DESC:Field of the Invention
The present invention relates to the method of preparing antibody drug conjugate, herein, brentuximab vedotin.
Background of the Invention
Antibody Drug Conjugates (ADCs) are complex engineered therapeutics that contain monoclonal antibodies (mAbs) directed toward disease-associated antigens to which highly potent cytotoxic agents are attached typically via linkers. The ADCs provide highly specific targeting ability from use of mAbs and highly potent killing effect from use of cytotoxic agents, thus eliminating only targeted cells.
The process of making ADC, for example, brentuximab vedotin, consists of preparation of brentuximab antibody, partial reduction of brentuximab antibody with Dithiothreitol (DTT) or Tris (2-carboxyethyl) phosphine (TCEP), and conjugating the reduced antibody with the cytotoxic agent monomethyl auristatin E (MMAE) through linker maleimidocaproyl-L-valine-L-citrulline-p-aminobenzyl carbamate (mc-vc-PABC).[1] Purification is one of the key steps in preparing brentuximab antibody. Certain prior arts show that purification processes of brentuximab require analytic techniques to detect the presence of contaminants, signifying that the known purification processes for brentuximab are not efficient enough to control the presence of contaminants. Thus, a novel purification method for brentuximab is required which is robust and does not require further analysis. For reduction, TCEP is preferred more over DTT because excess DTT readily reacts with vcMMAE and would compete with antibody-cysteines for the drug, thus, the excess DTT needs to be removed before adding mc-vc-PABC-MMAE. However, phosphines of TCEP react poorly with maleimides, and thus, any remaining TCEP does not have to be removed before adding mc-vc-PABC-MMAE.[1] Certain specific buffers, for example, borate buffer, is used to stabilize TCEP during reduction step.[2] Then, borate buffer is exchanged with other buffers such as phosphate buffer at conjugation step.[1] Also, the brentuximab antibody is generally observed to be prepared in phosphate buffer as per prior art. Thus, on reduction of brentuximab with TCEP in borate buffer, the reduced brentuximab moves to borate buffer from phosphate buffer and again, after conjugation, moves to phosphate buffer from borate buffer as per the processes disclosed in the prior art. This increases the cost, decreases the scalability and affects the purity of the antibody drug conjugate brentuximab vedotin as the buffers are changed in every step of the reaction. It is also noted in prior art that quenching agents for example, cysteine, N-acetyl cysteine are used to stop the conjugation reaction. This affects the environment as excess reagents are being used to obtain the final product. Thus, a novel process for preparing brentuximab vedotin is required, that mitigates some of the challenges posed by the prior arts.
Several existing patents disclose methods related to the purification and conjugation of antibody-drug conjugates. WO2004010957 describes an antibody-drug conjugate purified by elution over a G25 desalting column using PBS at pH 7.4, with partial reduction and conjugation occurring at 2-8°C. US202103308032 introduces a micro-reactor method. Furthermore, US 7,837,980 discloses a fully reduced antibody using reoxidation reagents such as DTNB, followed by desalting, quenching, and conjugation at 0°C. WO2015077605 discloses detection of oxidizing activity in antibody preparations, particularly when the antibody is intended for conjugation to a drug. To achieve this, an assay was developed using partially reduced SGN-30 (cAC10 antibody), which is also the antibody component of brentuximab vedotin, as a substrate. The choice of SGN-30 was based on the fact that the cAC10 antibody has been consistently purified without contamination from the enzyme QSOX1 (quiescin sulfhydryl oxidase 1). In this patent i.e. WO2015077605, brentuximab vedotin is utilized not as the primary antibody but as a reference for the detection of oxidizing activity by the QSOX1 enzyme. This is because brentuximab vedotin does not contain QSOX1 contamination, making it a reliable reference substrate for the assay. Importantly, WO2015077605 does not outline a process for manufacturing brentuximab vedotin itself; rather, it uses this antibody as a reference for assessing the oxidizing activity of QSOX1 in other antibody preparations.

The present invention reduces the number of reducing steps while maintaining the same pH and buffer conditions. Additionally, the present invention operates within a conjugation temperature range of 25-30°C, without the use of quenching and desalting, providing an efficient and streamlined approach compared to the methods described in the prior art patents.

Summary of the invention
The present invention provides method of preparing brentuximab vedotin comprising (a) brentuximab antibody kept in suitable buffer at pH 5 to pH 8.5, (b) partial reduction of the brentuximab antibody with TCEP in same buffer and at about same pH as used in step (a), and (c) conjugation of the reduced brentuximab antibody with mc-vc-PABC-MMAE in same buffer and at about same pH as used in step (a). In another aspect, the present invention provides method of purifying brentuximab antibody, wherein the method comprises recombinant Protein A chromatography with washes at suitable pH and/or conductance, alone or in combination with other chromatography and non-chromatography techniques, wherein the order of some of the steps may be altered as are well known to a person skilled in the art.

Brief description of figures
Figure 1: illustrates the purity of the recombinant Protein A affinity purified brentuximab by analytical HP- size exclusion chromatography (HP-SEC). The figure shows that more than 97% purity of brentuximab is achieved after first column purification.
Figure 2: illustrates the purity of the anion exchange purified brentuximab by analytical high performance – size exclusion chromatography (HP-SEC). The figure shows that more than 99% purity of brentuximab is achieved after second column purification.
Figure 3: illustrates the purity of the cation exchange purified brentuximab by analytical high performance – size exclusion chromatography (HP-SEC). The figure shows that more than 99% purity of brentuximab is achieved after third column purification.
Figure 4: illustrates the purity of brentuximab vedotin by analytical high performance – size exclusion chromatography (HP-SEC). The figure shows that more than 97% purity of brentuximab vedotin is achieved after conjugation.
Figure 5: illustrates the purity of the recombinant Protein A affinity purified brentuximab by analytical HP- size exclusion chromatography (HP-SEC). The figure shows that more than 97% purity of brentuximab is achieved after first column purification.
Figure 6: illustrates the purity of the cation exchange purified brentuximab by analytical high performance – size exclusion chromatography (HP-SEC). The figure shows that more than 99% purity of brentuximab is achieved after second column purification.
Figure 7: illustrates the purity of the anion exchange purified brentuximab by analytical high performance – size exclusion chromatography (HP-SEC). The figure shows that more than 99% purity of brentuximab is achieved after third column purification.
Figure 8: illustrates the purity of nano-filtrate brentuximab by – size exclusion chromatography (HP-SEC). The figure shows that more than 97% purity.
Figure 9: illustrates the purity of Critical Intermediate (CI), of brentuximab by HP-SEC – size exclusion chromatography (HP-SEC). The figure shows that more than 99% purity.
Figure 10: illustrates the purity of brentuximab vedotin– by size exclusion chromatography (HP-SEC). The figure shows that more than 97% purity of brentuximab vedotin is achieved.
Figure 11: illustrates the Drug antibody ratio (DAR) of brentuximab vedotin– by High pressure liquid chromatography hydrophobic interaction chromatography (HPLC-HIC). The figure shows the average DAR of brentuximab vedotin was observed to be about 4.34.
Figure 12: illustrates the Drug antibody ratio (DAR) of brentuximab vedotin– by High pressure liquid chromatography hydrophobic interaction chromatography (HPLC-HIC). The figure shows the average DAR of brentuximab vedotin was observed to be about 3.82.
Figure 13: illustrates the Drug antibody ratio (DAR) of brentuximab vedotin– by High pressure liquid chromatography hydrophobic interaction chromatography (HPLC-HIC). The figure shows the average DAR of brentuximab vedotin was observed to be about 3.68.

Abbreviations
ACN – Acetonitrile
ADC – antibody drug conjugate
AEX chromatography – Anion exchange chromatography
CEX chromatography – Cation exchange chromatography
Cond. – conductivity
Da - dalton
DAR – drug-antibody ratio
DEAE – Diethylamino ethyl
DMAC – Dimethylacetamide
DMAE – Dimethylamino ethyl
DMSO – Dimethyl sulfoxide
DTPA – Diethylenetriamine pentaacetate
DTT – Dithiothreitol
DV – dia-volumes
HP-HIC – High Performance – Hydrophobic Interaction Chromatography
HP-SEC – High performance – size exclusion chromatography
kDa – kilo-dalton
L/m2/hr - litres per square meter per hour
m2 – square meter
mAb – monoclonal antibody
mc-vc-PABC – maleimidocaproyl-L-valine-L-citrulline-p-aminobenzyl carbamate
mg/ml – milligram per millilitre
min. - minutes
mM – millimolar
MMAE – monomethyl auristatin E
mS / cm – milliSiemens per meter
MWCO – Molecular weight cutoff
NaCl – Sodium chloride
pH – potential of Hydrogen
QAE – quaternary aminoethyl
rPA chromatography – recombinant Protein A chromatography
rpm – revolutions per minute
SP – Sulphopropyl
TCEP – Tris (2-carboxyethyl) phosphine
TMAE – trimethylammonium ethyl
TMP – transmembrane pressure
Tris-HCl – Tris(hydroxymethyl)aminomethane hydrochloride
UF/DF – ultrafiltration-diafiltration
w / v – weight by volume
°C – degree celsius
µm – micrometer

Embodiments of the invention
In one embodiment, the present invention provides a method of preparing antibody drug conjugate, preferably, brentuximab vedotin, wherein the method comprises (a) brentuximab antibody kept in suitable buffer at about pH 5 to pH 8.5, wherein the suitable buffer is selected from phosphate buffer, citrate buffer, succinate buffer, borate buffer or citrate-phosphate buffer or suitable combinations thereof; (b) partial reduction of the brentuximab antibody obtained from step (a) with suitable reducing agent in same buffer and at about same pH as used in step (a); (c) conjugation of the reduced brentuximab antibody obtained from step (b) with the drug conjugate mc-vc-PABC-MMAE kept dissolved in a suitable organic solvent in same buffer and at about same pH as used in step (a).
In one of the embodiments, the present invention provides a method of preparing antibody drug conjugate, preferably, brentuximab vedotin, wherein the method comprises (a) brentuximab antibody kept in a suitable buffer at about pH 5 to pH 8.5, wherein the suitable buffer is selected from phosphate buffer, citrate buffer, succinate buffer, borate buffer or citrate-phosphate buffer; (b) partial reduction of the brentuximab antibody obtained from step (a) with reducing agent selected from, but not limited to, TCEP, DTT or Mercaptoethanol in same buffer and at about same pH as used in step (a); (c) conjugation of the reduced brentuximab antibody obtained from step (b) with mc-vc-PABC-MMAE drug conjugate kept dissolved in organic solvent in same buffer and at about same pH as used in step (a), wherein organic solvent can be, but not limited to, Dimethyl sulfoxide (DMSO), Dimethylacetamide (DMAC), or Acetonitrile (ACN).
In one of the embodiments, the present invention provides a method of preparing antibody drug conjugate, preferably, brentuximab vedotin, wherein the method comprises (a) brentuximab antibody kept in a suitable buffer at about pH 5 to pH 8.5, wherein the suitable buffer is selected from phosphate buffer, citrate buffer, succinate buffer, borate buffer or citrate-phosphate buffer; (b) partial reduction of the brentuximab antibody obtained from step (a) with reducing agent selected from, but not limited to, TCEP, DTT or Mercaptoethanol in same buffer and at about same pH as used in step (a); (c) conjugation of the reduced brentuximab antibody obtained from step (b) with mc-vc-PABC-MMAE drug conjugate kept dissolved in 5% to 30% organic solvent in same buffer and at about same pH as used in step (a) and temperature about 0 °C to 8 °C, wherein organic solvent can be, but not limited to, Dimethyl sulfoxide (DMSO), Dimethylacetamide (DMAC), or Acetonitrile (ACN).
In one of the embodiments, the present invention provides a method of preparing antibody drug conjugate, preferably, brentuximab vedotin, wherein the method comprises (a) brentuximab antibody kept in a suitable buffer at about pH 5 to pH 8.5, wherein the suitable buffer is selected from phosphate buffer, citrate buffer, succinate buffer, borate buffer or citrate-phosphate buffer; (b) partial reduction of the brentuximab antibody obtained from step (a) with 2.0 to 3.0 molar excess of TCEP in same buffer and at about same pH as used in step (a), (c) conjugation of the reduced brentuximab antibody obtained from step (b) with 5 to 10 molar excess of mc-vc-PABC-MMAE drug conjugate kept dissolved in 5% to 30% DMSO in same buffer and at about same pH as used in step (a) and temperature about 0 °C to 8 °C.
In one of the embodiments, brentuximab conjugated to the drug-conjugate mc-vc-PABC-MMAE, according to the present invention, is Diafiltered using UF/DF to atleast about 20 to 30 dia-volumes to obtain the concentrated antibody drug conjugate brentuximab vedotin.
In another embodiment, the present invention provides a method of purifying brentuximab antibody, wherein the method of purifying crude brentuximab comprises recombinant Protein A chromatography with first wash at suitable pH and/or conductivity, second wash at the same pH as of the first wash buffer and/or a conductivity higher than the first wash buffer and third wash at a pH and/or a conductivity lower than the second wash buffer, and elution of an antibody at lower pH and/or higher conductivity than third wash buffer, alone or in combination with other chromatography and non-chromatography techniques, wherein the order of some of the steps may be altered as are well known to a person skilled in the art.
In one of the embodiments, the present invention provides a method of purifying brentuximab antibody, wherein the method of purifying crude brentuximab comprises recombinant Protein A chromatography with first wash at suitable pH and/or conductivity, second wash at the same pH as of the first wash buffer and/or a conductivity higher than the first wash buffer and third wash at a pH and/or a conductivity lower than the second wash buffer, and elution of an antibody at lower pH and/or higher conductivity than third wash buffer, alone or in combination with other chromatography techniques such as, but not limited to, anion exchange chromatography, cation exchange chromatography and non-chromatography techniques such as, but not limited to, centrifugation, depth filtration, viral inactivation at low pH, pH neutralization, virus clearance by nano-filtration and terminal filtration, wherein the order of some of the steps may be altered as are well known to a person skilled in the art.
In one of the embodiments, the present invention provides a method of purifying brentuximab antibody, wherein the method comprises use of recombinant Protein A chromatography with first wash is at about pH 7.2 to pH 7.6 and/or conductivity at about 5 mS / cm to 30 mS / cm, second wash at about pH 7.2 to pH 7.6 and/or a conductivity at about 75 mS / cm to 100 mS / cm and third wash at a pH at 5 to 6.5 and/or a conductivity at about 1 mS/cm to 5 mS / cm; and elution of an antibody at about pH 3.3 to pH 3.7 and/or conductivity at about 5 mS / cm to 30 mS / cm, alone or in combination with other chromatography techniques such as, but not limited to, anion exchange chromatography, cation exchange chromatography and non-chromatography techniques such as, but not limited to, centrifugation, depth filtration, viral inactivation at low pH, pH neutralization, virus clearance by nano-filtration and terminal filtration, wherein the order of some of the steps may be altered as are well known to a person skilled in the art.
In one of the embodiments, the anion exchange chromatography is performed either in bind-elute mode or in flow-through-and-wash mode.
In one of the embodiments, the cation exchange chromatography is performed either in bind-elute mode or in flow-through-and-wash mode.
According to the present invention, TCEP and TCEP-HCl can be used interchangeably as TCEP is often prepared and used as a hydrochloride salt (TCEP-HCl)
According to the present invention, antibody-drug conjugate and drug conjugated antibody and conjugate can be used interchangeably. According to the present invention, drug conjugate and drug linker can be used interchangeably.
The present invention also includes a conjugate comprising an antibody chemically coupled to a cytotoxic drug prepared according to the processes described herein.

Definitions
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature used herein and the laboratory procedures in cell culture, chemistry, microbiology, molecular biology, cell science and cell culture described below are well known and commonly employed in the art. Conventional methods are used for these procedures, such as those provided in the art and various general references. Where a term is provided in the singular, the plural of that term is contemplated; and where a term is provided in the plural, the singular of that term is contemplated. The nomenclature used herein and the laboratory procedures described below are those well-known and commonly employed in the art. As employed throughout the disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings:
The term "antibody" herein is used in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired biological activity (Miller et al (2003) Jour, of Immunology 170:4854-4861). Antibodies may be murine, human, humanized, chimeric, or derived from other species. An antibody is a protein generated by the immune system that is capable of recognizing and binding to a specific antigen. (Janeway, C, Travers, P., Walport, M., Shlomchik (2001) Immuno Biology, 5th Ed., Garland Publishing, New York). A target antigen generally has numerous binding sites, also called epitopes, recognized by CDRs on multiple antibodies. Each antibody that specifically binds to a different epitope has a different structure. Thus, one antigen may have more than one corresponding antibody. An antibody includes a full-length immunoglobulin molecule or an immunologically active portion of a full-length immunoglobulin molecule, i.e., a molecule that contains an antigen binding site that immunospecifically binds an antigen of a target of interest or part thereof, such targets including but not limited to, cancer cell or cells that produce autoimmune antibodies associated with an autoimmune disease. The immunoglobulin disclosed herein can be of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass of immunoglobulin molecule. The immunoglobulins can be derived from any species. In one aspect, however, the immunoglobulin is of human, murine, or rabbit origin. "Antibody fragments" comprise a portion of a full-length antibody, generally the antigen binding or variable region thereof. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; minibodies (Olafsen et al (2004) Protein Eng. Design & SeI. 17(4):315-323), fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, CDR (complementary determining region), and epitope-binding fragments of any of the above which immunospecifically bind to cancer cell antigens, viral antigens or microbial antigens, single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.
The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations which include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al (1975) Nature 256:495, or may be made by recombinant DNA methods (see for example: US 4816567; US 5807715). The monoclonal antibodies may also be isolated from phage antibody libraries using the techniques described in Clackson et al (1991) Nature, 352:624-628; Marks et al (1991) J. MoI. Biol, 222:581-597; for example. The monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (US 4816567; and Morrison et al (1984) Proc. Natl. Acad. Sci. USA, 81 :6851-6855). Chimeric antibodies of interest herein include "primatized" antibodies comprising variable domain antigen-binding sequences derived from a non-human primate {e.g., Old World Monkey, Ape etc) and human constant region sequences.
Interchain Cysteine Residue: As used herein, "interchain cysteine residue" or "interchain cysteine" refer to a cysteine residue of an antibody chain that can be involved in the formation of an interchain disulfide bond with a cysteine residue of another chain of the antibody. The interchain cysteine residues are located in the CL domain of the light chain, the CH1 domain of the heavy chain, and in the hinge region. The number of interchain cysteine residues in an antibody can vary. For example, human IgGl, IgG2, IgG3 and IgG4 isotypes have 4, 6, 13 and 4 interchain cysteine bonds, respectively.
Interchain Disulfide Bond. The term "interchain disulfide bond," in the context of an antibody, refers to a disulfide bond between two heavy chains, or a heavy and a light chain.
The term "isomer", in the context of an immunoconjugate, refers to an antibody having a particular pattern of sites of conjugation of an active moiety or moieties. An isomer of an antibody can be referred to by the nomenclature C#v#, where C# indicates the number of interchain cysteine residues available for conjugation and v# refers to a particular pattern or order of interchain cysteine residues. An isomer of an immunoconjugate can be referred to by the nomenclature C#v#Y, where C# and v# have the same meaning as stated above and Y refers to the average number of diagnostic, preventative or therapeutic agents attached per antibody molecule.
Partially-Loaded. The term "partially-loaded" refers to an antibody in which only some of the predetermined points of conjugation of a particular type and/or of a similar reactivity are conjugated to an active moiety, resulting in formation of a certain isomer or isomers of the immunoconjugate (C# > Y).
Diagnostic, Preventative or Therapeutic Agent. As used herein, a "diagnostic, preventative or therapeutic agent" is an active moiety such as a macromolecule, molecule or atom which is conjugated to an antibody to produce an immunoconjugate which is useful for diagnosis, prevention and/or for therapy. Examples of diagnostic, preventative or therapeutic agents include drugs, toxins, and detectable labels.
Antibody drug conjugate (ADC): As used herein, an " Antibody drug conjugate” or “ADC" is a molecule comprising an antibody conjugated directly or indirectly to at least one diagnostic, preventative and/or therapeutic agent, or a chelating agent that binds the diagnostic, preventative and/or therapeutic agent. An ADC retains the immunoreactivity of the antibody, e.g., the antibody has approximately the same, or only slightly reduced, ability to bind the antigen after conjugation as before conjugation.
Linker: The term “linker" refers to any chemical moiety that links a drug covalently to an antibody. In some instances, part of the linker is provided by the drug. Therefore, the final linker is assembled from two pieces, the cross-linking reagent introduced into the antibody and the side chain from the drug. Linkers may broadly be either a cleavable linker or a non-cleavable linker.
Cleavable linkers are linkers that are sensitive to cleavage in the intracellular environment of a target cell but is not substantially sensitive to the extracellular environment, such that the drug is cleaved from the antibody when the ADC is internalized by the target cell (e.g., in the endosomal or, for example by virtue of pH sensitivity or protease sensitivity, in the lysosomal environment or in the caveolear environment). Typically, the ADC comprises a linker between the drug and the antibody. As noted supra, the linker may be cleavable under intracellular conditions, such that cleavage of the linker releases the drug from the antibody in the intracellular environment {e.g., within a lysosome or endosome or caveolea). Many known linkers fall in this category and are described below:
i) Disulfide containing linkers are linkers cleavable through disulfide exchange, which can occur under physiological conditions.
ii) Acid-labile linkers are linkers cleavable at acid pH. For example, certain intracellular compartments, such as endosomes and lysosomes, have an acidic pH (pH 4-5), and provide conditions suitable to cleave acid-labile linkers.
iii) Linkers that are photo-labile are useful at the body surface and in many body cavities that are accessible to light. Furthermore, infrared light can penetrate tissue. Some linkers can be cleaved by peptidases. Only certain peptides are readily cleaved inside or outside cells, see e.g. Trouet et al., 79 Proc. Natl. Acad. Sci. USA, 626-629 (1982) and Umemoto et al. 43 Int. J. Cancer, 677-684 (1989). Furthermore, peptides are composed of a- amino acids and peptidic bonds, which chemically are amide bonds between the carboxylate of one amino acid and the a-amino group of a second amino acid. Other amide bonds, such as the bond between a carboxylate and the e-amino group of lysine, are understood not to be peptidic bonds and are considered non-cleavable.
iv) Some linkers can be cleaved by esterases. Again, only certain esters can be cleaved by esterases present inside or outside cells. Esters are formed by the condensation of a carboxylic acid and an alcohol. Simple esters are esters produced with simple alcohols, such as aliphatic alcohols, and small cyclic and small aromatic alcohols.
The linker also can be a non-cleavable linker, such as an maleimido-alkylene- or maleimide-aryl linker that is directly attached to the drug (e.g., a drug) and released by degradation of the antibody. Thus, a non-cleavable linker is any chemical moiety that is capable of linking the drug to an antibody in a stable, covalent manner and does not fall under the categories listed above as cleavable linkers. And thus, the non-cleavable linkers are substantially resistant to acid-induced cleavage, light-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage.
Substantially resistant" to cleavage means that the chemical bond in the linker or adjoining the linker in at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 95%, and most preferably at least 99% of the antibody drug conjugate population remains non-cleavable by an acid, a photolabile-cleaving agent, a peptidase, an esterase, or a chemical or a physiological compound that cleaves the chemical bond (such as a disulfide bond) in a cleavable linker, for within a few hours to several days of treatment with any of the agents described above.
Furthermore, "non-cleavable" refers to the ability of the chemical bond in the linker or adjoining to the linker to withstand cleavage induced by an acid, a photolabile-cleaving agent, a peptidase, an esterase, or a chemical or a physiological compound that cleaves a disulfide bond, at conditions under which the drug or the cell binding agent does not lose its activity.
A person of ordinary skill in the art would readily distinguish non-cleavable from cleavable linkers.
The terms “patient” and “subject” are used interchangeably and are used in their conventional sense to refer to a living organism suffering from or prone to a condition that can be prevented or treated by administration of a composition of the present invention, and includes animals. The term “Animal” refers to a human or non-human animal, including, but not limited to, farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs; birds, including domestic, wild and game birds such as chickens, turkeys and other gallinaceous birds, ducks, geese and non-human primates, including, but not limited to, monkeys, chimpanzees and other apes and monkey species. The term does not denote a particular age. Thus, adult, juvenile and newborn individuals are of interest.
The term “formulated bulk” refers to preparations which are in such form as to permit the biological activity of the active ingredients to be unequivocally effective, and which contain no additional components which are significantly toxic to the subjects to which the formulation would be administered.
Detailed description of the present invention
The challenges posed by the prior arts included the use of additional analytical techniques to detect and control the presence of contaminants such as QSOX1 enzyme detection during the process of preparation, changing of buffers at each step of brentuximab vedotin preparation, and quenching through reagents after conjugation. Present inventors have surprisingly found a streamlined method of preparing brentuximab vedotin, wherein the purification is carried out without any such further analysis, reduction with TCEP is performed in the same buffer and pH as used in other reaction steps of preparing brentuximab vedotin and quenching with reagents is not required to stop the conjugation reaction. Therefore, the process according to the present invention is robust, cost-effective, scalable and eco-friendly giving final product with improved purity. The purity of brentuximab vedotin of marketed product – Adcetris, is 95.8%,[3] whereas the purity of brentuximab vedotin, obtained through the process of present invention is more than 97%. Thus, the method of preparing brentuximab vedotin according to the present invention is efficient and safe for production of the drug conjugated antibody brentuximab vedotin.
In one aspect, the present invention provides a method of preparing antibody drug conjugate, preferably, brentuximab vedotin, wherein the method comprises preparation of the brentuximab antibody, partial reduction of the brentuximab antibody and conjugation of the reduced brentuximab antibody with the drug conjugate mc-vc-PABC-MMAE. The crude antibody, i.e., crude brentuximab can be prepared by processes known in the art. In one of the aspects, the present invention provides a method of preparing brentuximab vedotin wherein brentuximab is optionally purified and subsequently reduced and conjugated with mc-vc-PABC-MMAE according to the reduction and conjugation process of the present invention. In one aspect, the present invention provides a method of preparing antibody drug conjugate, preferably, brentuximab vedotin, wherein the method comprises purification of the brentuximab antibody, partial reduction of the brentuximab antibody and conjugation of the reduced brentuximab antibody with the drug conjugate mc-vc-PABC-MMAE.
In one aspect, the present invention provides a method of preparing antibody drug conjugate, preferably, brentuximab vedotin, wherein the method comprises (a) brentuximab antibody kept in a suitable buffer at about pH 5 to pH 8.5, wherein the suitable buffer is selected from phosphate buffer, citrate buffer, succinate buffer, borate buffer or citrate-phosphate buffer; (b) partial reduction of the brentuximab antibody obtained from step (a) with 2.0 to 3.0 molar excess of TCEP in same buffer and at about same pH as used in step (a), (c) conjugation of the reduced brentuximab antibody obtained from step (b) with 5 to 10 molar excess of mc-vc-PABC-MMAE drug conjugate kept dissolved in 5% to 30% DMSO in same buffer and at about same pH as used in step (a) and temperature about 0 °C to 8 °C.
The brentuximab antibody kept in a suitable buffer at about pH 5 to pH 8.5, according to the present invention, comprises suitable buffer selected from phosphate buffer, citrate buffer, succinate buffer, borate buffer or citrate-phosphate buffer. In one of the aspects. In one of the aspects, the suitable buffer is phosphate buffer. In one of the aspects, the suitable buffer is phosphate buffer. In one of the aspects, the suitable buffer is citrate buffer. In one of the aspects, the suitable buffer is succinate buffer. In one of the aspects, the suitable buffer is borate buffer. In one of the aspects, the suitable buffer is citrate-phosphate buffer. The pH range of about pH 5 to pH 8.5, according to the present invention, includes each integer and non-integer present between them. For example, the range of about pH 5 to pH 8.5 includes, but not limited to, pH 5.0, pH 5.1, pH 5.2, pH 5.3, pH 5.4, pH 5.5, pH 5.6, pH 5.7, pH 5.8, pH 5.9, pH 6.0, pH 6.1, pH 6.2, pH 6.3, pH 6.4, pH 6.5, pH 6.6, pH 6.7, pH 6.8, pH 6.9, pH 7.0, pH 7.1, pH 7.2, pH 7.3, pH 7.4, pH 7.5, pH 7.6, pH 7.7, pH 7.8, pH 7.9, pH 8.0, pH 8.1, pH 8.2, pH 8.3, pH 8.4, pH 8.5, or any integer or non-integer between them.
The partial reduction and conjugation, according to the present invention, is performed in same buffer and at same pH as the suitable buffer and pH of the suitable buffer used in preparation of the brentuximab antibody, wherein the suitable buffer is selected from phosphate buffer, citrate buffer, succinate buffer, borate buffer or citrate-phosphate buffer.
The partial reduction of the brentuximab antibody, according to the present invention, comprises reduction of the brentuximab antibody with 2.0 to 3.0 molar excess of TCEP in the same buffer and at about same pH as used in step (a).
In one of the aspects, the range of about 2.0 to 3.0 molar excess of TCEP, according to the present invention, includes each integer and non-integer present between them. For example, the range of about 2.0 to 3.0 molar excess includes 2.0, 2.05, 2.1, 2.15, 2.2, 2.25, 2.3, 2.35, 2.4, 2.45, 2.5, 2.55, 2.6, 2.65, 2.7, 2.75, 2.8, 2.85, 2.9, 2.95, 3.0, or any integer or non-integer between them.
In one of the aspects, the conjugation step as in step (c) comprises conjugation of the partially reduced brentuximab antibody with mc-vc-PABC-MMAE in the same buffer and at about same pH as used in step (a) and at temperature conditions in the range of about 0 °C to 8 °C. The temperature range of about 0 °C to 8 °C, according to the present invention, includes each integer and non-integer present between them. For example, the temperature range of about 0 °C to 8 °C includes, but not limited to, 0 °C, 1 °C, 2 °C, 3 °C, 4 °C, 5 °C, 6 °C, 7 °C, or 8 °C.
In one of the aspects, conjugation of the partially reduced antibody is with 5 to 10 molar excess mc-vc-PABC-MMAE, wherein the range of 5 to 10 molar excess mc-vc-PABC-MMAE includes integer and non-integer between them. For example, the range of 5 to 10 molar excess includes 5.0, 5.3, 5.5, 5.8, 6.0, 6.3, 6.5, 6.8, 7.0, 7.3, 7.5, 7.8, 8.0, 8.3, 8.5, 8.8, 9.0, 9.3, 9.5, 9.8, 10.0, or any integer or non-integer between them.
In one of the aspects, the drug conjugate mc-vc-PABC-MMAE, according to the present invention, is kept dissolved in organic solvent during the conjugation, wherein, the organic solvent is in the range of 5% to 30% and is selected from, but not limited to, DMSO, DMAC, or ACN. The range of 5% to 30% organic solvent according to the present invention, includes integer and non-integer between them. For example, the range of 5% to 30% organic solvent includes 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30% or any integer or non-integer between them.
In one of the aspects, the removal of unreacted drug conjugate mc-vc-PABC-MMAE, according to the present invention, comprises UF/DF of the crude brentuximab vedotin fraction obtained from conjugation, wherein ultrafiltration concentrates the brentuximab vedotin and diafiltration removes the unreacted mc-vc-PABC-MMAE with exchange of buffers. The UF/DF is performed to atleast about 20 to 30 dia-volumes to obtain the concentrated ADC brentuximab vedotin, wherein the range of about 20 to 30 dia-volumes (DV), according to present invention, includes all the integers and non-integers present between them. For example, the UF/DF range of about 20 to 30 dia-volumes (DV) include 20 DV, 21 DV, 22, DV, 23 DV, 24 DV, 25 DV, 26 DV, 27 DV, 28 DV, 29 DV, 30 DV, or any integer or non-integer between them. The concentrated brentuximab vedotin was then formulated in a desired buffer to obtain the formulated bulk of brentuximab vedotin.
In one of the aspects, the terminal filtration of the brentuximab vedotin, according to the present invention, comprises terminal filtration of the formulated bulk of brentuximab vedotin with 0.2 µm filter to ensure purity of the brentuximab vedotin by removal of any contaminants, if present.
In some aspects, the conjugated brentuximab antibody species can be analyzed based on the characteristics of the antibody, the drug and/or the conjugate. For example, hydrophobic interaction chromatography (HIC) can be used to analyze species corresponding to 0, 2, 4, 6, and 8 drugs per antibody.
A variety of containers may be used to store and transport the formulated bulk of brentuximab vedotin, including pre-filled syringes, single-use vials, PETG bottles and plastic ampules. In some instances, plastic ampules can be manufactured using the blow-fill-seal manufacturing technique or method. In one of the aspects, the formulated bulk is stored in frozen or lyophilized form in sterile, non-pyrogenic, PETG bottles at temperature conditions minus -25 °C ± 5 °C.
The lyophilization process, according to the present invention, can be performed by a skilled person using the techniques available in the art, which includes freezing, primary drying, secondary drying and optionally annealing.
In one of the aspects, the formulated bulk of the present invention is stable either in frozen or lyophilized or liquid form.
In the aspects, the method of preparing brentuximab vedotin provides brentuximab vedotin with purity of at least more than 97%. In the aspects, the purity of antibody or ADC, according to the present invention, can be estimated through several analytical techniques including HP-SEC (High performance – size exclusion chromatography) and HP-HIC (High performance – hydrophobic interaction chromatography).
In another aspect, the method of purifying brentuximab antibody comprises recombinant Protein A chromatography with first wash is at about pH 7.2 to pH 7.6 and/or conductivity at about 5 mS / cm to 30 mS / cm, second wash at about pH 7.2 to pH 7.6 and/or a conductivity at about 75 mS / cm to 100 mS / cm and third wash at a pH at 5 to 6.5 and/or a conductivity at about 1 mS/cm to 5 mS / cm; and elution of an antibody at about pH 3.3 to pH 3.7 and/or conductivity at about 5 mS / cm to 30 mS / cm, alone or in combination with other chromatography techniques such as, but not limited to, anion exchange chromatography, cation exchange chromatography and non-chromatography techniques such as, but not limited to, centrifugation, depth filtration, viral inactivation at low pH, pH neutralization, virus clearance by nano-filtration and terminal filtration.
The first wash with equilibration buffer, according to the present invention, is in the range of about pH 7.2 to pH 7.6 and/or conductivity in the range of about 5 mS/cm to 30 mS / cm. The range of about pH 7.2 to pH 7.6 and the conductivity range of about 5 mS / cm to 30 mS / cm, includes each integer and non-integer between them. For example, the pH range of about pH 7.2 to pH 7.6 includes pH 7.2, pH 7.3, pH 7.4, pH 7.5, pH 7.6, or any integer or non-integer between them. For example, the conductivity range of about 5 mS/cm to 30 mS / cm includes 5 mS / cm, 6.1 mS / cm, 7.9 mS / cm, 8.4 mS / cm, 9.5 mS / cm 10.1 mS / cm, 11.6 mS / cm, 12.3 mS / cm, 13.7 mS / cm, 14.8 mS/cm, 15.2 mS / cm, 16.8 mS / cm, 17.7 mS / cm, 18.2 mS / cm, 19.5 mS / cm, 20.3 mS / cm, 21.6 mS / cm, 22.9 mS / cm, 23.4 mS / cm, 23.5 mS / cm, 24.2 mS / cm, 25 mS / cm, 26.1 mS / cm, 27.7 mS / cm, 28.9 mS / cm, 29.3 mS / cm, 30 mS / cm, or any integer or non-integer between them.
The second wash, according to the present invention, is in the range of about pH 7.2 to pH 7.6 and/or a conductivity in the range of about 75 mS / cm to 100 mS / cm. The range of about pH 7.2 to pH 7.6 and the conductivity range of about 75 mS / cm to 100 mS / cm includes each integer and non-integer between them. For example, the pH range of about pH 7.2 to pH 7.6 includes pH 7.2, pH 7.3, pH 7.4, pH 7.5, pH 7.6, or any integer or non-integer between them. For example, the conductivity range of about 75 mS / cm to 100 mS / cm includes 75 mS / cm, 75.7 mS / cm, 76.5 mS / cm, 77.3 mS / cm, 78.8 mS / cm, 79.2 mS / cm, 79.8 mS / cm, 80 mS / cm, 82.3 mS / cm, 84.8 mS / cm, 85 mS / cm, 87.8 mS / cm, 89 mS / cm, 89.6 mS / cm, 91.5 mS / cm, 92.3 mS / cm, 94.7 mS / cm, 95 mS / cm, 95.9 mS / cm, 96.4 mS / cm, 97.1 mS / cm, 97.9 mS / cm, 98.3 mS / cm, 98 mS / cm, 99.2 mS / cm, 100 mS / cm or any integer or non-integer between them.
The third wash is at a pH in the range of about pH 5 to pH 6.5 and/or a conductivity in the range of about 1 mS / cm to 5 mS / cm, wherein the pH range of about 5 to 6.5 and a conductivity range of about 1 mS / cm to 5 mS / cm, according to the present invention, includes each integer and non-integer present between them. For example, the pH range of about 5 to 6.5 includes pH 5, pH 5.1, pH 5.2, pH 5.3, pH 5.4, pH 5.5, pH 5.6, pH 5.7, pH 5.8, pH 5.9, pH 6, pH 6.1, pH 6.2, pH 6.3, pH 6.4, pH 6.5, or any integer or non-integer between them. For example, the conductivity range of about 1 mS / cm to 5 mS / cm includes 1 mS / cm, 1.6 mS / cm, 2.1 mS / cm, 2.3 mS / cm, 2.7 mS / cm, 3.2 mS / cm, 3.5 mS / cm, 3.9 mS / cm, 4.1 mS / cm, 4.4 mS / cm, 4.8 mS / cm, 5 mS / cm, or any integer or non-integer between them. The elution of an antibody is in the range of about pH 3.3 to pH 3.7 and/or conductivity in the range of about 5 mS / cm to 30 mS / cm, wherein the pH range of about 3.3 to pH 3.7 and/or conductivity in the range of about 5 mS / cm to 30 mS / cm, according to the present invention, includes each integer and non-integer present between them. For example, the pH range of about 3.3 to pH 3.7 includes pH 3.3, pH 3.4, pH 3.5, pH 3.6, pH 3.7, or any integer or non-integer present between them. For example, the conductivity range of about 5 mS / cm to 30 mS / cm includes 5.0 mS / cm, 5.4 mS / cm, 5.9 mS / cm, 6.3 mS / cm, 6.7 mS / cm, 7.2 mS / cm, 7.6 mS / cm, 8 mS / cm, 8.5 mS / cm, 9.2 mS / cm, 9.8 mS / cm, 10.4 mS / cm, 11.3 mS / cm, 11.9 mS / cm, 12.4 mS / cm, 12.9 mS / cm, 13.1 mS / cm, 13.7 mS / cm, 14.5 mS / cm, 15 mS / cm, 16.2 mS / cm, 17.4 mS / cm, 18 mS / cm, 18.8 mS / cm, 19.3 mS / cm, 19.9 mS / cm, 20.4 mS / cm, 21.2 mS / cm, 21.8 mS / cm, 22 mS / cm, 23.1 mS / cm, 23.7 mS / cm, 24.4 mS / cm, 25 mS / cm, 26.1 mS / cm, 26.8 mS / cm, 27.5 mS / cm, 28.4 mS / cm, 29.6 mS / cm, 30 mS / cm, or any integer or non-integer present between them.
In one of the aspects, the purification of brentuximab can be performed using recombinant Protein A (rPA) chromatography, anion exchange chromatography (AEX), and cation exchange chromatography (CEX).
The recombinant Protein A (rPA) column step is used to capture the monoclonal antibody – brentuximab from crude mixture and to elute the desired monoclonal antibody from the column step with high level of purity in bind-elute mode. The anion exchange (AEX) column step is useful for further removal of process- and product- related impurities in bind-elute mode. Cation exchange (CEX) chromatography is useful for further removal of process-related impurities in bind-elute mode.
In one of the aspects, the purification of brentuximab can also be performed using hydrophobic interaction chromatography (HIC), mixed-mode chromatography (MMC) and/or any other chromatographies known in the art.
In one of the aspects, the purification of brentuximab comprises recombinant Protein A (rPA) chromatography. In one of the aspects, the purification of brentuximab comprises anion exchange chromatography (AEX). In one of the aspects, the purification of brentuximab comprises cation exchange chromatography (CEX). In one of the aspects, the purification of brentuximab comprises hydrophobic interaction chromatography (HIC). In one of the aspects, the purification of brentuximab comprises mixed-mode chromatography (MMC). In one of the aspects, the non-chromatography techniques according to the present invention, comprises centrifugation, depth filtration, viral inactivation at low pH, pH neutralization, virus clearance by nano-filtration and terminal filtration.
In one of the aspects, viral inactivation at low pH is in the range of about pH 3.5 to pH 4, wherein the pH range of about pH 3.5 to pH 4, according to the present invention, includes each integer and non-integer present between them. For example, the pH range of about pH 3.5 to pH 4 includes pH 3.5, pH 3.6, pH 3.7, pH 3.8, pH 3.9, pH 4.0, or any integer or non-integer present between them.
In one of the aspects, pH neutralization after viral inactivation is in the pH range of about pH 5.5 to pH 6.5, wherein the pH range of about pH 5.5 to pH 6.5, according to the present invention, includes each integer and non-integer present between them. For example, the pH range of about pH 5.5 to pH 6.5 includes pH 5.5, pH 5.6, pH 5.7, pH 5.8, pH 5.9, pH 6.0, pH 6.1, pH 6.2, pH 6.3, pH 6.4, pH 6.5, or any integer or non-integer between them.
In one of the aspects, the inactivated viruses can be removed using various techniques, for example, filtration using ultrafilters, microfilters or nanofilters.
In one of the aspects, the anion exchange chromatography, according to the present invention, can use strongly basic resin, for example, quaternary aminoethyl (QAE), trimethylammonium ethyl (TMAE) or weakly basic, for example, Diethylamino ethyl (DEAE), or Dimethylamino ethyl (DMAE).
In one of the aspects, the cation exchange chromatography, according to the present invention, can use strongly acidic resin, for example, sulphopropyl (SP), sulphoethyl or sulphoisobutyl, or weakly acidic, for example, carboxyl group containing resin.
In one of the aspects, reconditioning of the antibody fraction with UF/DF at intervals in respective buffers, according to the present invention, concentrates antibody and allows exchange of buffers.
In one of the aspects, reconditioning the obtained rPA eluate, according to the present invention, is to match the equilibration conditions of the next column step. For example, the rPA eluate is reconditioned with buffer selected from, but not limited to Tris-HCl at pH range of about pH 7.2 to pH 7.6, wherein the pH range of about pH 7.2 to pH 7.6, according to the present invention includes all integers and non-integers present between them. For example, the pH range of about pH 7.2 to pH 7.6 includes pH 7.2, pH 7.3, pH 7.4, pH 7.5, pH 7.6, or any integers or non-integers between them.
In one of the aspects, reconditioning the obtained AEX eluate, according to the present invention, is to match the equilibration conditions of the next column step. For example, the AEX eluate is reconditioned with buffer selected from, but not limited to sodium acetate at pH range of about pH 4.5 to pH 5.7, wherein the pH range of about pH 4.5 to pH 5.7, according to the present invention includes all integers and non-integers present between them. For example, the pH range of about pH 4.5 to pH 5.7 includes pH 4.5, pH 4.6, pH 4.7, pH 4.8, pH 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, or any integers or non-integers between them.
In one of the aspects, reconditioning the obtained CEX eluate, according to the present invention, provides concentrated brentuximab antibody in buffer selected from, but not limited to, phosphate buffer, citrate buffer, succinate buffer, borate buffer or citrate-phosphate buffer at about pH 6.
In one of embodiment, the purified brentuximab antibody with concentration of 8 mg / mL was reduced with 2.5 molar excess of the reducing agent tris(carboxyethyl)phosphine hydrochloride (TCEP HCl) in 50 mM sodium phosphate buffer pH 6 and conductance 9.5 mS / cm, 50 mM NaCl, 1 mM DTPA at 37 °C for a maximum period of 120 ± 10 minutes with gentle mixing. In of the embodiment the interchain disulfide bonds are reduced by addition of the reducing agent tris(carboxyethyl)phosphine hydrochloride (TCEP HCl). After partial reduction, about 0 to 8 free thiol groups are accessible per molecules of brentuximab. Post-partial reduction, brentuximab protein solution was passed through a 0.22 µm filter and subjected to drug conjugation.
In of the aspect , the reduced brentuximab antibody with concentration of 4.5 ± 0.5 mg / mL was subjected to conjugation reaction with excess amount of (7.5 molar excess) drug-linker component mc-vc-PABC-MMAE in 50 mM sodium phosphate buffer pH 6 and conductance 9.5 mS / cm, 50 mM NaCl, 1 mM DTPA, under cold temperature conditions (4 °C to 8 °C) for 60 ± 10 minutes with gentle mixing. In one of other aspect the Organic solvent (10 % DMSO) was used during conjugation to maintain solubility of mc-vc-PABC-MMAE. Conjugation takes place with the formation of covalent thioether bond between free thiol (-SH) groups of partially reduced brentuximab and maleimide containing side chain of drug linker mc-vc-PABC-MMAE. Following conjugation of the mc-vc-PABC-MMAE to the brentuximab antibody, the conjugated drug-antibody species brentuximab vedotin is analyzed using HP-HIC technique. In another embodiment, At the end of conjugation reaction, the antibody-drug conjugate (brentuximab vedotin) crude reaction mixture was filtered through a 0.22 µm filter and was moved further for removal of unreacted mc-vc-PABC-MMAE.

In one of the aspects, terminal filtration of the obtained brentuximab antibody with 0.2 µm ensures purity of the antibody by removal of any contaminants, if present.
In some of the aspects, the purified brentuximab antibody is stored at minus -25 °C ± 5 °C, and thawed under room temperature conditions when required for ADC preparation. After thawing, the protein solution is reconditioned again.
In another aspect, the method of purifying brentuximab antibody comprises recombinant Protein A chromatography with first wash is at about pH 7.2 to pH 7.6 and/or conductivity at about 5 mS / cm to 30 mS / cm, second wash at about pH 7.2 to pH 7.6 and/or a conductivity at about 75 mS / cm to 100 mS / cm and third wash at a pH at 5 to 6.5 and/or a conductivity at about 1 mS/cm to 8 mS / cm; and elution of an antibody at about pH 3.3 to pH 3.7 and/or conductivity at about 5 mS / cm to 30 mS / cm, alone or in combination with other chromatography techniques such as, but not limited to, cation exchange chromatography, anion exchange chromatography and non-chromatography techniques such as, but not limited to, centrifugation, depth filtration, viral inactivation at low pH, pH neutralization, virus clearance by nano-filtration and terminal filtration.
The first wash with equilibration buffer, according to the present invention, is in the range of about pH 7.2 to pH 7.6 and/or conductivity in the range of about 5 mS/cm to 30 mS / cm. The range of about pH 7.2 to pH 7.6 and the conductivity range of about 5 mS / cm to 30 mS / cm, includes each integer and non-integer between them. For example, the pH range of about pH 7.2 to pH 7.6 includes pH 7.2, pH 7.3, pH 7.4, pH 7.5, pH 7.6, or any integer or non-integer between them. For example, the conductivity range of about 5 mS/cm to 30 mS / cm includes 5 mS / cm, 6.1 mS / cm, 7.9 mS / cm, 8.4 mS / cm, 9.5 mS / cm 10.1 mS / cm, 11.6 mS / cm, 12.3 mS / cm, 13.7 mS / cm, 14.8 mS/cm, 15.2 mS / cm, 16.8 mS / cm, 17.7 mS / cm, 18.2 mS / cm, 19.5 mS / cm, 20.3 mS / cm, 21.6 mS / cm, 22.9 mS / cm, 23.4 mS / cm, 23.5 mS / cm, 24.2 mS / cm, 25 mS / cm, 26.1 mS / cm, 27.7 mS / cm, 28.9 mS / cm, 29.3 mS / cm, 30 mS / cm, or any integer or non-integer between them.
The second wash, according to the present invention, is in the range of about pH 7.2 to pH 7.6 and/or a conductivity in the range of about 75 mS / cm to 100 mS / cm or 1 M Nacl. The range of about pH 7.2 to pH 7.6 and the conductivity range of about 75 mS / cm to 100 mS / cm includes each integer and non-integer between them. For example, the pH range of about pH 7.2 to pH 7.6 includes pH 7.2, pH 7.3, pH 7.4, pH 7.5, pH 7.6, or any integer or non-integer between them. For example, the conductivity range of about 75 mS / cm to 100 mS / cm includes 75 mS / cm, 75.7 mS / cm, 76.5 mS / cm, 77.3 mS / cm, 78.8 mS / cm, 79.2 mS / cm, 79.8 mS / cm, 80 mS / cm, 82.3 mS / cm, 84.8 mS / cm, 85 mS / cm, 87.8 mS / cm, 89 mS / cm, 89.6 mS / cm, 91.5 mS / cm, 92.3 mS / cm, 94.7 mS / cm, 95 mS / cm, 95.9 mS / cm, 96.4 mS / cm, 97.1 mS / cm, 97.9 mS / cm, 98.3 mS / cm, 98 mS / cm, 99.2 mS / cm, 100 mS / cm or any integer or non-integer between them.
The third wash is at a pH in the range of about pH 5 to pH 6.5 and/or a conductivity in the range of about 1 mS / cm to 5 mS / cm, wherein the pH range of about 5 to 6.5 and a conductivity range of about 1 mS / cm to 5 mS / cm, according to the present invention, includes each integer and non-integer present between them. For example, the pH range of about 5 to 6.5 includes pH 5.0, pH 5.1, pH 5.2, pH 5.3, pH 5.4, pH 5.5, pH 5.6, pH 5.7, pH 5.8, pH 5.9, pH 6, pH 6.1, pH 6.2, pH 6.3, pH 6.4 or pH 6.5, or any integer or non-integer between them. For example, the conductivity range of about 1 mS / cm to 5 mS / cm includes 1 mS / cm, 1.6 mS / cm, 2.1 mS / cm, 2.3 mS / cm, 2.7 mS / cm, 3.2 mS / cm, 3.5 mS / cm, 3.9 mS / cm, 4.1 mS / cm, 4.4 mS / cm, 4.8 mS / cm or 5 mS / cm, or any integer or non-integer between them. The elution of an antibody is in the range of about pH 3.3 to pH 3.7 and/or conductivity in the range of about 5 mS / cm to 30 mS / cm, wherein the pH range of about 3.3 to pH 3.7 and/or conductivity in the range of about 5 mS / cm to 30 mS / cm, according to the present invention, includes each integer and non-integer present between them. For example, the pH range of about 3.3 to pH 3.7 includes pH 3.3, pH 3.4, pH 3.5, pH 3.6, pH 3.7, or any integer or non-integer present between them. For example, the conductivity range of about 5 mS / cm to 30 mS / cm includes 5.0 mS / cm, 5.4 mS / cm, 5.9 mS / cm, 6.3 mS / cm, 6.7 mS / cm, 7.2 mS / cm, 7.6 mS / cm, 8 mS / cm, 8.5 mS / cm, 9.2 mS / cm, 9.8 mS / cm, 10.4 mS / cm, 11.3 mS / cm, 11.9 mS / cm, 12.4 mS / cm, 12.9 mS / cm, 13.1 mS / cm, 13.7 mS / cm, 14.5 mS / cm, 15 mS / cm, 16.2 mS / cm, 17.4 mS / cm, 18 mS / cm, 18.8 mS / cm, 19.3 mS / cm, 19.9 mS / cm, 20.4 mS / cm, 21.2 mS / cm, 21.8 mS / cm, 22 mS / cm, 23.1 mS / cm, 23.7 mS / cm, 24.4 mS / cm, 25 mS / cm, 26.1 mS / cm, 26.8 mS / cm, 27.5 mS / cm, 28.4 mS / cm, 29.6 mS / cm, 30 mS / cm, or any integer or non-integer present between them.
In one of the aspects, the purification of brentuximab can be performed using recombinant Protein A (rPA) chromatography, cation exchange chromatography (CEX), and anion exchange chromatography (AEX).
The recombinant Protein A (rPA) column step is used to capture the monoclonal antibody – brentuximab from crude mixture and to elute the desired monoclonal antibody from the column step with high level of purity in bind-elute mode. The Cation exchange (CEX) column and Anion exchange (AEX) chromatography steps is useful for further removal of process- and product- related impurities in bind-elute mode.
In one of the aspects, the purification of brentuximab can also be performed using mixed-mode chromatography (MMC) and/or any other chromatographies known in the art.
In one of the aspects, the purification of brentuximab comprises recombinant Protein A (rPA) chromatography. In one of the aspects, the purification of brentuximab comprises cation exchange chromatography (CEX). In one of the aspects, the purification of brentuximab comprises anion exchange chromatography (AEX). In one of the aspects, the purification of brentuximab comprises mixed-mode chromatography (MMC). In one of the aspects, the non-chromatography techniques according to the present invention, comprises centrifugation, depth filtration, viral inactivation at low pH, pH neutralization, virus clearance by nano-filtration and terminal filtration.
In one of the aspects, viral inactivation at low pH is in the range of about pH 3.5 to pH 4, wherein the pH range of about pH 3.5 to pH 4, according to the present invention, includes each integer and non-integer present between them. For example, the pH range of about pH 3.5 to pH 4 includes pH 3.5, pH 3.6, pH 3.7, pH 3.8, pH 3.9, pH 4.0, or any integer or non-integer present between them.
In one of the aspects, pH neutralization after viral inactivation is in the pH range of about pH 5.0 to pH 6.5, wherein the pH range of about pH 5.0 to pH 6.5, according to the present invention, includes each integer and non-integer present between them. For example, the pH range of about pH 5.0 to pH 6.5 includes pH 5.0, pH 5.1, pH 5.2, pH 5.3, pH 5.4, pH 5.5, pH 5.5, pH 5.6, pH 5.7, pH 5.8, pH 5.9, pH 6.0, pH 6.1, pH 6.2, pH 6.3, pH 6.4, pH 6.5, or any integer or non-integer between them.
In one of the aspects, the inactivated viruses can be removed using various techniques, for example, filtration using ultrafilters, microfilters or nanofilters.
In one of the aspects, reconditioning the obtained CEX eluate, according to the present invention, is to match the equilibration conditions of the next column step. For example, the CEX eluate is reconditioned with buffer selected from, but not limited to sodium acetate at pH range of about pH 4.5 to pH 5.7, wherein the pH range of about pH 4.5 to pH 5.7, according to the present invention includes all integers and non-integers present between them. For example, the pH range of about pH 4.5 to pH 5.7 includes pH 4.5, pH 4.6, pH 4.7, pH 4.8, pH 4.9, pH 5.0, pH 5.1, pH 5.2, pH 5.3, pH 5.4, pH 5.5, pH 5.6 or pH 5.7, or any integers or non-integers between them.
In one of the aspects, the cation exchange chromatography, according to the present invention, can use strongly acidic resin, for example, sulphopropyl (SP), sulphoethyl or sulphoisobutyl, or weakly acidic, for example, carboxyl group containing resin.
In one embodiment the method of preparing brentuximab includes partial reduction of the brentuximab antibody with suitable reducing agent in suitable buffer and at suitable pH then conjugation of the reduced brentuximab antibody obtained from partial reduction step with the drug conjugate mc-vc-PABC-MMAE in same buffer and at same pH as used in partial reduction step.
In one of the aspects, the anion exchange chromatography, according to the present invention, can use strongly basic resin, for example, quaternary aminoethyl (QAE), trimethylammonium ethyl (TMAE) or weakly basic, for example, Diethylamino ethyl (DEAE), or Dimethylamino ethyl (DMAE).
In one of the aspects, reconditioning the obtained AEX eluate, according to the present invention, provides concentrated brentuximab antibody in buffer selected from, but not limited to, phosphate buffer, citrate buffer, succinate buffer, borate buffer or citrate-phosphate buffer at about pH 6.
In one of the aspects, terminal filtration of the obtained brentuximab antibody with 0.2 µm ensures purity of the antibody by removal of any contaminants, if present.
In one of embodiment, the purified brentuximab antibody is stored at minus -25 °C ± 5 °C, and thawed under room temperature conditions when required for ADC preparation. After thawing, the protein solution is subjected for the partial reduction.
In second embodiment the brentuximab (12 mg / mL) was subjected to partial reduction (inter-chain S-S cross-links) in the presence of 2.5 molar excess of tris (carboxyethyl) phosphine [TCEP] in 50 mM phosphate buffer, about 0 to 50 mM NaCl preferably 50mM NaCl, and 1 mM DTPA, with a pH below 7. For example, the pH range of about below 7 more preferably between the range of 5.8-6.3 pH for NMT about 300 min. Incubation temperature was maintained at about 15?C to 40 ?C more preferably 37 ?C, under stirring condition (200 rpm), for the reaction pH was maintained between 5.8 to 6.3 during the partial reduction reaction.
In the other embodiment, after the reduction step, the conjugation reaction was carried out in the same reaction vessel at about 4?C to 37 ?C more preferably 25 ?C temperature. About 6 to 10 molar excess of vc-MMAE (valine-citrulline monomethyl auristatin E) preferably 8.25 was used relative to the protein, which was dissolved in 100% DMSO or 100 % DMAc . In of the aspects, the final concentration of DMSO or 100 % DMAc in the reaction mixture was adjusted to 10% (v/v). In other aspects, the protein concentration was maintained at 10 mg/mL in a reaction buffer containing 50 mM phosphate buffer, about 1 to 50 mM NaCl more preferably 50mM NaCl and 1 mM DTPA, with a pH of below 7, For example, the pH range of about below 7 more preferably between the range of 5.8-6.3 pH In another embodiment, incubation was performed under 4?C to 37 ?C more preferably 25 ?C temperature for NMT 120 min, under gentle stirring (200 rpm). At the end of conjugation reaction, the reaction mixture was passed through a 0.2 µm filter and subjected to UF / DF for the removal of unreacted drug-linker molecules and buffer exchange. Post UD / DF step, brentuximab protein solution was formulated in the 20 mM sodium citrate buffer pH 6.6 ± 0.1. + 70 mg / ml a, a trehalose and 0.2 mg / ml polysorbate-80. In of the embodiment the formulated bulk of brentuximab vedotin was passed through a 0.2 µm filter (150 cm2 surface area), under bio-safety cabinet, and collected in single-use, sterile, non-pyrogenic polyethylene terephthalate glycol (PETG) bottle with white high-density polyethylene (HDPE) screw closure. The filtered formulated bulk was labeled as brentuximab vedotin. The average drug antibody ratio (DAR) was found to be 4.34 as shown in Figure 11.
In the third embodiment, the brentuximab protein concentration was maintained at 12 mg/mL in a reaction buffer containing 50 mM phosphate buffer, about 1 to 350 mM NaCl, more preferably 250mM NaCl, and 1 mM DTPA, with a pH of below 7, more preferably between the range of 5.8-6.3 pH was subjected to partial reduction (inter-chain S-S cross-links) in the presence of 2.5 molar excess of tris (carboxyethyl) phosphine [TCEP] for NMT about 300 min. In one of the other aspects, Incubation temperature was maintained at 15?C to 40 ?C more preferably 37 ?C, under stirring condition (200 rpm), for the reaction. pH was maintained between 5.8 to 6.3 during the partial reduction reaction.
In one embodiment, after the reduction step, the conjugation reaction was carried out in the same reaction vessel at 4?C to 37 ?C more preferably 25 ?C temperature. About 6 to 10 molar excess of vc-MMAE (valine-citrulline monomethyl auristatin E) preferably 8.25 was used relative to the protein, which was dissolved in 100% DMSO or 100 % DMAc. In of the aspects, the final concentration of 100 % DMSO or 100 % DMAc in the reaction mixture was adjusted to 10% (v/v). In another embodiment, the protein concentration was maintained at 10 mg/mL in a reaction buffer containing 50 mM phosphate buffer, about 1 to 350 mM NaCl more preferably 250mM, and 1 mM DTPA, with a pH of below 7, For example, the pH range of about below 7 more preferably between the range of 5.8-6.3 pH . In another aspects, incubation was performed under 4?C to 37 ?C more preferably 25 ?C temperature for NMT 120 min, under gentle stirring (200 rpm). At the end of conjugation reaction, the reaction mixture was passed through a 0.2 µm filter and subjected to UF / DF for the removal of unreacted drug-linker molecules and buffer exchange. In one of the embodiment Post UD / DF step, brentuximab protein solution was formulated in the 20 mM sodium citrate buffer pH 6.6 ± 0.1. + 70 mg / ml a, a trehalose and 0.2 mg / ml polysorbate-80. The formulated bulk of brentuximab vedotin was passed through a 0.2 µm filter (150 cm2 surface area), under bio-safety cabinet, and collected in single-use, sterile, non-pyrogenic polyethylene terephthalate glycol (PETG) bottle with white high-density polyethylene (HDPE) screw closure. The filtered formulated bulk was labeled as brentuximab vedotin. The average drug antibody ratio (DAR) was found to be 3.82 as shown in Figure 12.
In fourth embodiment, the brentuximab protein concentration was maintained at 12 mg/mL in a reaction buffer containing 50 mM phosphate buffer, about 1 to 350 mM NaCl more preferably 250mM, and 1 mM DTPA, with a pH of below 7, For example, the pH range of about below 7 more preferably between the range of 5.8-6.3 pH was subjected to partial reduction (inter-chain S-S cross-links) in the presence of 2.5 molar excess of tris (carboxyethyl) phosphine [TCEP] for NMT about 300 min. In one of the other aspects, incubation temperature was maintained at 15 to 40 more preferably 37?C, under stirring condition (200 rpm), for the reaction. pH was maintained between 5.8 to 6.3 during the partial reduction reaction.
In one embodiment, after the reduction step, the conjugation reaction was carried out in the same vessel at 15?C to 40 ?C more preferably 37 ?C temperature . About 6 to 10 molar excess of vc-MMAE (valine-citrulline monomethyl auristatin E) more preferably 8.25 molar excess of vc-MMAE (valine-citrulline monomethyl auristatin E) was used relative to the protein, which was dissolved in 100% DMSO or 100 % DMAc . In of the aspects the final concentration of DMSO 100 % or 100 % DMAc in the reaction mixture was adjusted to 10% (v/v). In another embodiment, protein concentration was maintained at 10 mg/mL in a reaction buffer containing 50 mM phosphate buffer, more preferably 250mM NaCl , and 1 mM DTPA, with a pH of below 7, For example, the pH range of about below 7 more preferably between the range of 5.8-6.3 pH . In another aspect, incubation was performed under 15 ?C to 40 ?C more preferably 37 ?C temperature for NMT 120 min, under gentle stirring (200 rpm). At the end of conjugation reaction, the reaction mixture was passed through a 0.2 µm filter and subjected to UF / DF for the removal of unreacted drug-linker molecules and buffer exchange. In one of the embodiment Post UD / DF step, brentuximab protein solution was formulated in the 20 mM sodium citrate buffer pH 6.6 ± 0.1. + 70 mg / ml a, a trehalose and 0.2 mg / ml polysorbate-80. The formulated bulk of brentuximab vedotin was passed through a 0.2 µm filter (150 cm2 surface area), under bio-safety cabinet, and collected in single-use, sterile, non-pyrogenic polyethylene terephthalate glycol (PETG) bottle with white high-density polyethylene (HDPE) screw closure. The filtered formulated bulk was labeled as brentuximab vedotin. The average drug antibody ratio (DAR) was found to be 3.68 as shown in Figure 13.
In one of the aspect the pH of below 7 in the present invention includes pH range between 0 pH to 7 pH or any integer or non-integer present between them. , For example, the pH range of about below 7 includes pH 0, pH 1, pH 1.1, pH 1.2, pH 1.3, pH 1.4, pH 1.5, pH 1.6, pH 1.7, pH 1.8, pH 1.9, pH 2.0, pH 2.1, pH 2.2, pH 2.3, pH 2.4, pH 2.5, pH 2.6, pH 2.7, pH 2.8, pH 2.9, pH 3.0, pH 3.1, pH 3.2, pH 3.3, pH 3.4, pH 3.5, pH 3.6, pH 3.7, pH 3.8, pH 3.9, pH 4.0, pH 4.5, pH 4.6, pH 4.7, pH 4.8, pH 49, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7 pH 5.8, pH 5.9, pH 6.0, pH 6.1, pH 6.2, pH 6.3, pH 6.4, pH 6.5, pH6.6, pH 6.7, pH 6.8, pH 6.9, pH 7.0 or any integer or non-integer between them more preferably between the range of 5.8-6.3 pH.
In one embodiment about 6 to 10 molar excess of vc-MMAE includes 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9 or 10.0 molar excess of vc-MMAE.
In one of the aspects, Incubation temperature was maintained at 15?C to 40 ?C more preferably 37 ?C, under stirring condition (200 rpm), for the reaction. pH was maintained between 5.8 to 6.3 during the partial reduction reaction. The temperature range of about 15 °C to 40 °C, according to the present invention, includes each integer and non-integer present between them. For example, the temperature range of about 15 °C to 40 °C includes, but not limited to, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C 37°C, 38°C , 39°C or 40°C.
In one of the aspects, the conjugation step comprises conjugation of the partially reduced brentuximab antibody with mc-vc-PABC-MMAE in the same buffer and at about the same pH and at temperature conditions in the range of 4 ?C to 37 ?C more preferably 25?C .
The temperature range of about 4°C to 37°C, according to the present invention, includes each integer and non-integer present between them. For example, the temperature range of about 4 °C to 37 °C includes, but not limited to, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C 37°C, 38°C , 39°C or 40°C.
In one of the aspects, the conjugation step comprises conjugation of the partially reduced brentuximab antibody with mc-vc-PABC-MMAE in the same buffer and at about same pH and at temperature conditions in the range of 15?C to 40 ?C more preferably 37?C . The temperature range of about 15 °C to 40°C, according to the present invention, includes each integer and non-integer present between them. For example, the temperature range of about 15 °C to 40 °C includes, but not limited to, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C 37°C, 38°C , 39°C or 40°C.

In another aspect the elution of an antibody is in the range of about pH 3.3 to pH 3.7 and/or conductivity in the range of about 6 mS / cm to 30 mS / cm, wherein the pH range of about 3.3 to pH 3.7 and/or conductivity in the range of about 5 mS / cm to 30 mS / cm, according to the present invention, includes each integer and non-integer present between them. For example, the pH range of about 3.3 to pH 3.7 includes pH 3.3, pH 3.4, pH 3.5, pH 3.6, pH 3.7, or any integer or non-integer present between them. For example, the conductivity range of about 6 mS / cm to 30 mS / cm includes 6.0 mS / cm , 6.3 mS / cm, 6.7 mS / cm, 7.2 mS / cm, 7.6 mS / cm, 8 mS / cm, 8.5 mS / cm, 9.2 mS / cm, 9.8 mS / cm, 10.4 mS / cm, 11.3 mS / cm, 11.9 mS / cm, 12.4 mS / cm, 12.9 mS / cm, 13.1 mS / cm, 13.7 mS / cm, 14.5 mS / cm, 15 mS / cm, 16.2 mS / cm, 17.4 mS / cm, 18 mS / cm, 18.8 mS / cm, 19.3 mS / cm, 19.9 mS / cm, 20.4 mS / cm, 21.2 mS / cm, 21.8 mS / cm, 22 mS / cm, 23.1 mS / cm, 23.7 mS / cm, 24.4 mS / cm, 25 mS / cm, 26.1 mS / cm, 26.8 mS / cm, 27.5 mS / cm, 28.4 mS / cm, 29.6 mS / cm, 30 mS / cm, or any integer or non-integer present between them more preferably at conductance 6 mS / cm.

In one of the embodiment the NaCl range of about 1 to 350 mM or 1 to 350mM includes 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 21 mM, 22 mM, 23 mM, 24 mM, 25 mM, 26 mM, 27 mM, 28 mM, 29 mM, 30 mM, 31 mM, 32 mM, 33 mM, 34 mM, 35 mM, 36 mM, 37 mM, 38 mM, 39 mM, 40 mM, 41 mM, 42 mM, 43 mM, 44 mM, 45 mM, 46 mM, 47 mM, 48 mM, 49 mM, 50 mM, 51 mM, 52 mM, 53 mM, 54 mM, 55 mM, 56 mM, 57 mM, 58 mM, 59 mM, 60 mM, 61 mM, 62 mM, 63 mM, 64 mM, 65 mM, 66 mM, 67 mM, 68 mM, 69 mM, 70 mM, 71 mM, 72 mM, 73 mM, 74 mM, 75 mM, 76 mM, 77 mM, 78 mM, 79 mM, 80 mM, 81 mM, 82 mM, 83 mM, 84 mM, 85 mM, 86 mM, 87 mM, 88 mM, 89 mM, 90 mM, 91 mM, 92 mM, 93 mM, 94 mM, 95 mM, 96 mM, 97 mM, 98 mM, 99 mM, 100 mM, 101 mM, 102 mM, 103 mM, 104 mM, 105 mM, 106 mM, 107 mM, 108 mM, 109 mM, 110 mM, 111 mM, 112 mM, 113 mM, 114 mM, 115 mM, 116 mM, 117 mM, 118 mM, 119 mM, 120 mM, 121 mM, 122 mM, 123 mM, 124 mM, 125 mM, 126 mM, 127 mM, 128 mM, 129 mM, 130 mM, 131 mM, 132 mM, 133 mM, 134 mM, 135 mM, 136 mM, 137 mM, 138 mM, 139 mM, 140 mM, 141 mM, 142 mM, 143 mM, 144 mM, 145 mM, 146 mM, 147 mM, 148 mM, 149 mM, 150 mM, 151 mM, 152 mM, 153 mM, 154 mM, 155 mM, 156 mM, 157 mM, 158 mM, 159 mM, 160 mM, 161 mM, 162 mM, 163 mM, 164 mM, 165 mM, 166 mM, 167 mM, 168 mM, 169 mM, 170 mM, 171 mM, 172 mM, 173 mM, 174 mM, 175 mM, 176 mM, 177 mM, 178 mM, 179 mM, 180 mM, 181 mM, 182 mM, 183 mM, 184 mM, 185 mM, 186 mM, 187 mM, 188 mM, 189 mM, 190 mM, 191 mM, 192 mM, 193 mM, 194 mM, 195 mM, 196 mM, 197 mM, 198 mM, 199 mM, 200 mM, 201 mM, 202 mM, 203 mM, 204 mM, 205 mM, 206 mM, 207 mM, 208 mM, 209 mM, 210 mM, 211 mM, 212 mM, 213 mM, 214 mM, 215 mM, 216 mM, 217 mM, 218 mM, 219 mM, 220 mM, 221 mM, 222 mM, 223 mM, 224 mM, 225 mM, 226 mM, 227 mM, 228 mM, 229 mM, 230 mM, 231 mM, 232 mM, 233 mM, 234 mM, 235 mM, 236 mM, 237 mM, 238 mM, 239 mM, 240 mM, 241 mM, 242 mM, 243 mM, 244 mM, 245 mM, 246 mM, 247 mM, 248 mM, 249 mM, 250 mM, 251 mM, 252 mM, 253 mM, 254 mM, 255 mM, 256 mM, 257 mM, 258 mM, 259 mM, 260 mM, 261 mM, 262 mM, 263 mM, 264 mM, 265 mM, 266 mM, 267 mM, 268 mM, 269 mM, 270 mM, 271 mM, 272 mM, 273 mM, 274 mM, 275 mM, 276 mM, 277 mM, 278 mM, 279 mM, 280 mM, 281 mM, 282 mM, 283 mM, 284 mM, 285 mM, 286 mM, 287 mM, 288 mM, 289 mM, 290 mM, 291 mM, 292 mM, 293 mM, 294 mM, 295 mM, 296 mM, 297 mM, 298 mM, 299 mM, 300 mM, 301 mM, 302 mM, 303 mM, 304 mM, 305 mM, 306 mM, 307 mM, 308 mM, 309 mM, 310 mM, 311 mM, 312 mM, 313 mM, 314 mM, 315 mM, 316 mM, 317 mM, 318 mM, 319 mM, 320 mM, 321 mM, 322 mM, 323 mM, 324 mM, 325 mM, 326 mM, 327 mM, 328 mM, 329 mM, 330 mM, 331 mM, 332 mM, 333 mM, 334 mM, 335 mM, 336 mM, 337 mM, 338 mM, 339 mM, 340 mM, 341 mM, 342 mM, 343 mM, 344 mM, 345 mM, 346 mM, 347 mM, 348 mM, 349 mM, 350 mM or any integer or non-integer between them.
In one of the other embodiments the purification of brentuximab antibody does not involve phenyl membrane filtration and recovery yield of brentuximab vedotin in the present invention is more than 85 %.
In one embodiment the room temperature term includes range in between 25°C to 40°C temperature, including intermediate temperatures such as 25.1°C, 25.2°C, 25.3°C, 25.4°C, 25.5°C, 25.6°C, 25.7°C, 25.8°C, 25.9°C, 26.0°C, 26.1°C, 26.2°C, 26.3°C, 26.4°C, 26.5°C, 26.6°C, 26.7°C, 26.8°C, 26.9°C, 27.0°C, 27.1°C, 27.2°C, 27.3°C, 27.4°C, 27.5°C, 27.6°C, 27.7°C, 27.8°C, 27.9°C, 28.0°C, 28.1°C, 28.2°C, 28.3°C, 28.4°C, 28.5°C, 28.6°C, 28.7°C, 28.8°C, 28.9°C, 29.0°C, 29.1°C, 29.2°C, 29.3°C, 29.4°C, 29.5°C, 29.6°C, 29.7°C, 29.8°C, 29.9°C, 30.0°C, 30.1°C, 30.2°C, 30.3°C, 30.4°C, 30.5°C, 30.6°C, 30.7°C, 30.8°C, 30.9°C, 31.0°C, 31.1°C, 31.2°C, 31.3°C, 31.4°C, 31.5°C, 31.6°C, 31.7°C, 31.8°C, 31.9°C, 32.0°C, 32.1°C, 32.2°C, 32.3°C, 32.4°C, 32.5°C, 32.6°C, 32.7°C, 32.8°C, 32.9°C, 33.0°C, 33.1°C, 33.2°C, 33.3°C, 33.4°C, 33.5°C, 33.6°C, 33.7°C, 33.8°C, 33.9°C, 34.0°C, 34.1°C, 34.2°C, 34.3°C, 34.4°C, 34.5°C, 34.6°C, 34.7°C, 34.8°C, 34.9°C, 35.0°C, 35.1°C, 35.2°C, 35.3°C, 35.4°C, 35.5°C, 35.6°C, 35.7°C, 35.8°C, 35.9°C, 36.0°C, 36.1°C, 36.2°C, 36.3°C, 36.4°C, 36.5°C, 36.6°C, 36.7°C, 36.8°C, 36.9°C, 37.0°C, 37.1°C, 37.2°C, 37.3°C, 37.4°C, 37.5°C, 37.6°C, 37.7°C, 37.8°C, 37.9°C, 38.0°C, 38.1°C, 38.2°C, 38.3°C, 38.4°C, 38.5°C, 38.6°C, 38.7°C, 38.8°C, 38.9°C, 39.0°C, 39.1°C, 39.2°C, 39.3°C, 39.4°C, 39.5°C, 39.6°C, 39.7°C, 39.8°C, 39.9°C, or 40.0°C or any integer or non-integer present between them.
The present invention provides an efficient, robust, and time-saving process for synthesizing brentuximab vedotin. This process stands out compared to existing methods due to its innovative one-pot reaction, which eliminates the need for intermediate purification or pH adjustments. The commercial process developed in this invention achieves over 85% yield and >97% purity within a 24-hour to 48-hour batch cycle. This represents a significant improvement, reducing the batch cycle time from several days to just one day.
In this context, the invention provides synthesis method for brentuximab vedotin, ensuring high efficiency and quality, while also offering considerable time-saving advantages. Such advancements make the process commercially viable and beneficial for large-scale production.
The terms brentuximab antibody or brentuximab vedotin or brentuximab monoclonal antibody or brentuximab vedotin antibody drug conjugate are used interchangeably in the present invention. brentuximab vedotin sequence has been disclosed in International Nonproprietary Names for Pharmaceutical Substances (WHO Drug Information Vol. 24, No. 2, 2010) for brentuximab vedotin product4
ADCETRIS® (brentuximab vedotin) is a CD30-directed antibody-drug conjugate (ADC) consisting of three components: 1) the chimeric IgG1 antibody cAC10, specific for human CD30, 2) the microtubule disrupting agent MMAE, and 3) a protease-cleavable linker that covalently attaches MMAE to cAC10 having a structure as disclosed in 5
The term mc-vc-PABC-MMAE (maleimidocaproyl-L-valine-L-citrulline-p-aminobenzyl carbamate) used in the present invention is used interchangeably with the term vc-MMAE in the specification of present invention.

The term depth filtration used herein refers to remove impurities at the harvest stage. In addition to removal of material based on size exclusion, depth filters have been shown to remove soluble impurities by adsorption through hydrophobic, ionic, and other interactions to remove endotoxin and DNA6
The term terminal filtration used herein refers to remove microbial contamination or bioburden through the 0.2 µm filtration or aseptic filtration7.
In one embodiment the distribution of drug-linker (vcMMAE) or the average DAR (drug to antibody ratio) of brentuximab vedotin was assessed by analytical HIC-HPLC method as mentioned in the present invention. . The peaks are separated by HIC on the basis of relative hydrophobicity of the drug-conjugated-antibody (ADC) carrying 0 – 8 payloads (drug toxin)8.

Here, the present invention is illustrated with the following non-limiting examples which should not be interpreted as limiting the scope of the invention in any way:

Examples
Example 1: Preparation of brentuximab antibody (Batch No. Z3318T1008)
Step 1: Cell separation / clarification / reconditioning
After harvesting the batch, cells were separated from the culture broth, first by centrifugation followed by depth filtration in order to obtain clear supernatant containing the protein of interest along with other soluble contaminants. Centrifugation was carried out at 10,500 g x 30 minutes. Depth filtration was performed by using 0.45 µm + 0.22 µm membrane. The clarified supernatant was reconditioned to tune up with the recombinant Protein A (rPA) column equilibration buffer condition for pH and conductance.
Step 2: Recombinant Protein A (rPA) column chromatography
The clarified supernatant after reconditioning was passed through a recombinant Protein A affinity column to capture brentuximab by the affinity matrix followed by its elution from the column at low pH. Prior to loading, the column was equilibrated with a suitable buffer of pH 7.4 at a conductance of 17.7 mS / cm. Subsequent to loading, the column was washed with the same buffer (first wash). Following the first wash step, the column was washed with the same buffer of pH 7.4 but at conductance of 89 mS / cm. A third wash step was performed with a suitable buffer of pH 5.4 having a conductance of 3.9 mS / cm. After the third wash step, elution of the desired protein, brentuximab was conducted with a suitable buffer of pH 3.5 and conductance of 13.7 mS / cm. brentuximab eluted after this step shows about 97.59 % purity when analyzed by analytical HP-SEC shown in Figure 1.
Step 3: Viral inactivation at low pH and pH neutralization
Recombinant Protein A column-eluted desired protein fraction was incubated at the same elution pH condition for about 45 - 60 minutes under room temperature condition for viral inactivation, after which the protein solution was passed through a 0.22 µm filter. Following low-pH treatment, neutralization step was performed with the addition of alkaline solution in a controlled manner.
Step 4: Reconditioning
The protein solution was reconditioned with the adjustment of pH and conductance by UF / DF using 30 kDa MWCO membrane filter to match up to the next column equilibration conditions. For adjustment of pH and conductance, Tris-HCl solution was added to the protein solution. After reconditioning, protein solution was passed through 0.22 µm membrane filter and loaded on to an anion exchange column.
Step 5: Anion exchange (AEX) column chromatography
After reconditioning, the protein solution containing the brentuximab antibody was passed through a DEAE column in bind-elute mode. The column was equilibrated with a Tris-HCl buffer of pH 7.5 having a conductance of 2.2 mS / cm. Following binding to the column matrix, brentuximab was eluted from the column in a suitable buffer other than the equilibration buffer.
More than 99 % purity of brentuximab is achieved after this column step, as assessed by HP-SEC shown in Figure 2.
Step 6: Reconditioning
The AEX column eluate was reconditioned, substantially, by UF / DF using 30 kDa MWCO membrane filter against low ionic strength sodium-acetate buffer solution of pH 5.5 in order to match to equilibration buffer conditions (e.g. pH and conductance) of the next column (SP column) step. Diafiltered protein solution was passed through a 0.22 µm filter and loaded on to a SP column.
Step 5: Cation exchange (CEX) column chromatography
Diafiltered protein solution containing the desired monoclonal antibody was passed through a SP column in bind-and-elute mode with a suitable buffer of pH 5.5 at conductance of 2.09 mS / cm. After the SP-column step, purity of brentuximab is observed to be more than 99 %, as assessed by HP-SEC shown in Figure 3.
Step 6: Nano-filtration
After the SP-column step, the protein solution containing the desired monoclonal antibody underwent a nano-filtration step. After nano-filtration, purity of brentuximab is observed to remain more than 99 %.
Step 7: Ultrafiltration-diafiltration
After nano-filtration, protein solution was diafiltered with desired media for the preparation of a purified brentuximab antibody. The purified brentuximab antibody either can be directly used for preparation of ADC (brentuximab vedotin) or can be stored at -25 °C ± 5 °C, and thawed when required for ADC preparation, following reconditioning of the thawed brentuximab with the fresh media.
Example 2: Reduction of the purified brentuximab antibody (Batch No. Z3318T1008)
The purified brentuximab antibody with concentration of 8 mg / mL was reduced with 2.5 molar excess of the reducing agent tris(carboxyethyl)phosphine hydrochloride (TCEP HCl) in 50 mM sodium phosphate buffer pH 6 and conductance 9.5 mS / cm, 50 mM NaCl, 1 mM DTPA at 37 °C for a maximum period of 120 ± 10 minutes with gentle mixing. The interchain disulfide bonds are reduced by addition of the reducing agent tris(carboxyethyl)phosphine hydrochloride (TCEP HCl). After partial reduction, about 0 to 8 free thiol groups are accessible per molecules of brentuximab. Post-partial reduction, brentuximab protein solution was passed through a 0.22 µm filter and subjected to drug conjugation.
Example 3: Conjugation of the reduced brentuximab (Batch No. Z3318T1008)
The reduced brentuximab antibody with concentration of 4.5 ± 0.5 mg / mL was subjected to conjugation reaction with excess amount of (7.5 molar excess) drug-linker component mc-vc-PABC-MMAE in 50 mM sodium phosphate buffer pH 6 and conductance 9.5 mS / cm, 50 mM NaCl, 1 mM DTPA, under cold temperature conditions (4 °C to 8 °C) for 60 ± 10 minutes with gentle mixing. Organic solvent (10 % DMSO) was used during conjugation to maintain solubility of mc-vc-PABC-MMAE. Conjugation takes place with the formation of covalent thioether bond between free thiol (-SH) groups of partially reduced brentuximab and maleimide containing side chain of drug linker mc-vc-PABC-MMAE. Following conjugation of the mc-vc-PABC-MMAE to the brentuximab antibody, the conjugated drug-antibody species brentuximab vedotin is analyzed using HP-HIC technique. At the end of conjugation reaction, the antibody-drug conjugate (brentuximab vedotin) crude reaction mixture was filtered through a 0.22 µm filter and was moved further for removal of unreacted mc-vc-PABC-MMAE.
Example 4: Removal of unreacted mc-vc-PABC-MMAE drug linker with UF/DF and preparation of the formulated bulk
To remove unreacted mc-vc-PABC-MMAE, UF / DF with 30 kDa MWCO membrane filter was carried out at a maximum TMP of 0.50 bar with an average flux rate of about 30 L / m2 / h, under room temperature conditions. In the first step, a 3-fold concentration of crude brentuximab vedotin protein mixture was performed, after which constant volume diafiltration was carried out with the desired buffered solution of pH 6.6 ± 0.1; conductivity 4.5 ± 0.5 mS / cm (20 mM sodium-citrate), for about 30 dia-volumes. Excess / unreacted mc-vc-PABC-MMAE, was completely removed after 20-fold diafiltration with the said buffer. However, additional 10-fold diafiltration was performed. Diafiltration was monitored and controlled for pH and conductivity of the retentate to achieve the target values. After achieving the target pH and conductivity, brentuximab vedotin protein (Batch No. Z3318B2S001), at about 7 mg / mL, was recovered. The concentrated protein solution was collected, trehalose solution (from 30 % stock) was added to a final concentration of 7 % (w / v). Polysorbate 80 was also added to a final concentration of 0.02% and the final volume was adjusted with the addition of the same buffered solution, mentioned above. Finally, the brentuximab vedotin protein concentration was adjusted to about 5 mg / mL.
Example 5: Terminal filtration and storage (Batch No. Z3318B2S001),
The brentuximab vedotin solution was passed through a 0.22 µm filter and was analyzed with HP-SEC. The purity of brentuximab vedotin was observed to be more than 97 %, as shown in Figure 4 was aliquoted in non-pyrogenic, single-use, sterile PETG bottles and stored under frozen condition at minus -25 °C ± 5 °C. The therapeutic efficacy shall be assigned based on animal immunogenicity study data.
Example 6: Preparation of brentuximab antibody (Batch No. Z3318B1S018)
Step 1: Cell separation / clarification / reconditioning
After harvesting the batch, cells were separated from the culture broth, first by centrifugation followed by depth filtration in order to obtain clear supernatant containing the protein of interest along with other soluble contaminants. Centrifugation was carried out at 10,500 g x 30 minutes. Depth filtration was performed by using 0.45 µm + 0.22 µm membrane. The clarified supernatant was reconditioned to tune up with the recombinant Protein A (rPA) column equilibration buffer condition for pH and conductance.
Step 2: Recombinant Protein A (rPA) column chromatography
The clarified supernatant after reconditioning was passed through a recombinant Protein A affinity column to capture brentuximab by the affinity matrix followed by its elution from the column at low pH. Prior to loading, the column was equilibrated with a suitable buffer of pH 7.4 at a conductance of 17.7 mS / cm. Subsequent to loading, the column was washed with the same buffer (first wash). Following the first wash step, the column was washed with the same buffer of pH 7.4 but at conductance of 89 mS / cm. A third wash step was performed with a suitable buffer of pH 5.4 having a conductance of 3.9 mS / cm. After the third wash step, elution of the desired protein, brentuximab was conducted with a suitable buffer of pH 3.5 and conductance of 4.0 ± 1 mS / cm. brentuximab eluted after this step shows about 98.02 % purity when analyzed by analytical HP-SEC shown in Figure 5.
Step 3: Viral inactivation at low pH and pH neutralization
Recombinant Protein A column-eluted desired protein fraction was incubated at the same elution pH condition for about 45 - 60 minutes under room temperature condition for viral inactivation, after which the protein solution was passed through a 0.22 µm filter. Following low-pH treatment, neutralization step was performed with the addition of alkaline solution in a controlled manner.
Step 4: Reconditioning
Post-pH neutralization, brentuximab protein solution is reconditioned in order to tune up with the second column step equilibration buffer conditions in terms of mainly conductivity (3.0 ± 1.0 mS / cm) by dilution with WFI, at ambient temperature (between 18 to 27 ?C) condition.
After diluting the brentuximab protein solution with addition of WFI, if necessary, pH of the protein solution is adjusted to pH 5.0 ± 0.1 by addition of 2 M Tris-base (to increase the pH of the protein solution) or by addition of 1 M citric acid (to lower the pH of the protein solution).
Step 5:
Cation exchange (sCEX) column chromatography
sCEX column chromatography is performed in bind-elute mode for further purification of brentuximab with the removal of residual product-related and process-related impurities.
sCEX column matrix is equilibrated with sCEX equilibration buffer (25 mM sodium citrate, pH 5.0 ± 0.2, Cond. 3.0 ± 1.0 mS / cm) Equilibration is performed at a controlled flow rate considering a contact time of 5 min which should also be maintained during loading of sCEX load onto the column. Post-equilibration, the sCEX load is loaded (maximum of 30 g of brentuximab protein / L of matrix) onto the sCEX column, equilibrated with 25 mM sodium citrate, pH 5.0 ± 0.2, Cond. 3.0 ± 1.0 mS / cm, Following loading, the column is washed with the equilibration buffer for 3 column bed volumes. Post-loading and equilibration buffer wash, the column is washed with Wash I buffer, at a second conductivity 6.0 ± 1.0 mS / cm, higher than the equilibration buffer. Wash I step is performed for three column bed volumes, at the same flow rate, with 25 mM sodium citrate buffer of pH 5.0 ± 0.2 containing 25 mM NaCl, conductivity 6.0 ± 1.0 mS / cm for the removal of non-specifically bound proteins from the column. Post-wash I buffer wash, elution of brentuximab protein is carried out at a third higher conductivity (22.0 ± 2.0 mS / cm) with 25 mM sodium citrate buffer of pH 5.0 ± 0.1 containing 200 mM NaCl in linear gradient mode of 10 CV. brentuximab protein is observed to elute out of the sCEX column as a single peak. The eluted protein is collected in three fractions with pre-defined criteria
Step 6: Reconditioning
sCEX column purified major fraction (eluted FR 02) is reconditioned in order to tune up with equilibration buffer conditions of the third column step in terms of pH (7.4 ± 0.1) by addition of 2 M Tris-base (to increase the pH), After adjusting the pH of brentuximab protein solution, conductivity of the protein solution is adjusted to 7.4 ± 1.0 mS / cm by addition of WFI (to lower the conductivity of the protein solution), More than 98 % purity of brentuximab is achieved after this column step, as assessed by HP-SEC shown in Figure 6.

Step 7: Anion exchange (AEX) column chromatography
Anion exchange chromatography (AEX) is performed in bind-elute mode for further purification of brentuximab protein, mainly for the removal of residual product-related size species variants and process-related impurities. Post pre-Equilibration, wAEX column matrix is equilibrated with AEX column equilibration buffer (25 mM Tris-Cl + 50 mM NaCl, pH 7.4 ± 0.2, Conductivity 7.4 ± 1.0 mS / cm) by passing three column bed volumes, at which time it attains the desired pH (7.4 ± 0.1) and conductivity (7.4 ± 1.0 mS / cm). Post-equilibration, the reconditioned wAEX column load is loaded (NMT 30 g protein / L of matrix) onto the wAEX column, equilibrated with 25 mM Tris-Cl buffer of pH 7.4 ± 0.1 containing 50 mM NaCl, Conductivity 7.4 ± 1.0 mS / cm. Loading is conducted at a controlled flow rate to maintain a contact time of 5 min between the protein and column matrix. Following loading, the column is washed with the equilibration buffer for 3 column bed volumes at the same flow rate
Post-loading and equilibration buffer wash, the column is washed with Wash I buffer, at a second conductivity 12.0 ± 2.0 mS / cm, higher than the equilibration buffer. Wash I step is performed for three column bed volumes, at the same flow rate, with 25 mM Tris-Cl buffer of pH 7.4 ± 0.1 containing 100 mM NaCl, Conductivity 12.0 ± 2.0 mS / cm. Post-wash I buffer wash, elution of brentuximab protein is carried out at a third higher conductivity 48.0 ± 5.0 mS / cm) with 25 mM Tris-Cl buffer of pH 7.4 ± 0.1 containing 500 mM NaCl, in linear gradient mode of 15 CV. brentuximab protein is observed to elute out of the wAEX column as a single broad peak. The eluted protein is collected in three fractions with pre-defined criteria, purity of brentuximab is observed to be more than 99 %, as assessed by HP-SEC shown in Figure 7.
Step 8: Nano-filtration
After the anion-column step, the protein solution containing the desired monoclonal antibody underwent a nano-filtration step. After nano-filtration, purity of brentuximab is observed to remain more than 99 % as shown in Figure 8.
Step 9: Ultrafiltration-diafiltration
After nano-filtration, protein solution was diafiltered with desired media for the preparation of a purified brentuximab antibody. The purified brentuximab antibody either can be directly used for preparation of ADC (brentuximab vedotin) or can be stored at -25 °C ± 5 °C, and thawed when required for ADC preparation, following reconditioning of the thawed brentuximab with the fresh media.
Example 7: Reduction of the purified brentuximab antibody(Batch No. Z3318B1S013)
brentuximab (12 mg / mL) was subjected to partial reduction (inter-chain S-S cross-links) in the presence of 2.5 molar excess of tris (carboxyethyl) phosphine [TCEP] in 50 mM phosphate buffer, 50 mM NaCl, and 1 mM DTPA, with a pH of 5.8-6.3. for NMT about 300 min. Incubation temperature was maintained at 37 ?C, under stirring condition (200 rpm), for the reaction. pH was maintained between 5.8 to 6.3 during the partial reduction reaction.
Example 8: Conjugation of the reduced CI ( Batch No. Z3318B1S013)
After the reduction step in example 7, the conjugation reaction was carried out in the same or different reaction vessel at 25 ?C temperature, about 6 to 10 molar excess of vc-MMAE (valine-citrulline monomethyl auristatin E) more preferably 8.25 molar excess of vc-MMAE was used relative to the protein, which was dissolved in 100% DMSO or DMAc. However, the final concentration of DMSO or DMAc in the reaction mixture was adjusted to 10% (v/v). The protein concentration was maintained at 10 mg/mL in a reaction buffer containing 50 mM phosphate buffer, 50 mM NaCl, and 1 mM DTPA, with a pH of 5.8-6.3. Incubation was performed under 25 ?C for NMT 120 min, under gentle stirring (200 rpm). At the end of conjugation reaction, the reaction mixture was passed through a 0.2 µm filter and subjected to UF / DF for the removal of unreacted drug-linker molecules and buffer exchange. Post UF / DF step, brentuximab protein solution was formulated in the 20 mM sodium citrate buffer pH 6.6 ± 0.1. + 70 mg / ml a, a trehalose and 0.2 mg / ml polysorbate-80. The formulated bulk of brentuximab vedotin was passed through a 0.2 µm filter (150 cm2 surface area), under bio-safety cabinet, and collected in single-use, sterile, non-pyrogenic polyethylene terephthalate glycol (PETG) bottle with white high-density polyethylene (HDPE) screw closure. The filtered formulated bulk was labeled as brentuximab vedotin. Antibody Drug conjugated was subjected to DAR analysis as shown in figure 11. The purity of brentuximab vedotin was observed to be more than 97 %, as shown in Figure 10.
Example 9: Reduction of the purified brentuximab antibody (Batch No. Z3318B1S013)
Brentuximab protein concentration was maintained at 12 mg/mL in a reaction buffer containing 50 mM phosphate buffer, 250 mM NaCl, and 1 mM DTPA, with a pH of 5.8-6.3 was subjected to partial reduction (inter-chain S-S cross-links) in the presence of 2.5 molar excess of tris (carboxyethyl) phosphine [TCEP] for NMT about 300 min. Incubation temperature was maintained at 37 ?C, under stirring condition (200 rpm), for the reaction. pH was maintained between 5.8 to 6.3 during the partial reduction reaction.
Example 10: Conjugation of the reduced CI (Batch No. Z3318B1S013)
After the reduction step in example 9, the conjugation reaction was carried out in the same or different reaction vessel at 25 ?C temperature. About 6 to 10 molar excess of vc-MMAE (valine-citrulline monomethyl auristatin E) more preferably 8.25 molar excess of vc-MMAE was used relative to the protein, which was dissolved in 100% DMSO or DMAc. However, the final concentration of DMSO or DMAc in the reaction mixture was adjusted to 10% (v/v). The protein concentration was maintained at 10 mg/mL in a reaction buffer containing 50 mM phosphate buffer, 250 mM NaCl, and 1 mM DTPA, with a pH of 5.8-6.3. Incubation was performed under 25 ?C for NMT 120 min, under gentle stirring (200 rpm). At the end of conjugation reaction, the reaction mixture was passed through a 0.2 µm filter and subjected to UF / DF for the removal of unreacted drug-linker molecules and buffer exchange. Post UF / DF step, brentuximab protein solution was formulated in the 20 mM sodium citrate buffer pH 6.6 ± 0.1. + 70 mg / ml a, a trehalose and 0.2 mg / ml polysorbate-80. The formulated bulk of brentuximab vedotin was passed through a 0.2 µm filter (150 cm2 surface area), under bio-safety cabinet, and collected in single-use, sterile, non-pyrogenic polyethylene terephthalate glycol (PETG) bottle with white high-density polyethylene (HDPE) screw closure. The filtered formulated bulk was labeled as brentuximab vedotin. Antibody Drug conjugated was subjected to DAR analysis as shown in figure 12.
The purity of brentuximab vedotin was observed to be more than 97 %, as shown in Figure 10.
Example 11: Conjugation of the reduced CI (Batch No. Z3318B1S013)
After the reduction step in example 9, the conjugation reaction was carried out in the same or different reaction vessel at 37 ?C temperature. About 6 to 10 molar excess of vc-MMAE (valine-citrulline monomethyl auristatin E) more preferably 8.25 molar excess of vc-MMAE was used relative to the protein, which was dissolved in 100% DMSO or DMAc. However, the final concentration of DMSO or DMAc in the reaction mixture was adjusted to 10% (v/v). The protein concentration was maintained at 10 mg/mL in a reaction buffer containing 50 mM phosphate buffer, 250 mM NaCl, and 1 mM DTPA, with a pH of 5.8-6.3. Incubation was performed under 25 ?C for NMT 120 min, under gentle stirring (200 rpm). At the end of conjugation reaction, the reaction mixture was passed through a 0.2 µm filter and subjected to UF / DF for the removal of unreacted drug-linker molecules and buffer exchange. Post UF / DF step, brentuximab protein solution was formulated in the 20 mM sodium citrate buffer pH 6.6 ± 0.1. + 70 mg / ml a, a trehalose and 0.2 mg / ml polysorbate-80. The formulated bulk of brentuximab vedotin was passed through a 0.2 µm filter (150 cm2 surface area), under bio-safety cabinet, and collected in single-use, sterile, non-pyrogenic polyethylene terephthalate glycol (PETG) bottle with white high-density polyethylene (HDPE) screw closure. The filtered formulated bulk was labeled as brentuximab vedotin. Antibody Drug conjugated was subjected to DAR analysis as shown in figure 13.
The purity of brentuximab vedotin was observed to be more than 97 %, as shown in Figure 10.

Results and discussion
The purity of brentuximab vedotin obtained using the method of preparation according to the present invention is more than 97 % as shown in Figure 4 and Table 1. Figure 3 shows purity of brentuximab to be more than 99 % after performing purification steps comprising rPA chromatography, AEX chromatography, CEX chromatography and non-chromatography steps. This shows that the method of preparing brentuximab vedotin according to the present invention is a robust, cheaper, scalable and eco-friendly process that gives improved purity.

Table 1: Purity of brentuximab and brentuximab vedotin
Sr. No. Name of sample (Batch No. Z3318T1008) % Principal peak
1. rPA eluate of brentuximab 97.59
AEX eluate of brentuximab 99.41
CEX eluate of brentuximab 99.33
2. brentuximab vedotin 97.56

The purity of brentuximab vedotin obtained using the method of preparation according to the present invention is more than 97 % as shown in Figure 10 and Table 2. Figure 5 to Figure 9 shows purity of brentuximab to be more than 99 % after performing purification steps comprising rPA chromatography, sCEX chromatography, AEX chromatography and non-chromatography steps. This shows that the method of preparing brentuximab vedotin according to the present invention is a robust, cheaper, scalable and eco-friendly process that gives improved purity. The recovery or yield of brentuximab vedotin according to the process of the present invention was obtained about more than 85 %, in particular about 88 %.

Table 2: Purity of brentuximab and brentuximab vedotin
Sr. No. Name of sample (Batch No. Z3318B1S018) % Principal peak
1. rPA eluate of brentuximab 98.02
sCEX eluate of brentuximab 98.81
AEX eluate of brentuximab 99.66
Nano filtrate of brentuximab 99.57
Critical intermediate (CI), of brentuximab 99.44
2. brentuximab vedotin 97.14

Incorporation by reference
The entire disclosure of each of the patent documents and scientific articles referred to herein is incorporated by reference for all purposes.
Equivalents
The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting the invention described herein. Scope of the invention is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

References incorporated in current patent application:
[1] WO2005084390
[2] Han, J C, and G Y Han. “A procedure for quantitative determination of tris(2-carboxyethyl)phosphine, an odorless reducing agent more stable and effective than dithiothreitol.” Analytical biochemistry vol. 220,1 (1994): 5-10. doi:10.1006/abio.1994.1290
[3] Certificate of compliance for Adcetris®
[4] WHO Drug Information, Vol. 24, No. 2, 2010 Recommended INN: List 103 International Non-proprietary Names for Pharmaceutical Substances (INN).
[5] https://www.accessdata.fda.gov/drugsatfda_docs/label/2025/125388Orig1s108lbl.pdf.
[6] Parau M, Pullen J, Bracewell DG. Depth filter material process interaction in the harvest of mammalian cells. Biotechnol Prog. 2023 May-Jun;39(3):e3329. doi: 10.1002/btpr.3329. Epub 2023 Feb 23. PMID: 36775837; PMCID: PMC10909467.
[7] Karoline Bechtold-Peters, Stephen Chang, Andrew C. Lennard, Jeanne Mateffy, Marie Murphy, Melvyn Perry, David Roesti, Donald C. Singer, Friedrich von Wintzingerode, Harry Yang,
Risk-based approach to setting sterile filtration microbial bioburden limits – Focus on biotech-derived products, European Journal of Pharmaceutics and Biopharmaceutics, Volume 198,2024,114151,ISSN 0939-6411.
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,CLAIMS:We Claim,
1. A method of preparing brentuximab vedotin comprising;
(a) partial reduction of the brentuximab antibody with suitable reducing agent in suitable buffer and at suitable pH;
(b) conjugation of the reduced brentuximab antibody obtained from step (a) with the drug conjugate mc-vc-PABC-MMAE in same buffer and at same pH as used in step (a).
2. The method of preparing brentuximab vedotin as claimed in claim 1 further comprising purification of brentuximab before subjecting to step (a) of claim 1 wherein purification of brentuximab does not involve phenyl membrane filtration and does not involve pH adjustment or buffer exchange or removal of any excipients present in the brentuximab antibody solution prior to the process as claimed in claim 1.
3. The method of preparing brentuximab vedotin as claimed in claim 2 wherein purification of the brentuximab comprising chromatography and non-chromatography steps, wherein chromatography comprises protein A chromatography (rPA), cation exchange chromatography and anion exchange chromatography, either alone or in combination.
4. The method of preparing brentuximab vedotin as claimed in claim 3 wherein protein A chromatography (rPA) comprises three column wash steps prior to elution, wherein first wash with equilibration buffer at suitable pH and / or conductivity, second wash at the same pH as of the first wash buffer and /or a conductivity higher than the first wash buffer and third wash at a pH and / or a conductivity lower than the second wash buffer and elution of brentuximab at lower pH and / or higher conductivity than third wash buffer.
5. The method of preparing brentuximab vedotin as claimed in claim 4 wherein protein A chromatography (rPA) comprises three column wash steps prior to elution, wherein first wash with equilibration buffer at about pH 7.2 to pH 7.6 and / or conductivity of about 5 mS / cm to 30 mS / cm, second wash at about pH 7.2 to pH 7.6 and / or a conductivity of about 75 mS / cm to 100 mS / cm, third wash at a pH of about 5.0 to pH 6.5 and / or a conductivity of about 1 mS / cm to 5 mS / cm and elution of brentuximab at pH about 3.3 to pH about 3.7 and / or conductivity of about 6 mS / cm to 30 mS/cm.
6. The method of preparing brentuximab vedotin as claimed in claim 5 wherein protein A chromatography (rPA) comprises three column wash steps prior to elution, wherein first wash with equilibration buffer at about pH 7.4 and / or conductivity of about 17.7 mS / cm, second wash at about pH 7.4 and / or a conductivity of about 89 mS / cm, third wash at a pH of about 5.4 and / or a conductivity of about 3.9 mS / cm and elution of brentuximab at pH about 3.5 and / or conductivity of about 13.7 mS / cm.
7. The method of preparing brentuximab vedotin as claimed in claim 3 wherein purification of the brentuximab comprising chromatography and non-chromatography steps, wherein chromatography comprises protein A chromatography (rPA), cation exchange chromatography (CEX) and anion exchange chromatography (AEX) wherein rPA, CEX and AEX can be performed in any order.
8. The method of preparing brentuximab vedotin as claimed in claim 1 wherein reducing agent is selected from Tris (2-carboxyethyl) phosphine (TCEP) or Dithiothreitol (DTT) and buffer is selected from phosphate buffer, citrate buffer, succinate buffer, borate buffer or citrate-phosphate buffer or suitable combinations thereof at about pH between 5.8 and 6.3 and wherein conjugation is performed at temperature between 25 °C and 30 °C.
9. The method of preparing brentuximab vedotin as claimed in any preceding claim comprising;
a) Preparation of brentuximab antibody wherein brentuximab is purified by rPA, CEX and AEX as claimed in claim 3,
b) partial reduction of the brentuximab antibody obtained from step (a) with a reducing agent TCEP in phosphate buffer and at about pH 5.8 to pH 6.3 wherein partial reduction is performed at temperature about 15 °C to 40 °C,
c) conjugation of the reduced brentuximab antibody obtained from step (b) with mc-vc-PABC-MMAE drug conjugate in phosphate buffer and at about pH 5.8 to pH 6.3 wherein conjugation is performed at temperature about 4°C to 37°C and wherein mc-vc-PABC-MMAE drug conjugate kept dissolved in 5 % to 30% Dimethyl sulfoxide (DMSO) or Dimethylacetamide (DMAC).
10. The method of preparing brentuximab vedotin as claimed in any preceding claim, comprises;
a) Preparation of brentuximab antibody; wherein the preparation step comprises;
(i) clarification of harvested cell culture fluid (HCCF) by centrifugation and/ or depth filtration;
(ii) reconditioning of the clarified cell culture fluid (CCCF), optionally ;
(iii) loading and washing on a Protein A (rPA) column, wherein the column wash steps comprises;
i. First wash with equilibration buffer at about pH 7.4 and/or conductivity of about 17.7 mS/cm.
ii. Second wash at about pH 7.4 and/or a conductivity of about 89 mS/cm.
iii. Third wash at a pH of about 5.4 and/or a conductivity of about 3.9 mS/cm.
(iv) Elution of an antibody at pH about 3.5 and/or conductivity of about 13.7 mS/cm. viral inactivation at pH 3.5 to 4 and its followed by pH neutralization;
(v) performing cation exchange chromatography with the antibody obtained in step (iv)
(vi) performing anion exchange chromatography with the antibody obtained in step (v);
(vii) virus clearance of antibody obtained in step (vi) by nano-filtration;
(viii) terminal filtration; and optionally
(ix) reconditioning by UF/DF before steps (v), (vi) and (viii);
b) Partial reduction of the antibody produced in step (a) with TCEP;
c) Conjugation of reduced antibody obtained from step (b) with vc-MMAE drug conjugate kept dissolved in 5% to 20% DMSO or DMAc;
d) Removal of unreacted drug conjugate formed in step (c) with UF/DF;
e) Terminal filtration of the brentuximab vedotin obtained from step (d).
11. The method as claimed in any preceding claims, wherein the preparation of brentuximab vedotin comprises;
a) Preparation of brentuximab antibody; wherein the preparation step comprises (i) clarification of harvested cell culture fluid (HCCF) by centrifugation and/or depth filtration; (ii) reconditioning of the clarified cell culture fluid (CCCF); (iii) loading and washing on a recombinant Protein A (rPA) column, wherein CCCF is loaded and washed on a recombinant Protein A (rPA) column:
i. The first wash uses an equilibration buffer at approximately pH 7.4 and/or a conductivity of about 17.7 mS/cm, comprising 25 mM Tris-Cl buffer with 150 mM NaCl.
ii. The second wash is at about pH 7.4 and/or a conductivity of about 89 mS/cm, with 25 mM Tris-Cl buffer containing 1M NaCl.
iii. The third wash occurs at a pH of about 5.4 and/or a conductivity of about 3.9 mS/cm, using 25 mM Sodium citrate .
iv. The antibody is then eluted at pH about 3.5 and/or a conductivity of about 13.7 mS/cm, using 25 mM Sodium citrate with /and without 100 mM NaCl.
(iv) viral inactivation at pH lower than that of rPA eluate and its pH neutralization; (v) The fraction obtained in (iv) is subjected to cation exchange chromatography using a DEAE column at pH 4.5 to 5.5, with a conductivity of 2 to 4 mS/cm, wherein the Equilibration buffer comprises 20 mM Sodium citrate at pH 5.5, while the elution buffer consists of 20 mM Sodium acetate at pH 5.5 with 200 mM NaCl and a conductivity of 20 to 25 mS/cm; (vi) performing anion exchange chromatography obtained in step (v) at pH 7.5, with a conductivity of 6.5 to 8.5 mS/cm, using an equilibration buffer of 25 mM Tris-Cl with 50 mM NaCl (Tris pH 7.5) and an elution buffer of 25 mM Tris-Cl at pH 7.3 to 7.5 with 500 mM NaCl on a DEAE column, achieving a conductivity of 45 to 50 mS/cm, (vii) virus clearance of fraction obtained in step (vi) by nano-filtration; (viii) terminal filtration; and (ix) reconditioning by UF/DF before steps (v), (vi) and (viii), Depth filtration is performed using NaCl concentration of 150mM;
b) Partial reduction of the antibody produced in step (a) with about 2.5 molar excess TCEP wherein the concentration of NaCl is in the range of 0- 50mM and pH is in the range of 5.8-6.3;
c) Conjugation of reduced antibody obtained from step (b) with vc-MMAE drug conjugate kept dissolved in 5% to 20% DMSO or DMAc;
d) Removal of unreacted drug conjugate formed in step (c) with UF/DF;
e) Terminal filtration of the brentuximab vedotin obtained from step (d).
12. The method as claimed in any preceding claims comprises brentuximab vedotin wherein the purity of brentuximab vedotin is more than 97 % and recovery yield of brentuximab vedotin is more than 85 %.