phosphate buffered Saline (PBS), pH 6.5 was used as mobile phase.
Results:
Rabies mab formulation did not show any time dependent aggregation and purity/monomer content was found to be >99%.
13.2 SDS Page Analysis Batch 1—Test Sample at 2-8° C.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2016621044139 | Dec 2016 | IN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IB2017/058194 | 12/20/2017 | WO | 00 |
