A process for the purification of polyclonal antibodies

Abstract

The present invention relates to a process of purifying a protein. Specifically, the present invention relates to a process of purification of a polyclonal antibody and / or antibody fragments. The present invention employs an adsorptive separation using anion exchange chromatography and cation exchange chromatography along with ultrafiltration and diafiltration to achieve high-purity antibody / or antibody fragments. The process provided in the present invention is applicable for polyclonal antibody and / or antibody fragments, which are associated with conditions but not limited to snake envenomation, scorpion envenomation, diphtheria infection, tetanus infection, rabies virus infection and gas gangrene infection due to Clostridium species.

Description

[0001] PT / 2025 / 17145

[0002] A PROCESS FOR THE PURIFICATION OF POLYCLONAL ANTIBODIES FIELD OF INVENTION

[0003] The present invention relates to a process for the purification of proteins. In particular this invention relates to a process of purification of a polyclonal antibody and / or antibody fragments. More Particularly, the present invention relates to a chromatography-assisted purification of polyclonal antibody and / or antibody fragments.

[0004] BACKGROUND OF THE INVENTION

[0005] Polyclonal antibody therapies (pAB’s) have a long history of use since the 19th century to protect humans against various infectious agents, toxins, and disease conditions such as an autoimmune disorder. Polyclonal antibody (pAb) represents a collection of antibodies produced from B-cells that recognize many epitopes and have differing specificities against the various antigens. The complex population of polyclonal antibodies includes different subclasses of antibodies such as IgG, IgM, IgE, IgA, and IgD.

[0006] Polyclonal therapeutic antibodies are produced by hyper-immunizing animals with varying concentrations of antigens. Overtime, the animal's serum neutralizes the antigens by producing polyclonal antibodies specific to them. These antibodies are then purified from the serum of hyper-immunized animals and used to treat various disease conditions. Commercially available polyclonal antibodies are frequently obtained from various animal sources such as goats, rabbits, mice, camels, ovine, and equine.

[0007] Polyclonal antibodies have been widely used to treat multiple diseases such as venomous bites (Snake or scorpion) and manage other disease conditions such as serum sickness, anaphylaxis, and drug-related adverse effects. They also play a crucial role in combating infectious diseases like Rabies, Nipah, Hendra, Tetanus, Dengue, and Gas-gangrene. One such example is Antisnake venom (ASV), which is a polyvalent antivenom (polyclonal antibodies) effective against "the big four" snake species: the common krait (Bungarus ceruleus), Cobra (Naja naja), Russell's viper (Daboia russelii), and saw-scaled viper (Echis carinatus). On large scale, most commonly used industrial purification procedure for ASV involves salting-out andPT / 2025 / 17145

[0008] precipitating non-immunoglobulin proteins from hyper-immunized equine plasma against the "big four" snakes.

[0009] These polyclonal antibodies are produced by periodic immunization of animals with a gradual increase of sub-lethal doses of specific antigens or cocktails of antigens in combination with various adjuvants such as potent complete Freund’s adjuvant (CFA), incomplete Freund’s adjuvant (IF A), Bentonite, and alum. The low dose, low volume, multi-site immunization of animals is a widely used method in producing potent polyclonal antibodies.

[0010] After immunizing animals, the titer (potency) levels of polyclonal antibodies from animal plasma are analyzed at regular intervals until the desired titer (potency) is achieved. The immunization is designed to stimulate the immune system of the animal to produce a high amount of neutralizing immunoglobulins. Once the desired titer (potency) of pAB’s is achieved, animals are either bled (1.5% of the animal's total body weight) or sacrificed to collect blood under sterile conditions with anticoagulants. The collected blood is kept under cold conditions overnight, and plasma is separated using the decanting method into sterile bottles, while separated blood cells are reinfused into the respective animals.

[0011] Two commonly used methods were employed to purify polyclonal antibodies from different sources (i) Salt-based fractionation, a simple but crude method for polyclonal antibody purification that results in partial purification, and (ii) Caprylic acid-based precipitation of impurities in hyper-immunized serums leading to partially purified antibody solution. Precipitation based methods always have limitations in clearance of various process and product related impurities in serum. Significant effort has been made for the development of the purification process of polyclonal antibodies.

[0012] Despite achieving higher IgG purity through single stage caprylic acid precipitation, the recovery values remain low, approximately 67%. Varying concentrations of caprylic acid alone do not achieve a higher purity level yielding not more than 70% of the recovered product (A. Fernandes et al., 2008-Chromatographic purification of equine immunoglobulin GF(ab)2 from plasma). A. Fernandes et al., demonstrated the efficient separation of active F(ab)2 from Fc fragments and other serum proteins. In this investigation, equine F(ab)2 fragments from horse serum were purified using a combination of ion exchange and protein A-based affinity purification chromatography. However, the integration of affinity -based matrices for purifyingPT / 2025 / 17145

[0013] polyclonal antibodies is limited due to their high cost, especially when high doses are required, and product costs are too low. This investigation does not report clearance of product related impurities such as aggregated form of the polyclonal antibodies.

[0014] EP 2399613 Al and US 5164487A describe methods for plasma protein fractionation using caprylic acid and highlight challenges when applied to equine plasma containing polyclonal antibodies against snake venom. EP2399613A1 describes plasma protein fractionation, specifically immunoglobulin using caprylic acid. However, precipitation using caprylic acid of equine plasma comprising polyclonal antibodies against snake venom resulted in a recovery of less than 50 to 65 % of total immunoglobulin. US5164487A describes the manufacturing of intravenous immunoglobulin-G preparation process for separating target proteins from nontarget proteins. However, the precipitation of non-immunoglobulin proteins causes the coprecipitation of target proteins in plasma and the formation of soluble aggregates due to vigorous mixing during the formation of plasma and caprylic acid emulsion, resulting in a supernatant with product purity of less than 70%. This invention does not describe the purity levels of polyclonal antibody therapeutics.

[0015] Despite its significant advantages this method has other problems when dealing with the large plasma volume. It generates large amounts of solid waste that forms after non-IgG precipitation. This must be removed either by filtration or centrifugation. This investigation does not explain the clearance of low molecular mass impurities associated with polyclonal antibody therapeutics.

[0016] Despite their efficacy in neutralizing toxins, the production and purification of polyclonal antibodies have several limitations. The purification of polyclonal antibodies is a complex process due to the presence of a multi-component mixture of host serum proteins, including albumin and fibrinogen. Conventional methods, such as ammonium sulfate precipitation and caprylic acid-based precipitation, often result in protein aggregation, reducing antibody yield and activity. Additionally, these methods do not remove host-specific impurities, leading to low-purity target protein products. Another critical challenge in pAB’s production is endotoxin contamination, which can compromise the safety of therapeutic pAB’s preparations. Purification using precipitation methods results in significant loss of polyclonal antibody and / or antibody fragments.PT / 2025 / 17145

[0017] Accordingly, there is a clear need to provide a process of purification of polyclonal antibodies and / or antibody fragments used in the treatment of but not limited to snake envenomation, scorpion envenomation, diphtheria infection, tetanus infection, rabies virus infection and gas gangrene infection due to Clostridium species.

[0018] OBJECTIVES OF THE INVENTION

[0019] Main objective of the present invention is to provide a process of purification of polyclonal antibodies and / or antibody fragments.

[0020] Yet another objective of the present invention is to provide a process of purification of polyclonal antibodies and / or antibody fragments that not only increases the process yield but also produces a higher purity product of over 90% with an increase in potency.

[0021] Yet another objective of the present invention is to provide chromatography-assisted purification of polyclonal antibodies and / or antibody fragments.

[0022] Yet another objective of the present invention is to provide a one-step chromatography-assisted purification of polyclonal antibodies and / or antibody fragments.

[0023] Yet another objective of the present invention is to provide a two-step chromatography-assisted purification of polyclonal antibodies and / or antibody fragments.

[0024] Yet another objective of the present invention is to provide purified polyclonal antibodies and / or antibody fragments for the conditions but not limited to snake envenomation, scorpion envenomation, diphtheria infection, tetanus infection, rabies virus infection and gas gangrene infection due to Clostridium species.

[0025] SUMMARY OF THE INVENTION

[0026] Accordingly, the present invention provides a process for the purification of proteins.

[0027] In an aspect of the present invention, the process for purifying a protein comprising the steps of:PT / 2025 / 17145

[0028] a) providing a sample containing the protein of interest; wherein the sample is blood, plasma or serum;

[0029] b) diluting the sample adjusting its pH, digesting the proteins in the sample preferably by enzymatic digestion with pepsin, followed by quenching;

[0030] c) ultrafiltration of the sample obtained in step (b) followed by diafiltration;

[0031] d) optionally subjecting the sample obtained from step (c) to anion exchange chromatography;

[0032] e) subjecting the sample obtained from step (c) or the flowthrough obtained from step (d) to cation exchange chromatography;

[0033] f) subjecting the eluate from step (e) to ultrafiltration followed by diafiltration to obtain the purified protein;

[0034] wherein the protein is preferably a polyclonal antibody and / or antibody fragment.

[0035] In an embodiment of the present invention, the process comprises the steps of:

[0036] a) providing a sample containing the protein of interest; wherein the sample is blood, plasma or serum;

[0037] b) diluting the sample, adjusting its pH, enzymatic digestion of the sample with pepsin, followed by quenching;

[0038] c) ultrafiltration of the digested sample obtained in step (b) followed by diafiltration;

[0039] d) subjecting the sample obtained from step (c) to anion exchange chromatography;

[0040] e) subjecting the flowthrough from step (d) to a cation exchange chromatography to obtain the protein of interest;

[0041] f) subjecting the eluate of step (e) to ultrafiltration followed by diafiltration to obtain the purified protein;PT / 2025 / 17145

[0042] wherein the protein is preferably a polyclonal antibody and / or antibody fragment.

[0043] In an embodiment of the present invention, the process comprises the steps of:

[0044] a) providing a sample containing the protein of interest; wherein the sample is blood, plasma or serum;

[0045] b) diluting the sample, adjusting its pH, enzymatic digestion of the sample with pepsin, followed by quenching;

[0046] c) ultrafiltration of the digested sample obtained in step (b) followed by diafiltration;

[0047] d) subjecting the sample from step (c) to a cation exchange chromatography to obtain the protein of interest;

[0048] e) subjecting the eluate of step (d) to ultrafiltration followed by diafiltration to obtain the purified protein;

[0049] wherein the protein is preferably a polyclonal antibody and / or antibody fragment.

[0050] In an aspect, the antibody or antibody fragment is an antibody or antibody fragment against snake venom, scorpion venom, diphtheria toxin, tetanus toxin, rabies virus, or Clostridium toxin and belongs to one or more subclasses, including but not limited to IgG, IgM, IgE, IgA, and IgD.

[0051] In an embodiment, the sample is diluted with water in a ratio of 1:1 to 1:2 and the enzymatic digestion of the sample with pepsin is performed at pH 2.0 to 4.0, preferably pH 3.0 ± 0.2 at 30-40°C for 2-4 hrs, preferably at 37 ± 1°C for 3 hrs and neutralized at pH 5.0 ± 0.2.

[0052] In an embodiment, the ultrafiltration membrane has a molecular weight cut-off (MWCO) of 30 kDa - 50 kDa and ultrafiltration & diafiltration are carried out in a tangential flow direction, arranged in series or in parallel with a suitable pressure, time, volume, or protein concentrationbased flow and flow path controller.PT / 2025 / 17145

[0053] In another embodiment of the present invention, anion exchange chromatography is performed in either bind-elute mode or flow-through mode, preferably in flow-through mode to obtain protein of interest in flow-through and to capture process and product related impurities.

[0054] In an embodiment of the present invention, the loading capacity for the product of interest is more than 100 mg / mL for anion exchange chromatography and performed at pH 4 - 6 with conductivity 0.001 to 20 mS / cm and the elution is performed with conductivity 0.001 to 200 mS / cm.

[0055] In an embodiment of the present invention, cation exchange chromatography is performed in either bind-elute mode or flow-through mode, preferably in bind-elute mode to bind protein of interest and separate impurities in flow-through.

[0056] In an embodiment of the present invention, the loading capacity for the product of interest is more than 30 mg / mL for cation exchange chromatography and performed at pH 4 - 6 with conductivity 0.001 to 20 mS / cm and the elution is performed with conductivity 0.001 to 200 mS / cm

[0057] In an embodiment of the present invention, cation exchange chromatography is performed in bind-elute mode at a breakthrough capacity at greater than 0.1% of the product of interest.

[0058] In an embodiment of the present invention, the number of chromatography columns used for anion exchange chromatography and cation exchange chromatography is at least one.

[0059] In an embodiment of the present invention, the anion exchange chromatography or cation exchange chromatography is performed using the axial or radial flow chromatography column. In an embodiment of the present invention, the elution is performed with a salt-based linear gradient and / or a stepwise gradient over 5 to 15 column volumes.

[0060] In an embodiment of the present invention, the residence time for anion and / or cation exchange chromatography is more than 5 seconds but less than 30 minutes, preferably 4- 8 minutes, and more preferably, 6 minutes.PT / 2025 / 17145

[0061] In an embodiment, the anion exchange chromatography is performed using anion exchange functional groups selected from diethylaminoethyl, quaternary ammonium polyethyleneimine, and trimethylammoniumethyl, linked to bead based, membrane based, hydrogel based or fibrebased chromatography matrix.

[0062] In an embodiment, the cation exchange chromatography is performed using a cation exchange functional group selected from a group consisting of sulfonate group, sulfopropyl group and sulphonic acid linked to bead based, membrane based, hydrogel based or fibre-based chromatography matrix.

[0063] In an embodiment of the present invention, the ultrafiltration and diafiltration of step (c) is carried out at pH 4 - 6.

[0064] In an embodiment of the present invention, the ultrafiltration of step (f) equilibration and concentration is carried out at pH 4 - 6 and the diafiltration of step (f) is carried out at pH 6 -8, preferably at pH 7.4.

[0065] In an embodiment of the present invention, the process can be performed in a batch or in an integrated continuous operation.

[0066] In an embodiment of the present invention, the purity of polyclonal antibody is greater than 90%, preferably 95%, and more preferably about 99%; and the recoveryof the purified polyclonal antibody and / or antibody fragments is more than (>) 50%, preferably 80% and more preferably about 90 %and is independent of the initial concentration of the polyclonal antibody in the sample.

[0067] In an embodiment of the present invention, the purified polyclonal antibody and / or antibody fragment contains not more than 1% of animal albumin protein in purified drug substance and comprises less than 10% soluble aggregate.

[0068] In an embodiment of the present invention, the present invention provides a composition comprising the purified polyclonal antibody and / or antibody fragments obtained by the process of the present invention.PT / 2025 / 17145

[0069] In an embodiment of the present invention, the polyclonal antibody and / or polyclonal antibody fragment are produced from animals, including but not limited to horses, donkeys, mules, ponies, sheep, mice, guinea pigs, goats, and camels.

[0070] BRIEF DESCRIPTION OF THE DRAWINGS

[0071] Figure 1 depicts the processes of purifying polyclonal antibody and / or polyclonal antibody fragments F(ab)2.

[0072] Figure 2 indicates the enzyme digestion kinetics of polyclonal antibody to polyclonal antibody fragments F(ab)2 at different pH (A, B) at pH 3.0 and (C and D) at pH 3.5. : (A): - Lane 1: Plasma 1:2 diluted , Lane 2: Blank, Lane 3: Plasma pH 3.0 adjusted, Lane 4: Blank, Lane 5: Pepsin digestion ‘0’ hour, Lane 6: Blank, Lane 7: Pepsin digestion ‘0.5’ hour, Lane 8: Blank, Lane 9: Pepsin digestion ‘1’ hour, Lane 10: Ladder. (B): - Lane 1: Pepsin digestion ‘2’ hour, Lane 2: Blank, Lane 3: Pepsin digestion ‘3’ hour, Lane 4: Blank, Lane 5: Pepsin digestion ‘4’ hour, Lane 6: Blank, Lane 7: Pepsin digestion ‘5’ hour, Lane 8: Blank, Lane 9: Pepsin digestion ‘6’ hour, Lane 10: Ladder. (C): - Lane 1: Plasma 1:2 diluted, Lane 2: Blank, Lane 3: Plasma pH 3.5 adjusted, Lane 4: Blank, Lane 5: Pepsin digestion ‘0’ hour, Lane 6: Blank, Lane 7: Pepsin digestion ‘0.5’ hour, Lane 8: Blank, Lane 9: Pepsin digestion ‘1’ hour, Lane 10: Ladder.

[0073] (D): - Lane 1: Pepsin digestion ‘2’ hour, Lane 2: Blank, Lane 3: Pepsin digestion ‘3’ hour, Lane 4: Blank, Lane 5: Pepsin digestion ‘4’ hour, Lane 6: Blank, Lane 7: Pepsin digestion ‘5’ hour, Lane 8: Blank, Lane 9: Pepsin digestion ‘6’ hour, Lane 10: Ladder.

[0074] Figure 3 indicates the pepsin binding capacity on anion exchange resin using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. (SDS-PAGE). (A): - Lane 1 : Pepsin, Lane 2: Anion exchange chromatography wash, Lane 3: Anion exchange chromatography elute, Lane 4: Ladder, Lane 5: Anion exchange chromatography flowthrough fraction 2, Lane 6: Anion exchange chromatography flowthrough fraction 4, Lane 7: Anion exchange chromatography flowthrough fraction 7, Lane 8: Anion exchange chromatography flowthrough fraction 10, Lane 9: Anion exchange chromatography flowthrough fraction 11, Lane 10: Anion exchange chromatography flowthrough fraction 12. (B): - Lane 1: Ladder, Lane 2: Anion exchange chromatography flowthrough fraction 14, Lane 3: Anion exchange chromatography flowthrough fraction 15, Lane 4: Anion exchange chromatography flowthrough fraction 18, Lane 5: Anion exchange chromatography flowthrough fraction 20, Lane 6: Anion exchangePT / 2025 / 17145

[0075] chromatography flowthrough fraction 23, Lane 7: Blank, Lane 8: Anion exchange chromatography flowthrough fraction 28, Lane 9: Anion exchange chromatography flowthrough fraction 31, Lane 10: Anion exchange chromatography flowthrough fraction 34.

[0076] Figure 4 depicts the chromatograms (A,B,C) for the Anion exchange chromatography of three lab-scale consistency batches for Process-I of polyclonal antibody or antibody fragments F(ab)2 operated in flow-through mode, and chromatograms (D,E,F) represent the Cation exchange chromatography for Process-I operated in bind-and-elute mode with step-gradient elution.

[0077] Figure 5 depicts the chromatograms (A,C,E) for the Anion exchange chromatography profiles of pilot-scale consistency batches for Process-I of polyclonal antibody or antibody fragments F(ab)2 operated in flow-through mode, and chromatograms (B,D,F) represent the Cation exchange chromatography profiles for Process-I operated in bind-and-elute mode with stepgradient elution.

[0078] Figure 6 depicts the chromatograms (A,B,C) for the Cation exchange chromatography of three lab-scale consistency batches for Process-II operated in bind-and-elute mode with step-gradient elution.

[0079] Figure 7 depicts the chromatograms (A) and (B) of the Anion exchange and Cation exchange chromatography, respectively for the purification of anti-scorpion polyclonal antibody fragments using process-I, wherein the Cation exchange chromatography was performed using linear gradient elution.

[0080] Figure 8 depicts the chromatograms (A) and (B) of the Anion exchange and Cation exchange chromatography, respectively, for the purification of anti-diphtheria polyclonal antibody fragments using process-I, wherein the Cation exchange chromatography was performed using linear gradient elution.

[0081] Figure 9 depicts the analytical Protein A chromatogram used for quantitative estimation of polyclonal antibody concentration in plasma.

[0082] Figure 10 depicts the analytical RP-HPLC chromatogram (A, B) used for quantitative estimation of polyclonal antibody fragments F(ab)2 in digested plasma and also depicts thePT / 2025 / 17145

[0083] analytical RP-HPLC chromatogram (C, D) used for quantitative estimation of animal albumin impurities in plasma and purified drug substance.

[0084] Figure 11 depicts the analytical SEC-HPLC chromatogram used for quantitative estimation of high and low molecular mass species (HMMS and LMMS) associated with polyclonal antibody or antibody fragment F(ab)2 therapeutics.

[0085] Figure 12 depicts the mass spectra for intact mass analysis of plasma and purified drug substance obtained using MALDI-TOF mass spectrometer.

[0086] Figure 13 indicates the SDS-PAGE analysis of in process samples of chromatographic assisted purification (A): - Lane 1 : Plasma, Lane 2: Plasma 1 : 2 diluted, Lane 3 : Plasma pH 3.0 adjusted, Lane 4: UFDF-I Load, Lane 5: UFDF-I retentate, Lane 6: Protein marker, Lane 7: Anion exchange chromatography flowthrough, Lane 8: Anion exchange chromatography wash, Lane 9: Anion exchange chromatography elute, Lane 10: Cation exchange chromatography flow-through. (B): - Lane 1: Cation exchange chromatography elution 1, Lane 2: Cation exchange chromatography elution 2, Lane 3: UFDF-II elution 1 retentate, Lane 4: UFDF-II elution 2 retentate, Lane 5 : Ladder.

[0087] DETAILED DESCRIPTION OF THE INVENTION

[0088] As used herein, the term volumetric concentration factor, “VCF value,” when used in the context of the present invention, refers to the volumetric concentration factor as the amount that the feed stream has been reduced in volume from the initial volume. Accordingly, to accomplish the objectives of the present invention, the inventors propose a process of purifying a protein of interest using chromatographic process from hyper-immunized animal plasma. The term "animal" used herein refers to warm-blooded vertebrate animals of the class 'Mammalia', including humans, characterized by a covering of hair on the skin and, in the female, milkproducing mammary glands for nourishing the young, the term mammal includes animals such as cat, dog, rabbit, bear, fox, wolf, monkey, deer, mouse, pig, horse, camel, human, and the like.

[0089] In an embodiment of the present invention, a process for the purification of a protein of interest. The process can comprise steps of providing a sample containing protein of interest; digestingPT / 2025 / 17145

[0090] the proteins in the sample followed by quenching; ultrafiltration of the sample followed by diafiltration; subjecting the sample to chromatographic purification followed by ultrafiltration and diafiltration.

[0091] In an embodiment, of the present invention, the process for purifying a protein may comprise the steps of:

[0092] a) providing a sample containing the protein of interest; wherein the sample is blood, plasma or serum;

[0093] b) diluting the sample, adjusting its pH, digesting the proteins in the sample preferably by enzymatic digestion with pepsin, followed by quenching;

[0094] c) ultrafiltration of the sample obtained in step (b) followed by diafiltration;

[0095] d) optionally subjecting the sample obtained from step (c) to anion exchange chromatography;

[0096] e) subjecting the sample obtained from step (c) or the flowthrough obtained from step (d) to cation exchange chromatography;

[0097] f) subjecting the eluate from step (e) to ultrafiltration followed by diafiltration to obtain the purified protein.

[0098] In an embodiment of the present invention, the process may comprise the steps of:

[0099] a) providing a sample containing the protein of interest; wherein the sample is blood, plasma or serum;

[0100] b) diluting the sample, adjusting its pH, enzymatic digestion of the sample with pepsin, followed by quenching;

[0101] c) ultrafiltration of the digested sample obtained in step (b) followed by diafiltration;PT / 2025 / 17145

[0102] d) subjecting the sample obtained from step (c) to anion exchange chromatography;

[0103] e) subjecting the flowthrough from step (d) to a cation exchange chromatography to obtain the protein of interest;

[0104] f) subjecting the eluate of step (e) to ultrafiltration followed by diafiltration to obtain the purified protein.

[0105] In an embodiment of the present invention, the process may comprise the steps of:

[0106] a) providing a sample containing the protein of interest; wherein the sample is blood, plasma or serum;

[0107] b) diluting the sample, adjusting its pH, enzymatic digestion of the sample with pepsin, followed by quenching;

[0108] c) ultrafiltration of the digested sample obtained in step (b) followed by diafiltration;

[0109] d) subjecting the sample from step (c) to a cation exchange chromatography to obtain the protein of interest;

[0110] e) subjecting the eluate of step (d) to ultrafiltration followed by diafiltration to obtain the purified protein.

[0111] In an embodiment of the present invention, the protein of interest in the sample may be a polyclonal antibody and / or antibody fragment.

[0112] In an embodiment of the present invention, the sample may be selected from blood, plasma or serum. Preferably, the sample can be plasma.

[0113] For illustration purpose, the above process may be termed Process II. The ultrafiltration-diafiltration of step (c) may be termed as UFDF-I. The ultrafiltration-diafiltration of step (e) may be termed as UFDF-II.PT / 2025 / 17145

[0114] In an embodiment, the sample may be collected by bleeding of a hyper-immunized animal. The animal may be selected from horses, donkeys, mules, ponies, sheep, mice, guinea pigs, goats, and camels.

[0115] In yet another embodiment of the present invention, the process of purifying polyclonal antibodies includes collection of hyper-immunized animal blood in a sterile container containing anticoagulant solution / salt to avoid coagulation and followed by for separation of blood cells and plasma containing polyclonal antibody using centrifugation, dilution of hyperimmunized plasma, pH adjustment and enzymatic digestion and quenching, concentration and diafiltration, subsequently followed by adsorptive separation using anion and cation exchange chromatography finally ultrafiltration and diafiltration of purified antibody or antibody fragment drug substance in formulation buffer.

[0116] In yet another embodiment of the present invention, the antibody or antibody fragment may be an antibody or antibody fragment against snake venom, scorpion venom, diphtheria toxin, tetanus toxin, rabies virus, or Clostridium toxin and belongs to one or more subclasses, including but not limited to IgG, IgM, IgE, IgA, and IgD.

[0117] In an embodiment of the present invention, the plasma may be separated from the hyperimmunized blood by centrifugation at an RPM of 5000-11000 for 4 to 40 min.

[0118] In an embodiment of the present invention, the turbidity of the centrifuged plasma may be not more than 20.0 NTU.

[0119] In an embodiment of the present invention, the separation of blood cells and plasma containing polyclonal antibody may be effected by either by decanting or centrifugation to remove blood cells and remove process-related impurities, specifically originating from host cell breakage (insoluble cell components) during blood collection.

[0120] In an embodiment of the present invention, the plasma may be diluted using water in step (b) in a ratio of 1 : 1 to 1 :2 to reduce viscosity.

[0121] In an embodiment of the present invention, digestion may be carried out with one or more enzymes, preferably pepsin, at pH 2.0 to 4.0, preferably pH 3.0 ± 0.2 at 30-40°C for 2-4 hrs,PT / 2025 / 17145

[0122] preferably at 37 ± 1°C for 3 hrs. In some embodiments, the pH may be adjusted using HC1, acetic acid and citric acid. In some embodiments, the pH is adjusted using 1-5 N HC1.

[0123] In an embodiment of the present invention, neutralization after enzymatic digestion may be effected at pH 5.0 ± 0.2 using 1-3 M Tris base.

[0124] In an embodiment of the present invention, the ultrafiltration and diafiltration of step (c) may be performed using 1 mM - 100 mM of sodium acetate buffer having a pH of 4.0 to 6.0.

[0125] In another embodiment of the present invention, the ultrafiltration membrane may have a molecular weight cut-off (MWCO) of 30 kDa - 50 kDa and ultrafiltration & diafiltration may be carried out in a tangential flow direction, arranged in series or in parallel with a suitable pressure, time, volume, or protein concentration-based flow and flow path controller.

[0126] In an embodiment of the present invention, the tangential flow ultrafiltration may be performed at a pH range from 4.0 to 6.0 with conductivity of 0.001 to 20 mS / cm to an anion or cation exchange chromatography.

[0127] In an embodiment of the present invention, the volumetric concentration factor (VCF) achieved using tangential flow ultrafiltration units is not less than 1.1X, where X is the initial protein concentration of the feed.

[0128] In an embodiment of the present invention, the concentration of the polyclonal antibody and antibody fragments ultrafiltration and diafiltration step performed before chromatography is always higher than in hyper-immunized plasma.

[0129] In an embodiment of the present invention, the ultrafiltration and diafiltration-I increases the concentration of enzymatically digested polyclonal antibody or fragments and diafiltration into the chromatography equilibration buffer removes any product and process-related impurities such as turbidity, polyclonal antibody fragments or peptides, enzymes like pepsin, host cell proteins (HCP), and host cell nucleic acids (HC-DNA).

[0130] In yet another embodiment of the present invention, anion exchange chromatography may be performed in either bind-elute mode or flow-through mode, preferably in flow-through mode.PT / 2025 / 17145

[0131] The process and product-related impurities are captured in the resin while polyclonal antibody and / or polyclonal antibody fragments are obtained in flow-through.

[0132] In an embodiment of the present invention, the loading capacity for the product of interest is more than 100 mg / mL for anion exchange chromatography and performed at pH 4 - 6 with conductivity 0.001 to 20 mS / cm and the elution is performed with conductivity 0.001 to 200 mS / cm.

[0133] In yet another embodiment of the present invention, cation exchange chromatography may be performed in either bind-elute mode or flow-through mode, preferably in bind-elute mode. The protein of interest is bound to resin and various product-related impurities are obtained in flow-through.

[0134] In an embodiment of the present invention, the loading capacity for the product of interest is more than 30 mg / mL for cation exchange chromatography and performed at pH 4 - 6 with conductivity 0.001 to 20 mS / cm and the elution is performed with conductivity 0.001 to 200 mS / cm

[0135] In an embodiment of the present invention, cation exchange chromatography is performed in bind-elute mode at a breakthrough capacity at greater than 0.1% of the product of interest.

[0136] In one embodiment, the present invention provides an equilibration buffer used for binding the process and product related impurities to the anion exchange chromatography resin comprising 1-100 mM sodium acetate, with pH in the range of 4.0 to 6.0.

[0137] In one embodiment, the present invention provides an elution buffer used for eluting the process and product related impurities from the anion exchange chromatography resin comprising 1-100 mM sodium acetate buffer, with 0.1-1 M sodium chloride, with pH in the range of 4.0 to 6.0.

[0138] In one embodiment, the present invention provides a binding buffer used for binding the protein of interest to the cation exchange chromatography resin comprising 1-100 mM sodium acetate buffer, with pH in the range of 4.0 to 6.0.PT / 2025 / 17145

[0139] In an embodiment of the present invention, the elution of process-related impurities in anion exchange chromatography may be performed using either pH or salt gradient.

[0140] In an embodiment of the present invention, the elution of product-related impurities in cation exchange chromatography may be performed using either pH or salt-based linear gradient or in-step gradient. In some embodiments, the elution may be performed using both either linear or step gradients from 0% to 100% of the elution buffer.

[0141] In another embodiment, the present invention provides an elution buffer used for eluting the protein of interest to the cation exchange chromatography resin into an elution pool comprising 1-100 mM sodium acetate buffer with 0.1-1 M sodium chloride, with pH in the range of 4.0 to 6.0.

[0142] In an embodiment of the present invention, the cation exchange chromatography for separation of various process and product-related impurities, specifically aggregated form of polyclonal antibody or fragment.

[0143] In yet another embodiment of the present invention, the anion exchange chromatography or cation exchange may be performed using the axial or radial flow chromatography column. In an embodiment of the present invention, the anion exchange chromatography may be performed using anion exchange functional groups selected from diethylaminoethyl, quaternary ammonium, polyethyleneimine and trimethylammoniumethyl, linked to bead based, membrane based, hydrogel based or fibre-based chromatography matrix.

[0144] In an embodiment of the present invention, the anion exchange chromatography may include a resin / membrane selected from a group consisting of DEAE Sepharose® Fast Flow, Q Sepharose® Fast Flow, SOURCE™ 15Q, SOURCE™ 30Q, Fractogel®EMD DEAE, POROS® 50 HQ, Nuvia™ Q, Capto™ ImpRes Q. Capto™ Q, Capto™ DEAE, Fractogel®EMD TMAE, Fractogel®EMD DMAE, Natrix® Q, Sartobind® Q, and Mustang® Q.

[0145] In another embodiment of the present invention, the total amount of polyclonal antibody and / or antibody fragments passed through the single anion exchange chromatography column may be not less than 30 g / L resin.PT / 2025 / 17145

[0146] In another embodiment of the present invention, the cation exchange chromatography may be performed using a cation exchange functional group selected from a group consisting of sulfonate group, sulfopropyl group and sulphonic acid linked to bead based, membrane based, hydrogel based and fibre-based chromatography matrix.

[0147] In another embodiment of the present invention, the cation exchange chromatography may include a resin / membrane selected from the group consisting of Fractogel® EMD SOs'fM), Capto™ SP ImpRes, POROS® XS, Nuvia™ S, SOURCE™ 15S, SOURCE™ 30S, SP Sepharose® Fast Flow, Natrix® HD-Sb, Sartobind® S, and Mustang® S.

[0148] In an embodiment of the present invention, the cation exchange chromatography column may have a dynamic binding capacity for the polyclonal antibody or its fragment is not less than 10 g / L resin / membrane.

[0149] In yet another embodiment of the present invention, the elution may be performed with a saltbased linear gradient and / or a stepwise gradient over 5 to 15 column volumes, which can play an important role in the removal of impurity and optimization of purification process.

[0150] In yet another embodiment of the present invention, the residence time for anion and / or cation exchange chromatography is more than 5 seconds but less than 30 minutes, preferably, 4 - 8 minutes, and more preferably, 6 minutes.

[0151] In yet another embodiment of the present invention, the final / end ultrafiltration of UFDF-II equilibration and concentration may be carried out at pH 4 - 6 and the diafiltration of UFDF-II may be carried out at pH 6 - 8, preferably at pH 7.4.

[0152] In an embodiment the ultrafiltration of UFDF-II may be performed using 10-100 mM sodium acetate buffer and the diafiltration of UFDF-II may be performed using phosphate buffer saline pH 7.4 ±0.2 as formulation buffer.

[0153] In yet another embodiment of the present invention, the purity of polyclonal antibody may be greater than 90%, preferably 95%, and more preferably about 99% and the recovery of the purified polyclonal antibody and / or antibody fragments may be more than (>) 50%, preferablyPT / 2025 / 17145

[0154] 80% and more preferably about 90% and may be independent of the initial concentration of the polyclonal antibody in plasma.

[0155] In yet another embodiment of the present invention, the purified polyclonal antibody and / or antibody fragment may contain not more than 1% of animal albumin protein in purified drug substance and comprises less than 10% soluble aggregate.

[0156] In an embodiment of the present invention, the selected polyclonal antibody and / or polyclonal antibody fragment may be produced using different animals, including but not limited to donkeys, mules, ponies, ovine / sheep, equine / horse, mice, guinea pigs, goats, rabbit and camels.

[0157] In yet another embodiment of the present invention, the present invention provides a composition comprising the purified polyclonal antibody and / or antibody fragments obtained by the present purification process. The composition may be a pharmaceutical composition or diagnostic composition.

[0158] In still another embodiment, the present invention provides a kit comprising the purified polyclonal antibody and / or antibody fragments obtained by the process of the present invention.

[0159] EXAMPLES

[0160] The following examples are given by way of illustration and therefore should not be construed to limit the scope of the invention.

[0161] Materials and methods

[0162] Tri-Sodium Citrate dehydrate, Glacial Acetic Acid, Trifluoroacetic acid and Citric Acid Monohydrate from Thomas Baker, India; EDTA Disodium Salt Dihydrate, Ammonium Perusulphate (APS) and Acrylamide from SRL Chemicals, India; Dextrose, Sodium Acetate Anhydrous, Sodium Hydroxide pellets, Sodium Chloride, Potassium Chloride, Potassium Phosphate Monobasic, Sodium dihydrogen phosphate monohydrate, Eshmonu® CPS, Fractogel® EMD SO3-, di-Sodium hydrogen phosphate anhydrous, Pellicon® 3 Cassette, andPT / 2025 / 17145

[0163] Hydrochloric acid 37% from Merck Millipore, India; Pepsin and Tris, Ultra-Pure Grade (MP Biomedicals); Water LC-MS, BAKERBOND® XWP 500 PolyCSX- 35, and Acetonitrile LC-MS from Avantor Performance, India; Sodium Dodecyl sulphate, Bromophenol Blue sodium salt, & N,N,N',N'- Tetramethyl ethyl enediamne from Sigma-Aldrich; Glycine, Bis-Acrylamide & Glycerol from Himedia, India; Poros™ XS from Thermo Scientific™, India; UniSP-50XS from Suzhou Nanomicro technology Ltd. India; and DEAE sepharose FF from Cytiva, India.

[0164] Example 1: Pretreatment of animal blood for plasma separation

[0165] This example illustrates the use of various anticoagulants at variable concentrations on blood clotting. Blood from hyper-immunized equine / horse was collected in autoclaved glass bottles containing various anticoagulants. The collection was done in Pune, Maharashtra, India

[0166] The samples were incubated at room temperature for 60 minutes. Five anticoagulants were selected for the study: tri-sodium citrate, Anticoagulant Citrate Dextrose Solution A (ACD-A), Anticoagulant Citrate Dextrose Solution B (ACD-B), Ethylenediaminetetraacetic acid (EDTA salt), and EDTA (liquid solution). In this example, effect of anti-coagulants at variable concentration was performed on two different experimental scales of 40 and 200 mL of blood. Experimental observations and results are shown in table indicating the amount of plasma volume recovered and total proteins in plasma (optical density values at 280 nm are normalized with respect to volume of the solution). Except in case of 1 % Tri-Sodium Citrate in all other experiments no blood clotting was observed for 60 minutes allowing efficient separation of plasma. Out of the five different anticoagulants, EDTA Disodium salt dihydrate powder and trisodium citrate provided the highest recovery for plasma proteins containing polyclonal antibody. Post-collection of blood and separation of hyper-immunized plasma, containers were kept in a cold room and checked for coagulation if any. Tri-sodium citrate offers efficient anticoagulant effect due to lower cost and easy availability, similarly EDTA Disodium salt Dihydrate with 2mg / mL in powder form provides the highest plasma protein content based on OD at 280 nm in both experiments’ scales.

[0167] Table 1 lists the anticoagulants: their optimal concentrations and impact on protein recovery.

[0168] Table 1: - Shows the effect of various anticoagulants and their concentrations on blood clotting and plasma protein recovery at 40 mL scale of blood.PT / 2025 / 17145

[0169]

[0170] Example 2: CentrifugationPT / 2025 / 17145

[0171] Separation of hyper-immunized plasma comprising polyclonal antibodies derived from animal blood was performed using centrifugation. Centrifugation helps in removal of various process related impurities such as blood cells (RBCs and WBCs) and cell debris. Plasma containing polyclonal antibodies was separated using centrifugation at 5000 RPM - 11000 RPM for 10 to 20 minutes. Centrifugation at higher rotational speed results in blood cell breaking leading to change in color of separated plasma due to presence of released hemoglobin from RBCs. Conventionally the industries are using settling methods for plasma separation requiring excessive time and leading to turbid plasma (NTU=275-300). The centrifugation results in yield of 70% hyper-immunized plasma resulting in the requirement of lesser horse bleeding. Moreover, the NTU obtained for plasma through this process is less than 20 showing very clear plasma without any insoluble form of the impurities. (NTU - Nephelometric Turbidity Unit) Table 2 lists the NTU values obtained for plasma after centrifugation of animal blood.

[0172] Table 2: - Turbidity (NTU) measurement post centrifugation of animal blood

[0173]

[0174] Example 3: Optimization of enzymatic digestion of polyclonal antibody

[0175] Antibody fragment formation is a crucial step in the processing of anti-snake venom sera or other polyclonal antibodies used in treatment of various diseases. For pepsin digestion of antibody, plasma was diluted using water (1:2) to reduce viscosity. Optimization of pepsin digestion was performed at pH 3.0 ± 0.2 and pH 3.5 ± 0.2. For both the experiments, 50 mL of the centrifuged plasma was used and diluted using 100 mL of water. pH of the diluted plasma was reduced to 3.0 ± 0.2 and 3.5 ± 0.2 using 2 N HC1. Based on the OD obtained at 280 nm for the diluted and clarified plasma; 33 mg of pepsin was added to the solution for digestion. PepsinPT / 2025 / 17145

[0176] digestion was performed at 37 °C degrees for 3 hours and then the solution was neutralized to pH 5.0 ± 0.2 using Tris base. Kinetics of antibody digestion was monitored using SDS-PAGE analysis to ensure the complete digestion of antibody. 2.00 mL of sample from reaction mixture was aliquoted at various time intervals to understand the digestion status of the antibody. Figure 2 shows the SDS PAGE analysis of digested samples at various time points for pH 3.0 ± 0.2 and pH 3.5 ± 0.2 experiments. In case of enzymatic digestion performed at pH 3.0 ± 0.2 complete conversion of antib ody-to-antibody fragment was observed in 3 hours, whereas at pH 3.5 ± 0.2 partial digestion of antibody was observed even after 5 hours of digestion. In both the experiments complete digestion of horse serum albumin was also observed in 30 minutes time duration. Figure 2 indicates the enzyme digestion kinetics of polyclonal antibody to polyclonal antibody fragments F(ab)2 at different pH (A, B) at pH 3.0 and (C and D) at pH 3.5: (A): - Lane 1: plasma 1:2 diluted , Lane 2: Blank, Lane 3: Plasma pH 3.0 adjusted, Lane 4: Blank, Lane 5: Pepsin digestion ‘0’ hour, Lane 6: Blank, Lane 7: Pepsin digestion ‘0.5’ hour, Lane 8: Blank, Lane 9: Pepsin digestion ‘1’ hour, Lane 10: Ladder. (B): - Lane 1: Pepsin digestion ‘2’ hour, Lane 2: Blank, Lane 3: Pepsin digestion ‘3’ hour, Lane 4: Blank, Lane 5: Pepsin digestion ‘4’ hour, Lane 6: Blank, Lane 7: Pepsin digestion ‘5’ hour, Lane 8: Blank, Lane 9: Pepsin digestion ‘6’ hour, Lane 10: Ladder. (C): - Lane 1: plasma 1:2 diluted, Lane 2: Blank, Lane 3: Plasma pH 3.5 adjusted, Lane 4: Blank, Lane 5: Pepsin digestion ‘0’ hour, Lane 6: Blank, Lane 7: Pepsin digestion ‘0.5’ hour, Lane 8: Blank, Lane 9: Pepsin digestion ‘1’ hour, Lane 10: Ladder. (D): - Lane 1: Pepsin digestion ‘2’ hour, Lane 2: Blank, Lane 3: Pepsin digestion ‘3’ hour, Lane 4: Blank, Lane 5: Pepsin digestion ‘4’ hour, Lane 6: Blank, Lane 7: Pepsin digestion ‘5’ hour, Lane 8: Blank, Lane 9: Pepsin digestion ‘6’ hour, Lane 10: Ladder.

[0177] Example 4: Caprylic acid-based precipitation of polyclonal antibodies and / or polyclonal antibody fragments (Comparative Example)

[0178] To determine optimal conditions for caprylic acid precipitation of non-immunoglobulin proteins from hyperimmunized plasma containing polyclonal antibodies against snake venom, a Box-Behnken design was created in JMP® Pro 17.0.0. The design considered caprylic acid concentration (1-5%), pH (5-7), and stirring speed (200-600 rpm) as process parameters, each at three levels and three center point and 1 replicate of each experiment resulting in a total of 30 optimization experiments. The starting material of the experiment was generated in three parts. Hyperimmunized plasma containing polyclonal antibodies against snake venom was diluted 3-fold with water and the pH was adjusted to 3.0 ± 0.2 using 6N HC1. Pepsin was addedPT / 2025 / 17145

[0179] to the pH adjusted plasma in an enzyme to whole antibody ratio of 1 : 100. Pepsin digestion was carried out for 3 hours at 37 °C. Post 3 hours the pH of the pepsin digested plasma was neutralized using 2M Tris base. One part of the material was neutralized to pH 5.0 ± 0.2. The other part was neutralized to pH 7.0 ± 0.2 and the remaining part was neutralized to pH 6.0 ± 0.2. The caprylic acid-based precipitation of enzymatically digested plasma was carried out at different pH (5.0, 6.0, and 7.0) using caprylic acid concentrations ranging from 1% to 5%, and stirring speeds between 200 and 600 rpm, as mentioned above, on a scale of 150 mL of neutralized plasma. Following caprylic acid precipitation, the non-immunoglobulin precipitate was separated by centrifugation at 25 °C for 40 minutes at 7000 rpm, and the supernatant was collected. The F(ab)2 fragment rich supernatant obtained after caprylic acid precipitation from each experiment was analyzed using RP-HPLC for F(ab)2 fragment recovery and SEC-HPLC for purity analysis. SEC-HPLC was performed only for the 2 maximum recovery samples based on RP-HPLC at each pH. In this investigation, among 30 experiments the maximum recovery of F(ab)2 fragment of 65.33 % was observed in the experiment with parameters of caprylic acid concentration (3%), pH (5) and stirring speed (200 rpm). Purity analysis determined by SEC-HPLC showed 61.59 ± 3.02 % of monomeric species (F(ab)2 fragment), 0.35 ± 0.26 % of high molecular mass species (HMMS) and 38.07 ± 3.04 % of lower molecular mass species (LMMS). The resulting supernatant had purity not exceeding 65%. However, according to the WHO guidelines for the production, control, and regulation of snake antivenom immunoglobulins (Annex 5, TRS No. 1004, Section 17.1.8), the purity requirements for antivenom immunoglobulins are as follows: whole immunoglobulins and their fragments must achieve >90% purity, and albumin content should be <1% of the total protein.

[0180] Example 5: Ultrafiltration and diafiltration of polyclonal antibodies and antibody fragments

[0181] The pepsin-digested plasma containing polyclonal antibody and fragments (F(ab)2) was concentrated using 30 kDa ultrafiltration membrane in tangential flow direction. A starting volume of 1800 mL of pepsin-digested plasma was concentrated to 570 mL, achieving a >3X volumetric concentration factor (VCF). The desired volumetric concentration factor (VCF) was obtained by adjusting the feed flow rate and transmembrane pressure. The ultrafiltration and diafiltration operation were performed at a feed flux of 300 LMH, with a process throughput of 30 L / m2A 20 mM sodium acetate buffer containing 100 mM NaCl at pH 5.0 ± 0.2 was used as the equilibration buffer for the ultrafiltration step. Post concentration the retentate wasPT / 2025 / 17145

[0182] diafiltered using the buffer containing 20 mM sodium acetate at pH 5.0 ± 0.2. This was performed by passing 5-7 times the diafiltration volume of the buffer.

[0183] Table 3: - F(ab)2 based recovery (%) and purity profile of ultrafiltration and diafiltration out using HPLC

[0184]

[0185] Example 6: Anion exchange chromatography optimization for pepsin and albumin impurity clearance from plasma.

[0186] The dynamic binding capacity (DBC) for polyclonal antibody fragments was estimated at a 6-minute residence time using anion exchange chromatography with DEAE Sepharose® Fast Flow resin. The ultrafiltration and diafiltration -I retentate, containing impurities such as pepsin and digested albumin, was loaded onto the column at pH 5.0 ± 0.5 with conductivity of 1.5 ± 0.5mS / cm. These impurities bound to the resin, while the antibody fragments remained in the flow-through. A 20 mM sodium acetate buffer (pH 5.0 ± 0.2) was used for equilibration and washing, applied for 5column volumes (CV), while elution was performed with the same buffer containing 1.0 M NaCl. Elution was carried out over 5CV using both linear and step gradients. Resin cleaning was conducted using 0.5 M NaOH, followed by regeneration with the equilibration buffer. Post pepsin digestion intact horse serum albumin and pepsin were the critical process related impurities. To determine the binding capacity for pepsin (10 mg / mL) solution was prepared in equilibration buffer and loaded onto a 2 mL anion exchange column. Flow through fractions were collected, concentrated, and analyzed using SDS-PAGE to confirm the pepsin binding to anion exchange chromatography. Based on the determined binding capacities the experiment was performed to confirm the removal of pepsin and albumin impurities. The tangential flow ultrafiltration process output from example 5 was used as the feed material. Approximately, 90 ± 10 mg of antibody fragment was loaded onto the anion exchange chromatography resin DEAE Sepharose® Fast Flow (20 mL column volume). Flow-PT / 2025 / 17145

[0187] through from chromatography experiment was collected and concentrated. The product recovery and purity were estimated using RP-HPLC and SEC-HPLC.

[0188] Figure 3 indicates the pepsin binding capacity on anion exchange resin using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. (A): - Lane 1: Pepsin (5mg / mL), Lane 2: Anion exchange chromatography wash, Lane 3: Anion exchange chromatography elute, Lane 4: Ladder, Lane 5: Anion exchange chromatography flowthrough fraction 2, Lane 6: Anion exchange chromatography flowthrough fraction 4, Lane 7: Anion exchange chromatography flowthrough fraction 7, Lane 8: Anion exchange chromatography flowthrough fraction 10, Lane 9: Anion exchange chromatography flowthrough fraction 11, Lane 10: Anion exchange chromatography flowthrough fraction 12. (B): - Lane 1: Ladder, Lane 2: Anion exchange chromatography flowthrough fraction 14, Lane 3: Anion exchange chromatography flowthrough fraction 15, Lane 4: Anion exchange chromatography flowthrough fraction 18, Lane 5: Anion exchange chromatography flowthrough fraction 20, Lane 6: Anion exchange chromatography flowthrough fraction 23, Lane 7: Blank, Lane 8: Anion exchange chromatography flowthrough fraction 28, Lane 9: Anion exchange chromatography flowthrough fraction 31, Lane 10: Anion exchange chromatography flowthrough fraction 34.

[0189] Table 4: - F(ab)2 based recovery (%) and purity profile of anion exchange chromatography output using HPLC

[0190]

[0191] Example 7: Dynamic bind capacity estimation of polyclonal antibody fragments on various cation exchange resins

[0192] Dynamic binding capacities (DBC) for polyclonal antibody fragments were determined on five different cation exchange (CEX) resins at a 6-minute residence time. The flow-through solution containing immunoglobulin antibody fragments (F(ab)2) obtained from anion exchange chromatography was used as feed material for DBC estimation. The concentration of the antibody fragment in flow-through from anion exchange chromatography was determinedPT / 2025 / 17145

[0193] using RP-HPLC method. The preparative chromatography column was packed with the selected cation exchange resin (CEX) resins and were equilibrated with 5 column volumes (CV) of 20 mM sodium acetate buffer (pH 5.00 ± 0.20) and conductivity of 1.5 ± 0.5 mS / cm. The polyclonal antibody fragments were continuously loaded until saturation, followed by a wash step with 3 CV of the same equilibration buffer. Elution was performed using a buffer containing 20 mM sodium acetate with 1 M NaCl with conductivity 85 ± 5.0 mS / cm, applied over 5 CV using both either linear or step gradients. During DBC estimation, 1 mL fractions were collected, and breakthrough curves were generated using OD at 280 nm and RP-HPLC analysis. These curves were used to calculate the maximum binding capacities of the selected cation exchange resins. Among the five tested CEX resins, three showed higher dynamic binding capacities. The highest loading capacity was estimated to 59.69 g / L at 10% breakthrough in Poros XS resin.

[0194] Table 5: - Dynamic binding capacity for antibody and antibody fragment determined for various cation exchange resins

[0195]

[0196] Example 8: Cation exchange chromatography for separation of various product-related impurities

[0197] Based on the data obtained for dynamic binding capacity, cation exchange chromatography was performed at a loading capacity of 55 mg / mL at 10% breakthrough using Fractogel® EMD SOs'. resin. Approximately 500 mL of the flow-through polyclonal antibody fragment (F(ab)2) solution obtained from anion exchange chromatography was loaded onto a 40 mL Fractogel® EMD SO3‘ column. The column was equilibrated with 5 column volumes (CV) of 20 mMPT / 2025 / 17145

[0198] sodium acetate buffer (pH 5.0 ± 0.2) with conductivity of 1.5 ± 0.5mS / cm as the equilibration buffer. After loading, the column was washed with 3 CV of the same equilibration buffer, followed by elution with 15 CV of 20 mM sodium acetate containing 1 M NaCl (pH 5.0 ± 0.2) with conductivity 85 ± 5.0 mS / cm. Elution was performed using a linear salt gradient from 0% to 100% of the elution buffer. All process steps were conducted with a six-minute residence time. Samples were collected and analyzed using RP-HPLC for recovery calculation and SEC-HPLC for purity estimation.

[0199] Table 6: - F(ab)2 based recovery (%) and purity values obtained for the cation exchange chromatography experiment.

[0200]

[0201] Example 9: Dynamic bind capacity estimation of polyclonal antibody fragments on cation exchange resin for process II

[0202] Dynamic binding capacities (DBC) for polyclonal antibody fragments were determined on five different cation exchange (CEX) resins at a 6-minute residence time. The UFDF-1 retentate solution containing immunoglobulin antibody fragments (F(ab)2) was used as feed material for DBC estimation. The concentration of the antibody fragment in UFDF-1 retentate solution was determined using RP-HPLC method. The preparative chromatography column was packed with the selected cation exchange resin (CEX) resins and were equilibrated with 5 column volumes (CV) of 20 mM sodium acetate buffer (pH 5.0 ± 0.2) with conductivity of 1.5 ± 0.5mS / cm. The polyclonal antibody fragments were continuously loaded until saturation, followed by a wash step with 3 CV of the same equilibration buffer. Elution was performed using a buffer containing 20 mM sodium acetate with 1 M NaCl of conductivity 85 ± 5.0 mS / cm, applied over 5CV using step gradients. During DBC estimation, 1 mL fractions were collected, and breakthrough curves were generated using OD at 280 nm and RP-HPLC analysis. These curves were used to calculate the maximum binding capacities of the selected cation exchange resin.PT / 2025 / 17145

[0203] Table 7: - Determination of the 10% load capacity for cation exchange chromatography resins based on the breakthrough curve.

[0204] "

[0205]

[0206] Example 10: Cation exchange chromatography for separation of various product-related impurities

[0207] Based on the data obtained for dynamic binding capacity, cation exchange chromatography was performed at a loading capacity of 39 mg / mL at 10% breakthrough using Fractogel® EMD SO3". resin. Approximately 200 mL of the ultrafiltration and diafiltration-1 retentate solution was loaded onto a 40 mL Fractogel® EMD SO3" column. The column was equilibrated with 5 column volumes (CV) of 20 mM sodium acetate buffer (pH 5.0 ± 0.2) as the equilibration buffer. After loading, the column was washed with 3 CV of the same equilibration buffer, followed by elution with 15 CV of 20 mM sodium acetate containing 1 MNaCl (pH 5.0 ± 0.2). Elution was performed using step gradients from 0% to 100% of the elution buffer. All process steps were conducted with a six-minute residence time. Samples were collected and analyzed using RP-HPLC for recovery calculation and SEC-HPLC for purity estimation.

[0208] Table 8: - F(ab)2 based recovery (%) and purity profile of cation exchange chromatography output using HPLC and OD at 280 nm

[0209]

[0210] Example 11: Ultrafiltration and Diafiltration-II for concentration and diafiltration of polyclonal antibodies or immunoglobulin fragments in formulation buffer

[0211] The cation-exchange chromatography output containing highly pure polyclonal antibody fragments F(ab)? obtained from both process I (anion-exchange followed by cation-exchangePT / 2025 / 17145

[0212] chromatography) and process II (cation-exchange chromatography alone) was subsequently concentrated using a 30 kDa ultrafiltration membrane arranged in series. For process I, a starting volume of 1200 mL of cation-exchange output was reduced to 140 mL. A similar ultrafiltration operation was performed for process II, wherein the eluate generated from only the cation exchange chromatography, was subjected to identical concentration conditions using the 30 kDa membrane to obtain a comparable volumetric concentration factor VCF.

[0213] The desired concentration of the immunoglobulin fragment (2X, 5X or 10X) was achieved by adjusting the feed flow rate and transmembrane pressure. The ultrafiltration and diafiltration operation were performed at a feed flux of 300 LMH, with a process throughput of 30 L / m2A 20 mM sodium acetate buffer containing 150 mM NaCl at pH 5.0 ± 0.2 was used as the equilibration buffer for the ultrafiltration step. Post concentration the retentate was diafiltered using the buffer containing phosphate buffer saline (PBS) at pH 7.4 ± 0.2. This was performed by passing 5-7 times the diafiltration volume of the buffer.

[0214] Table 9: - F(ab)2 based recovery (%) and purity profile of ultrafiltration and diafiltration- II (UFDF-II) output for process I using HPLC

[0215]

[0216] Table 10: - F(ab)2 based recovery (%) and purity profile of ultrafiltration and diafiltration-II (UFDF-II) output for process II using HPLC

[0217]

[0218] Example 12: Lab-scale consistency batches for process-IPT / 2025 / 17145

[0219] Post optimization experiments of the ion exchange chromatography and ultrafiltration experiments, three replicate experiments were performed to test the reproducibility of the developed purification process. Experiments were performed using 20.0 mL packed column volume of DEAE Sepharose FF for anion exchange chromatography and 35 mL Fractogel® EMD SO3" (M) for cation exchange chromatography. Ultrafiltration experiments were performed using 0.11m230 KDa MWCO membrane. 1.0 L animal blood was collected in sterile glass bottle containing 2.0 g of EDTA disodium salt dihydrate as an anticoagulant. Post confirmation of non-coagulation, blood was centrifuged at 5000 RPM for 15 mins and plasma supernatant was separated and collected. Post centrifugation, 600 mL of plasma was obtained. Mice assay-based titer(potency) of plasma was obtained to be 0.2 mg / mL against cobra venom for all three replicate experiments. 600 mL plasma was diluted using 1200 mL water. pH of the diluted plasma was adjusted using 6N HC1 to 3.0 ± 0.2. Pepsin (1 : 100, enzyme: antibody ratio) was added to solution and digestion was initiated at 37 °C. Digestion reaction was stopped by increasing the pH of solution to 5.0 using Tris post 3 hr of enzymatic digestion. The solution was filtered and tangential flow-based ultrafiltration and diafiltration unit operation was performed. 1800 mL solution of digested plasma solution was concentrated to 600 mL (Plasma volume) and not less than 5 diafiltration volumes of the buffer exchange was performed. Retentate from the ultrafiltration step was subjected to anion exchange chromatography using DEAE Sepharose chromatography column. For each experiment six cycles of anion exchange chromatography experiments were performed with 100 mL feed material loading per cycle. Concentration of F(ab)2 content was estimated for flowthrough obtained for each batch and accordingly number of experiments for cation exchange chromatography were performed based on observed dynamic binding capacity values. Elution in cation exchange chromatography was performed using salt-based step elution. Peak 1 elute of each CEX experiment was pooled and final formulation buffer exchange was performed in phosphate buffer saline in ultra-diafiltration step II. Quantitative estimation of product recovery and purity analysis at each process step was performed using RP-HPLC and SEC-HPLC respectively. Summary of the obtained results are tabulated below in Table 11. Final drug substances of all three experiments were subjected to mice assay -based potency estimation. The three experiments yielded 101.7 ± 24.56 mL of purified drug substance with 0.65 ± 0.16 mg / mL of potency against cobra venom from 600 mL plasma. Similarly based on RP-HPLC analysis recoveries of F(ab)2 content at the end of overall process was calculated as 52.83 ± 9.65 %. Figure 4 depicts the chromatograms (A,B,C) for the anion exchange chromatography of three lab-scale consistency batches for Process-I of polyclonal antibody or antibody fragments F(ab)2PT / 2025 / 17145

[0220] operated in flow-through mode, and chromatograms (D,E,F) represent the cation exchange chromatography for Process I operated in bind-and-elute mode with step-gradient elution.

[0221] Table 11: - F(ab)2 based recovery (%) and purity profile for three replicate experiments performed to generate the drug substance of anti-snake venom using RP-HPLC and SEC-HPLC analysis.

[0222]

[0223] Example 13: Lab-scale consistency batches for process-II

[0224] Post optimization experiments of the ion exchange chromatography and ultrafiltration experiments, three replicate experiments were performed to test the reproducibility of the developed purification process. Experiments were performed using 35 mL Fractogel® EMD SO3" (M) for cation exchange chromatography. Ultrafiltration experiments were performed using 0.11 m230 KDa MWCO membrane. Three replicate experiments were performed at a scale of 200mL plasma obtained through centrifugation. Mice assay-based titer (potency) of plasma was obtained to be 0.2 mg / mL against cobra venom for all three replicate experiments.

[0225] 200 mL plasma was diluted using 400 mL water. pH of the diluted plasma was adjusted using 6N HC1 to 3.0 ± 0.2. Pepsin (1 : 100, enzyme: antibody ratio) was added to solution and digestion was initiated at 37°C. Digestion reaction was stopped by increasing the pH of solution to 5.0 using Tris post 3 hrs. of enzymatic digestion. The solution was filtered and tangential flow-PT / 2025 / 17145

[0226] based ultrafiltration and diafiltration unit operation was performed. 600 mL solution of digested plasma solution was concentrated to 200 mL (plasma volume) and not less than 5 diafiltration volumes of the buffer exchange was performed. A sample of retentate from the ultrafiltration step was subjected RP-HPLC based concentration estimation to define number of cycles of cation exchange chromatography to be performed based on observed dynamic binding capacity values. Elution in cation exchange chromatography was performed using salt-based step elution. Peak 1 elute of each CEX experiment was pooled and final formulation buffer exchange was performed in phosphate buffer saline in ultra-diafiltration step II. Quantitative estimation of product recovery and purity analysis at each process step was performed using RP-HPLC and SEC-HPLC respectively. Summary of the obtained results are tabulated below in Table 12. Final drug substances of all three experiments were subjected to mice assay -based potency estimation. The three experiments yielded 20.5 ± 0.5 mL of purified drug substance with 0.60 ± 0.06 mg / mL of potency against cobra venom from 200 mL plasma. Similarly based on RP-HPLC analysis recoveries of F(ab)2 content at the end of overall process was calculated as 51.36 ± 0.98 %. Figure 6 depicts the chromatograms (A, B, C) for the cation exchange chromatography of three lab-scale consistency batches for Process II operated in bind-and-elute mode with step-gradient elution.

[0227] Table 12: - F(ab)2 based recovery (%) and purity profile for three replicate experiments performed to generate the drug substance of anti-snake venom using RP-HPLC and SEC-HPLC analysis.

[0228]

[0229] PT / 2025 / 17145

[0230] Example 14: Pilot scale trials for process-I

[0231] Three pilot scale consistency batches for developed process-I were performed to test the reproducibility of the developed purification process at higher scale. Scale up was performed from 0.6 L of plasma as feed material at lab scale experiments to 5.0 L of plasma as feed material at pilot scale. Batches were performed using 0.82 L packed column volume of DEAE Sepharose FF for anion exchange chromatography and 1.57 L Fractogel® EMD SO3" (M) for cation exchange chromatography. Ultrafiltration experiments were performed using four 0.5 m230 KDa MWCO membrane arranged in series mode. 10.0 L animal blood was collected in sterile glass bottle containing sterile anticoagulant. Post confirmation of non-coagulation, blood was centrifuged at 5000 RPM for 15 mins and plasma supernatant was separated and collected. Post centrifugation, 6.32 ± 0.17 L of plasma was obtained from 10.0 L blood. From the obtained plasma, 5.0 L was taken out for batch processing. The titer (potency) of the plasma was estimated by injecting a complex of direct challenge between plasma and anti-venom into mice. The titer (potency) of the plasma was obtained to be 0.21 ± 0.08 mg / mL against cobra venom. 5.0 L plasma was diluted using 10.0 L water. pH of the diluted plasma was adjusted using 6.0 N HC1 to 3.0 ± 0.2. Pepsin (1 : 100, enzyme: antibody ratio) was added to solution and digestion was initiated at 37 °C. Digestion reaction was stopped by increasing the pH of solution to 5.0 using 2.0 M Tris base post 3.0 hrs. of enzymatic digestion. The solution was filtered and tangential flow-based ultrafiltration and diafiltration unit operation was performed.

[0232] 15.0 L solution of digested plasma solution was concentrated to 5.0 L (plasma volume) and not less than seven diafiltration volumes of the buffer exchange was performed. Filtered retentate from the ultrafiltration step was subjected to anion exchange chromatography using DEAE Sepharose chromatography column. For each batch, four cycles of anion exchange chromatography experiments. Based on the concentration of F(ab)2 content estimated for flowthrough obtained for each batch during lab-scale consistency batches, number of experiments for cation exchange chromatography were performed. Elution in cation exchange chromatography was performed using salt-based step elution. Peak 1 elute of each CEX experiment was pooled and final formulation buffer exchange was performed in phosphate buffer saline in ultra-diafiltration step II. Quantitative estimation of product recovery and purity analysis at each process step was performed using RP-HPLC and SEC-HPLC respectively. Summary of the obtained results are tabulated below in Table 13. Final drug substances of all three experiments were subjected to mice assay -based potency estimation. The three experiments yielded 403 ± 122.35 mL of purified drug substance with 1.65 ± 0.58 mg / mL ofPT / 2025 / 17145

[0233] potency against cobra venom from 5.0 L plasma. Similarly based on RP-HPLC analysis recoveries of F(ab)2 content at the end of overall process was calculated as 66.77 ± 13.89 %.

[0234] Figure 5: depicts the chromatograms (A,C,E) for the anion exchange chromatography profiles of pilot-scale consistency batches for Process-I of polyclonal antibody or antibody fragments F(ab)2 operated in flow-through mode, and chromatograms (B,D,F) represent the cation exchange chromatography profiles for Process-I operated in bind-and-elute mode with stepgradient elution.

[0235] Table 13: - F(ab)2 based recovery (%) and purity profile for three replicate experiments performed to generate the drug substance of anti-snake venom using RP-HPLC and SEC-HPLC analysis.

[0236]

[0237] Example 15: Anti-scorpion polyclonal antibody purification

[0238] To validate the performance of the developed process for other polyclonal antibody therapeutics, anti-scorpion venom plasma was obtained using similar process of that of anti-snake venom plasma processing. 100 mL of anti-scorpion venom equine plasma was diluted with 200 mL water and solution was adjusted to pH 3.0. Three hours of pepsin digestion wasPT / 2025 / 17145

[0239] performed at pH 3.0. Post neutralization, pH was increased to 5.0 and solution was filtered using 0.22 pm filter. Tangential flow based ultra and diafiltration was performed using 30 kDa ultrafiltration membrane. Ultrafiltration and diafiltration process output was subjected to 20mL DEAE Sepharose FF for anion exchange chromatography and 35 mL Fractogel® EMD SO3" (M) for cation exchange chromatography in flowthrough mode and bind and elute mode respectively and elution was performed in linear gradient with elution buffer. Cation exchange chromatography eluate was finally ultrafiltered and diafiltered in formulation buffer before subjecting to bioassay and other physicochemical testing. The process resulted in an overall recovery of 53.54 ± 9.65 % of polyclonal antibody therapeutic. Figure 7: depicts the anion exchange chromatogram (A) and cation exchange chromatogram (B) for purification of antiscorpion polyclonal antibody fragments.

[0240] Example 16: Anti- Diphtheria polyclonal antibody purification

[0241] To validate the performance of the developed process for other polyclonal antibody therapeutics, anti-diphtheria plasma was obtained using similar process of that of anti-snake venom plasma processing. 100 mL of anti -diphtheria equine plasma was diluted with 200 mL water and solution was adjusted to pH 3.0. Three hours of pepsin digestion was performed at pH 3.0. Post neutralization, pH was increased to 5.0 and solution was filtered using 0.22 pm filter. Tangential flow based ultra and diafiltration was performed using 30 kDa ultrafiltration membrane. Ultrafiltration and diafiltration process output was subjected to 20mL DEAE Sepharose FF for anion exchange chromatography and 35 mL Fractogel® EMD SO3" (M) for cation exchange chromatography in flowthrough mode and bind and eluate mode respectively and elution was performed in linear gradient with elution buffer. Cation exchange chromatography eluate was finally ultrafiltered and diafiltered in formulation buffer before subjecting to bioassay and other physicochemical testing. The process resulted in overall recovery of 51.37 ± 9.50 % of polyclonal antibody therapeutic.

[0242] Figure 8: depicts the anion exchange chromatogram (A) and cation exchange chromatogram (B) for purification of anti -diphtheria polyclonal antibody fragments.

[0243] Example 17: Analytical characterization of polyclonal antibody fragments (F(ab)2) using various analytical techniquesPT / 2025 / 17145

[0244] i. Protein A HPLC analysis for quantification of polyclonal antibody product Concentration of polyclonal antibody product in various chromatography outputs was determined using 2.1 mm x 30 mm of particle size 20 pm POROS™ A 20 column using Agilent 1200 HPLC system. The mobile phase consisted of IX phosphate buffered saline pH 7.2 ± 0.20 (mobile phase A) and 3 % glacial acetic acid pH 2.5 ± 0.20 (mobile phase B). Flow rate was maintained at 1.0 mL / min. Method involving a gradient of A to B for 5 minutes at a wavelength of 280 nm was used. Quantitative estimation of the polyclonal antibody product in various process outputs was determined using this analytical method. Representative chromatogram for this analytical method is further shown in Figure 9.

[0245] ii. RP-HPLC analysis to quantify the F(ab)2 fragments product.

[0246] The concentration of the F(ab)2 fragments product in various chromatography outputs was determined using 4.6 mm x 100 mm of particle size 5 pm Bio resolve™ RP mAb polyphenyl, 450A, 2.7 -micron column using Agilent 1200 HPLC system. The mobile phase consisted of 0.1% Trifluoroacetic acid - Milli Q (mobile phase A) and 0.1 % Trifluoroacetic acid -Acetonitrile (mobile phase B). Flow rate was maintained at 1.0 mL / min. Method involving a gradient of A to B for 10 minutes at a wavelength of 280 nm was used. Quantitative estimation of the F(ab)2 fragments product in various process outputs was determined using this analytical method. A representative chromatogram for this analytical method is further shown in Figure 10 (A- F(ab)2 reference standard, B- CEX Eluate I ).

[0247] iii. RP-HPLC analysis to quantify the animal albumin product.

[0248] The presence of animal albumin product in plasma and final output (UFDF-II output) was determined using 4.6 mm x 150 mm TSKgel Protein C4-300, 3-micron column on Agilent 1200 HPLC system. The mobile phase consisted of 0.1% Trifluoroacetic acid in 100% Milli Q (Mobile phase A) and 0.1 % Trifluoroacetic acid in 100% Acetonitrile (Mobile phase B). Flow rate was maintained at 1.0 mL / min. Method involving a gradient of A to B for 30 minutes at a wavelength of 280 nm was used. Quantitative estimation of the animal albumin product in process inputs and outputs was determined using this analytical method. A representative chromatogram for this analytical method is further shown in Figure 10 (C- overlay of horse plasma albumin and albumin, D- overlay of horse plasma albumin and anion exchange chromatography load).PT / 2025 / 17145

[0249] iv. Size exclusion chromatography HPLC analysis of polyclonal antibody fragments product

[0250] Aggregation and fragments in polyclonal antibody fragments F(ab)2 products were determined using a 7.8 mm x 300 mm TSKgel G2000SWXL column of particle size 5 pm on Agilent 1200 HPLC system. The mobile phase consisted of sodium phosphate buffer pH 7.2 ± 0.2 (Buffer A). The flow rate was maintained at 1.0 mL / min using an isocratic method at a wavelength of 280 nm. High molecular mass species (HMMS (%)), the Purity (Monomer), and low molecular mass species (LMMS (%)) of purified polyclonal antibody products were determined using this method. Representative chromatogram for this analytical method is further shown in Figure 11.

[0251] v. Intact mass analysis of purified polyclonal antibody and / or fragments using Matix- Assisted Laser Desorption Ionization - Time of Flight analysis

[0252] Intact mass analysis of purified polyclonal antibody and / or fragments was performed using Matix Assisted Laser Desorption ionization -Time of Flight analysis. Horse plasma and purified polyclonal antibody and / or fragments were mixed in a 1:1, 1:5 and 1:10 ratio with sinapinic acid to perform Matix Assisted Laser desorption ionization -Time of Flight analysis. Matrix sinapinic acid (10 mg / ml) was prepared in Acetonitrile: purified water: Trifluoroacetic acid (TFA) (50:50:0.1). 1 pL of a homogenized mixture of sample and matrix was spotted on a clean 384 well Matix Assisted Laser Desorption ionization -Time of Flight plate. The plate was inserted into AB SCIEX TOF / TOFTM 5800 instruments. The instrument was used in positive ion linear mode. The MALDLTOF range was lOkDa to 400 kDa, and laser intensity between 5000 to 6000 was used for the analysis of samples depicted in Figure 12 (A- whole immunoglobulin present in horse plasma and B- purified F(ab)2 fragments. Bovine Serum Albumin was used as a positive control. Result analysis was performed using Data Explorer software.

[0253] vi. SDS-PAGE analysis of polyclonal antibody and / or fragments after enzymatic digestion

[0254] SDS-PAGE analysis for identification of plasma protein comprising polyclonal antibody and albumin, fibrinogens, and others and after pepsin digestion was carried out using 12 %PT / 2025 / 17145

[0255] (Thickness 1.5 mm) of the resolving gel under reducing conditions (at the stacking gel constant voltage 100V and resolving gel constant voltage 100V conditions. Each sample was boiled for 10 min in the starting buffer before being loaded into the gel. 0.05% (w / v) Coomassie brilliant blue G-250 in 4.5: 1:4.5 (Water: Glacial Acetic acid: Methanol) was used to detect proteins after electrophoretic separation on polyacrylamide gels. Figure 13: indicates the SDS-PAGE analysis of in process samples of chromatographic assisted purification (A): - Lane 1: Plasma, Lane 2: Plasma 1: 2 diluted, Lane 3: Plasma pH 3.0 adjusted, Lane 4: UFDF-I Load, Lane 5: UFDF-I retentate, Lane 6: Protein marker, Lane 7: Anion exchange chromatography flowthrough, Lane 8: Anion exchange chromatography wash, Lane 9: Anion exchange chromatography elute, Lane 10: Cation exchange chromatography flow-through. (B): - Lane 1: Cation exchange chromatography elution 1, Lane 2: Cation exchange chromatography elution 2, Lane 3: UFDF-II elution 1 retentate, Lane 4: UFDF-II elution 2 retentate, Lane 5: Ladder.

[0256] vii. Potency estimation of drug substance by Reed and Muench method

[0257] The Reed and Muench method are a widely recognized statistical tool used to evaluate the potency of anti-snake venom (ASV) sera against the venom of the "big four" snakes: Indian cobra, common krait, Russell's viper, and saw-scaled viper. This method determines the median effective dose (ED50), which is the ASV dilution required to neutralize a fixed dose of venom, achieving 50% survival in animal models. The process begins with the determination of the venom's LD50 (the lethal dose that kills 50% of a test group). Once the LD50 is established, a fixed dose, typically 2-5x LD50, is prepared and mixed with serial dilutions of the ASV. These mixtures are incubated at 37°C to allow venom-ASV interaction before being administered to groups of mice, with at least five mice per group. A control group receiving venom without ASV is included to validate the results. After 24-48 hours, the survival data are recorded for each dilution, and the percentage survival is calculated. Using the Reed and Muench formula, the ED50 is identified from survival transitions near 50%, offering a reliable measure of ASV potency. The protocol ensures rigorous testing to assess the efficacy of ASV. It emphasizes precise preparation of venom and ASV dilutions, accurate dosing, and consistent monitoring of parameters like temperature and incubation time. The data derived from this method guide the optimization of ASV production and help establish quality standards for neutralizing snake venom toxins effectively. This methodology is critical for producing high-potency ASVPT / 2025 / 17145

[0258] formulations that can neutralize the lethal effects of the "big four" snake venoms, ensuring better clinical outcomes in snakebite treatment.

[0259] Table 13: - Potency-based titer estimation and comparative process recovery across both process-I and process-II

[0260]

[0261] ADVANTAGES OF THE INVENTION

[0262] • The developed process covers one step / two-step chromatography purification platform for obtaining higher yield and better purity of polyclonal antibody therapeutics used in the treatment of various diseases.

[0263] • The process of the invention provides greater than 90%, preferably 95%, and more preferably about 99% product purity and 1.61X more yield in comparison with the traditional salt precipitation process of polyclonal antibody purification.

[0264] • The purification process for anti-snake venom sera achieves an exceptional level of host cell protein (HCP) clearance, with horse serum albumin being reduced by over 99.0%.

[0265] • The purified anti-snake venom drug substance demonstrates aggregate content below 5.0%, a benchmark for high-quality biotherapeutics.

[0266] • Animal studies prove the higher potency of the purified drug substance as compared to precipitation-based products.

Claims

PT / 2025 / 17145WE CLAIM1. A process for purifying a protein comprising the steps of:(a) providing a sample containing a protein of interest, wherein the sample is blood, plasma or serum;(b) diluting the sample, adjusting pH, enzymatically digesting the proteins using pepsin, followed by quenching;(c) subjecting the digested sample to ultrafiltration followed by diafiltration;(d) optionally subjecting the sample obtained in step (c) to anion exchange chromatography;(e) subjecting the sample obtained from step (c) or the flow-through obtained from step (d) to cation exchange chromatography; and(f) subjecting the eluate from step (e) to ultrafiltration followed by diafiltration to obtain a purified protein,wherein the protein is a polyclonal antibody and / or antibody fragment.

2. The process as claimed in claim 1, wherein the process comprises the steps of:a) providing a sample containing the protein of interest; wherein the sample is blood, plasma or serum;b) diluting the sample, adjusting its pH, enzymatic digestion of the sample with pepsin, followed by quenching;c) ultrafiltration of the digested sample obtained in step (b) followed by diafiltration;d) subjecting the sample obtained from step (c) to anion exchange chromatography;e) subjecting the flowthrough from step (d) to a cation exchange chromatography to obtain the protein of interest;PT / 2025 / 17145f) subjecting the eluate of step (e) to ultrafiltration followed by diafiltration to obtain the purified protein;wherein the protein is preferably a polyclonal antibody and / or antibody fragment.

3. The process as claimed in claim 1, wherein the process comprises the steps of:a) providing a sample containing the protein of interest; wherein the sample is blood, plasma or serum;b) diluting the sample, adjusting its pH, enzymatic digestion of the sample with pepsin, followed by quenching;c) ultrafiltration of the digested sample obtained in step (b) followed by diafiltration;d) subjecting the sample from step (c) to a cation exchange chromatography to obtain the protein of interest;e) subjecting the eluate of step (d) to ultrafiltration followed by diafiltration to obtain the purified protein;wherein the protein is preferably a polyclonal antibody and / or antibody fragment.

4. The process as claimed in claim 1, wherein the sample is diluted with water in a ratio of 1 : 1 to 1:2, and the enzymatic digestion of the sample with pepsin is performed at pH 2.0 to 4.0, preferably pH 3.0 ± 0.2 at 30-40°C for 2-4 hrs, preferably at 37 ± 1°C for 3 hrs and neutralized at pH 5.0 ± 0.2.

5. The process as claimed in claim 1, wherein the ultrafiltration membrane has a molecular weight cut-off of 30-50 kDa and ultrafiltration and diafiltration are performed in tangential flow direction, arranged in series or in parallel with a suitable pressure, time, volume, or protein concentration-based flow and flow path controller.

6. The process as claimed in claim 1, wherein the anion exchange chromatography is performed in flow-through or bind-elute mode, preferably in flow-through mode to obtain protein of interest and to capture process and product related impurities; wherein the loading capacity forPT / 2025 / 17145the product of interest is more than 100 mg / mL; and performed at pH 4-6 with conductivity 0.001-20 mS / cm and elution is performed with conductivity 0.001 to 200 mS / cm, using an anion exchange functional group selected from diethylaminoethyl, quaternary ammonium polyethyleneimine or trimethylammoniumethyl, linked to bead based, membrane based, hydrogel based or fibre-based chromatography matrix.

7. The process as claimed in claim 1, wherein the cation exchange chromatography is performed in bind-elute or flow-through mode, preferably in bind-elute mode to bind protein of interest and separate impurities in flow-through; wherein the loading capacity for the product of interest is more than 30 mg / mL and performed at pH 4-6 and conductivity 0.001-20 mS / cm, and elution is performed with conductivity 0.001 to 200 mS / cm; and performed at a breakthrough capacity at greater than 0.1% of the product of interest, using a cation exchange functional group selected from sulfonate, sulfopropyl or sulphonic acid linked to bead based, membrane based, hydrogel based or fibre-based chromatography matrix.

8. The process as claimed in claim 1, wherein elution in the anion exchange and / or cation exchange chromatography is performed using a salt-based linear or stepwise gradient over 5-15 column volumes with a residence time of more than 5 seconds but less than 30 minutes, preferably 4 - 8 minutes, and more preferably, 6 minutes.

9. The process as claimed in claim 1, wherein the ultrafiltration and diafiltration of step (c) is carried out at pH 4 - 6; the ultrafiltration of step (f) is carried out at pH 4 - 6; and the diafiltration of step (f) is carried out at pH 6 - 8, preferably at pH 7.4.

10. The process as claimed in claim 1, wherein the purity of polyclonal antibody is greater than 90%, preferably 95%, and more preferably about 99%; recovery of the purified polyclonal antibody and / or antibody fragments is more than (>) 50%, preferably 80% and more preferably about 90 % and is independent of the initial concentration of the polyclonal antibody in the sample; the purified polyclonal antibody and / or antibody fragment contains not more than 1% of animal albumin protein in purified drug substance; and comprises less than 10% soluble aggregate.PT / 2025 / 1714511. The process as claimed in claim 1, wherein the antibody or antibody fragment is directed against snake venom, scorpion venom, diphtheria toxin, tetanus toxin, rabies virus or Clostridium toxin and belongs to IgG, IgM, IgA, IgE or IgD subclasses.

12. The process as claimed in claim 1, wherein the process is performed in a batch or in an integrated continuous operation.

13. A composition comprising the purified polyclonal antibody and / or antibody fragments obtained by the process of the present invention.

14. The composition as claimed in claim 18, wherein it is a pharmaceutical composition or diagnostic composition.

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