DESC:RELATED APPLICATIONS
This application is related to Indian provisional application IN202321036862 filed 29th May. 2023 and is incorporated herein in its entirety
FIELD OF THE INVENTION
The present invention relates to method for conjugation and purification of antibody drug conjugate wherein, the method controls drug to antibody ratio (DAR) species distribution of antibody drug conjugate, such as Trastuzumab emtansine.
BACKGROUND OF THE INVENTION
Antibody drug conjugates (ADC) are an emerging class of potent anti-cancer agents, which have recently demonstrated remarkable clinical benefit. ADCs are comprised of a cytotoxic agent attached to an antibody via a stable linker. Putatively, by a series of events, including antigen binding at the cell surface, endocytosis, trafficking to the lysosome, ADC degradation, release of payload, interruption of cellular processing (e.g. mitosis) and apoptosis, ADCs may destroy cancer cells possessing an over-expression of cell-surface proteins. ADCs combine the antigen-driven targeting properties of monoclonal antibodies with the potent anti-tumor effects of cytotoxic agents.
Trastuzumab emtansine (TDM1) is a full-length recombinant humanised IgG1 monoclonal antibody (mAb), Trastuzumab, conjugated with cytotoxic drug, DM1, via SMCC linker. This antibody drug conjugate (ADC) is used for the treatment of breast cancer with metastasized HER 2+ cells. TDM1 binds to the subdomain IV of HER 2 receptor through Trastuzumab mAb. After its internalization, it is degraded which results in release of DM1 which binds to microtubules of the targeted cells and thus inhibits their proliferation.
Studies have demonstrated deleterious effects of a high drug loaded ADCs. These deleterious effects of higher levels of conjugation include increased propensity towards aggregate formation.
Controlling the drug load of an ADC has been attempted using various methods, including: (i) limiting the molar excess of drug-linker intermediate or linker reagent relative to antibody, (ii) limiting the conjugation reaction time or temperature, and . While reduction methods that limit the number of attachment sites on the antibody have been used to achieve ADCs with fewer drugs per antibody, there remains a need for methods and compositions that can provide optimal drug loaded species.
OBJECTS OF THE INVENTION
The main object of the present invention is to provide a method for purification of an antibody drug conjugate, wherein the method comprises hydrophobic interaction chromatography operated in flow through mode.
Another object of the present invention is to provide a method for purification of an antibody drug conjugate, wherein the method comprises steps of:
a) Antibody DS thawing;
b) Carboxypeptidase B (CpB) treatment;
c) Tangential flow filtration – I;
d) Conjugation reaction;
e) Tangential flow filtration – II;
f) Hydrophobic interaction chromatography (HIC);
g) Tangential flow filtration – III; and
h) 0.2 µm filtration.
Another object of the present invention is to provide a method for purification of an antibody drug conjugate, wherein the method comprises steps of:
a) Antibody DS thawing;
b) Carboxypeptidase B (CpB) treatment;
c) Tangential flow filtration – I;
d) Conjugation reaction;
e) Tangential flow filtration – II;
f) Hydrophobic interaction chromatography (HIC) wherein, HIC is operated in flow through mode;
g) Tangential flow filtration – III; and
h) 0.2 µm filtration.
Another object of the present invention is to provide a method for purification of Trastuzumab Emtansine, wherein the method comprises steps of:
a) Trastuzumab DS thawing;
b) Carboxypeptidase B (CpB) treatment;
c) Tangential flow filtration – I;
d) Conjugation reaction;
e) Tangential flow filtration – II;
f) Hydrophobic interaction chromatography (HIC) wherein, HIC is operated in flow through mode;
g) Tangential flow filtration – III; and
h) 0.2 µm filtration.
Another object of the present invention is to provide a method for purification of Trastuzumab Emtansine, wherein the method comprises steps of:
a) Trastuzumab DS thawing;
b) Carboxypeptidase B (CpB) treatment;
c) Tangential flow filtration – I;
d) Conjugation reaction;
e) Tangential flow filtration – II;
f) Hydrophobic interaction chromatography (HIC) wherein, HIC is operated in flow through mode with phosphate and sodium chloride (NaCl) as a buffer system;
g) Tangential flow filtration – III; and
h) 0.2 µm filtration.
Another object of the present invention is to provide a method for purification of Trastuzumab Emtansine, wherein the method comprises steps of:
a) Trastuzumab DS thawing;
b) Carboxypeptidase B (CpB) treatment;
c) Tangential flow filtration – I;
d) Conjugation reaction wherein, the conjugation reaction is carried out at pH about 6 to 8 for about 2 to 10hours with organic solvent DMSO at about 10-20% v/v.
e) Tangential flow filtration – II;
f) Hydrophobic interaction chromatography (HIC) wherein, HIC is operated in flow through mode with phosphate and sodium chloride (NaCl) as a buffer system;
g) Tangential flow filtration – III; and
h) 0.2 µm filtration.
Another object of the present invention is to provide a method for purification of Trastuzumab Emtansine, wherein the method comprises hydrophobic interaction chromatography operated on flow through mode comprising steps of:
a) Equilibration with phosphate and sodium chloride buffer system
b) Loading of Trastuzumab Emtansine
c) Collection of purified Trastuzumab Emtansine in flow through mode, 10 mAU onwards (ascending; 2 mm path length chromatography system) to 3-5 CV of post load wash
d) Post load wash with equilibration buffer
e) Regeneration with water
f) Sanitization with sodium hydroxide
Another object of the present invention is to provide a method for preparing an antibody maytansinoid conjugate comprising the steps of:
contacting a linker with a maytansinoid to form a first mixture comprising the linker and the maytansinoid, then contacting the first mixture with an antibody, in a solution having about 10-20% v/v of organic solvent DMSO at pH of about 6 to about 8, for about 2 to 10hours to provide a second mixture comprising (i) the antibody maytansinoid conjugate, wherein the antibody is chemically coupled through the linker to the maytansinoid, (ii) free maytansinoid, and (iii) reaction by-products.
Another object of the present invention is to provide a method for preparing Trastuzumab Emtansine comprising the steps of:
contacting a SMCC linker with a DM1 to form a first mixture comprising the SMCC and the DM1, then contacting the first mixture with an Trastuzumab, in a solution having about 10-20% v/v of organic solvent DMSO at pH of about 6 to about 8, for about 2 to 10 hours to provide a second mixture comprising (i) the Trastuzumab Emtansine, wherein the Trastuzumab is chemically coupled through the SMCC to DM1, (ii) free DM1, and (iii) reaction by-products.
Another object of the present invention is to provide a method for preparing an antibody drug conjugate comprising the steps of:
(a) contacting a drug with a bifunctional crosslinking reagent to covalently attach a linker to the drug and thereby prepare a first mixture comprising drug having linkers bound thereto,
(b) optionally, subjecting the first mixture to tangential flow filtration, selective precipitation, adsorptive filtration, or an adsorptive chromatography resin and thereby prepare a purified first mixture of cell-binding agents having linkers bound thereto,
(c) conjugating an antibody to the drug having linkers bound thereto in the purified first mixture by reacting the drug having linkers bound thereto with an antibody in a solution having about 10-20% v/v of organic solvent DMSO at pH of about 6 to about 8, for about 2 to 10 hours to prepare a second mixture comprising (i) antibody chemically coupled through the linker to the drug, (ii) free drug, and (iii) reaction by-products, and
(d) subjecting the second mixture to a tangential flow filtration, selective precipitation, adsorptive filtration, or an adsorptive chromatography resin to purify the antibody chemically coupled through the linkers to the drug from the other components of the second mixture and thereby obtain final purified antibody drug conjugate.
SUMMARY OF THE INVENTION
The main aspect of the present invention is to provide a method for purification of an antibody drug conjugate, wherein the method comprises hydrophobic interaction chromatography operated in flow through mode.
Another aspect of the present invention is to provide a method for purification of an antibody drug conjugate, wherein the method comprises steps of:
a) Antibody DS thawing;
b) Carboxypeptidase B (CpB) treatment;
c) Tangential flow filtration – I;
d) Conjugation reaction;
e) Tangential flow filtration – II;
f) Hydrophobic interaction chromatography (HIC);
g) Tangential flow filtration – III; and
h) 0.2 µm filtration.
Another aspect of the present invention is to provide a method for purification of an antibody drug conjugate, wherein the method comprises steps of:
a) Antibody DS thawing;
b) Carboxypeptidase B (CpB) treatment;
c) Tangential flow filtration – I;
d) Conjugation reaction;
e) Tangential flow filtration – II;
f) Hydrophobic interaction chromatography (HIC) wherein, HIC is operated in flow through mode;
g) Tangential flow filtration – III; and
h) 0.2 µm filtration.
Another object of the present invention is to provide a method for purification of Trastuzumab Emtansine, wherein the method comprises steps of:
a) Trastuzumab DS thawing;
b) Carboxypeptidase B (CpB) treatment;
c) Tangential flow filtration – I;
d) Conjugation reaction;
e) Tangential flow filtration – II;
f) Hydrophobic interaction chromatography (HIC) wherein, HIC is operated in flow through mode;
g) Tangential flow filtration – III; and
h) 0.2 µm filtration.
Another aspect of the present invention is to provide a method for purification of Trastuzumab Emtansine, wherein the method comprises steps of:
a) Trastuzumab DS thawing;
b) Carboxypeptidase B (CpB) treatment;
c) Tangential flow filtration – I;
d) Conjugation reaction;
e) Tangential flow filtration – II;
f) Hydrophobic interaction chromatography (HIC) wherein, HIC is operated in flow through mode with phosphate and sodium chloride (NaCl) as a buffer system;
g) Tangential flow filtration – III; and
h) 0.2 µm filtration.
Another aspect of the present invention is to provide a method for purification of Trastuzumab Emtansine, wherein the method comprises steps of:
a) Trastuzumab DS thawing;
b) Carboxypeptidase B (CpB) treatment;
c) Tangential flow filtration – I;
d) Conjugation reaction wherein, the conjugation reaction is carried out at pH about 6 to 8 for about 6 to 8 hours with organic solvent DMSO at about 12-18% v/v;
e) Tangential flow filtration – II;
f) Hydrophobic interaction chromatography (HIC) wherein, HIC is operated in flow through mode with phosphate and sodium chloride (NaCl) as a buffer system;
g) Tangential flow filtration – III; and
h) 0.2 µm filtration.
Another aspect of the present invention is to provide a method for purification of Trastuzumab Emtansine, wherein the method comprises hydrophobic interaction chromatography operated on flow through mode comprising steps of:
a) Equilibration with phosphate and sodium chloride buffer system
b) Loading of Trastuzumab Emtansine
c) Collection of purified Trastuzumab Emtansine in flow through mode, 10 mAU onwards (ascending; 2 mm path length chromatography system) to 3-5 CV of post load wash
d) Post load wash with equilibration buffer
e) Regeneration with water
f) Sanitization with sodium hydroxide
Another aspect of the present invention is to provide a method for preparing an antibody maytansinoid conjugate comprising the steps of:
contacting a linker with a maytansinoid to form a first mixture comprising the linker and the maytansinoid, then contacting the first mixture with an antibody, in a solution having about 10-20% v/v of organic solvent DMSO at pH of about 6 to about 8, for about 2 to 10 hours to provide a second mixture comprising (i) the antibody maytansinoid conjugate, wherein the antibody is chemically coupled through the linker to the maytansinoid, (ii) free maytansinoid, and (iii) reaction by-products.
Another aspect of the present invention is to provide a method for preparing Trastuzumab Emtansine comprising the steps of:
contacting a SMCC linker with a DM1 to form a first mixture comprising the SMCC and the DM1, then contacting the first mixture with an Trastuzumab, in a solution having about 10-20% v/v of organic solvent DMSO at pH of about 6 to about 8, for about 2 to 10 hours to provide a second mixture comprising (i) the Trastuzumab Emtansine, wherein the Trastuzumab is chemically coupled through the SMCC to DM1, (ii) free DM1, and (iii) reaction by-products.
Another aspect of the present invention is to provide a method for preparing an antibody drug conjugate comprising the steps of:
(a) contacting a drug with a bifunctional crosslinking reagent to covalently attach a linker to the drug and thereby prepare a first mixture comprising drug having linkers bound thereto,
(b) optionally, subjecting the first mixture to tangential flow filtration, selective precipitation, adsorptive filtration, or an adsorptive chromatography resin and thereby prepare a purified first mixture of cell-binding agents having linkers bound thereto,
(c) conjugating an antibody to the drug having linkers bound thereto in the purified first mixture by reacting the drug having linkers bound thereto with an antibody in a solution having about 10-20% v/v of organic solvent DMSO at pH of about 6 to about 8, for about 2 to 10 hours to prepare a second mixture comprising (i) antibody chemically coupled through the linker to the drug, (ii) free drug, and (iii) reaction by-products, and
(d) subjecting the second mixture to a tangential flow filtration, selective precipitation, adsorptive filtration, or an adsorptive chromatography resin to purify the antibody chemically coupled through the linkers to the drug from the other components of the second mixture and thereby obtain final purified antibody drug conjugate.
BRIEF DESCRIPTION OF DRAWINGS
In order that disclosure may be readily understood and put into practical effect, reference will now be made to exemplary embodiments as illustrated with reference to the accompanying figures. The figure with a detailed description below, are incorporated in and form part of the specification, and serve to further illustrate the embodiments and explain various principles and advantages, in accordance with the present disclosure wherein:
Figure - 1: Process flow of TDM1
Figure - 2: Representative chromatogram of HIC in Flow through mode
Figure - 3: Representative TIC of DS with DAR species distribution
Figure - 4: Representative image of static mixer with DAR species distribution
Figure - 5: Conjugation reaction process flow diagram using static mixer.
Figure - 6: DAR distribution post static mixer of the conjugation reaction (A)-5L scale, (B) – 18L scale and (C) – 50L scale.
Figure - 7: DAR distribution post static mixer of the conjugation reaction (A)-5L scale, (B) – 18L scale and (C) – 50L scale.
Figure - 8: TDM1 HIC purification profile -5L scale.
Figure - 9: TDM1 HIC purification profile -18L scale.
Figure - 10: TDM1 HIC purification profile -50L scale.
DETAILED DESCRIPTION OF THE INVENTION
The following is a detailed description of embodiments of the invention. The embodiments are in such details as to clearly communicate the invention. However, the amount of details offered is not intended to limit the anticipated variations of embodiments; on the contrary, the intention is to cover all modifications, equivalents of embodiments, and alternative falling within the spirit and scope of the present invention.

DEFINITION
The following definitions are provided to facilitate understanding of certain terms used throughout the specification.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of particular embodiments, preferred embodiments of compositions, methods and materials are described herein. For the purposes of the present disclosure, the following terms are defined below.
The articles "a," "an," and "the" are used herein to refer to one or to more than one (i.e., to at least one, or to one or more) of the grammatical object of the article. By way of example, "an element" means one element or one or more elements.
The term "about" as used in the present patent specification is meant to specify that the specific value provided may vary to a certain extent.
The words "comprise", "comprises", and "comprising" are to be interpreted inclusively rather than exclusively. The words "consist", "consisting", and its variants, are to be interpreted exclusively, rather than inclusively. While various embodiments in the specification are presented using “comprising” language, under other circumstances, a related embodiment is also intended to be interpreted and described using “consisting of’ or “consisting essentially of’ language.
The term "antibody-drug-conjugate" or "ADC" refers to a binding protein, such as an antibody or antigen binding fragment thereof, chemically linked to one or more chemical drug(s) (also referred to herein as agent(s)) that may optionally be therapeutic or cytotoxic agents. In a preferred embodiment, an ADC includes an antibody, a cytotoxic or therapeutic drug, and a linker that enables attachment or conjugation of the drug to the antibody. An ADC typically has anywhere from 1 to 8 drugs conjugated to the antibody, including drug loaded species of 2, 4, 6, or 8. Non-limiting examples of drugs that may be included in the ADCs are mitotic inhibitors, antitumor antibiotics, immunomodulating agents, vectors for gene therapy, alkylating agents, antiangiogenic agents, antimetabolites, boron-containing agents, chemoprotective agents, hormones, antihormone agents, corticosteroids, photoactive therapeutic agents, oligonucleotides, radionuclide agents, topoisomerase inhibitors, tyrosine kinase inhibitors, and radiosensitizers.
The main embodiment of the present invention is to provide a method for purification of an antibody drug conjugate, wherein the method comprises hydrophobic interaction chromatography operated in flow through mode.
Another embodiment of the present invention is to provide a method for purification of an antibody drug conjugate, wherein the method comprises steps of:
a) Antibody DS thawing;
b) Carboxypeptidase B (CpB) treatment;
c) Tangential flow filtration – I;
d) Conjugation reaction;
e) Tangential flow filtration – II;
f) Hydrophobic interaction chromatography (HIC);
g) Tangential flow filtration – III; and
h) 0.2 µm filtration.
Another embodiment of the present invention is to provide a method for purification of an antibody drug conjugate, wherein the method comprises steps of:
a) Antibody DS thawing;
b) Carboxypeptidase B (CpB) treatment;
c) Tangential flow filtration – I;
d) Conjugation reaction;
e) Tangential flow filtration – II;
f) Hydrophobic interaction chromatography (HIC) wherein, HIC is operated in flow through mode;
g) Tangential flow filtration – III; and
h) 0.2 µm filtration.
Another embodiment of the present invention is to provide a method for purification of Trastuzumab Emtansine, wherein the method comprises steps of:
a) Carboxypeptidase B (CpB) treatment;
b) Tangential flow filtration – I;
c) Conjugation reaction;
d) Tangential flow filtration – II;
e) Hydrophobic interaction chromatography (HIC) wherein, HIC is operated in flow through mode;
f) Tangential flow filtration – III; and
g) 0.2 µm filtration.
In another embodiment of the present invention HIC is operated in flow through mode with buffer system selected from the group of buffer: phosphate, acetate, tris, MOPS, MES, HEPES & NaCl, KCl, MgSO4, NH4SO4, and K2HPO4.
Another embodiment of the present invention is to provide a method for purification of Trastuzumab Emtansine, wherein the method comprises steps of:
a) Trastuzumab DS thawing;
b) Carboxypeptidase B (CpB) treatment;
c) Tangential flow filtration – I;
d) Conjugation reaction;
e) Tangential flow filtration – II;
f) Hydrophobic interaction chromatography (HIC) wherein, HIC is operated in flow through mode with phosphate and sodium chloride (NaCl) as a buffer system;
g) Tangential flow filtration – III; and
h) 0.2 µm filtration.
Another embodiment of the present invention is to provide a method for purification of Trastuzumab Emtansine, wherein the method comprises steps of:
a) Trastuzumab DS thawing;
b) Carboxypeptidase B (CpB) treatment;
c) Tangential flow filtration – I;
d) Conjugation reaction wherein, the conjugation reaction is carried out at pH about 6 to 8 for about 6 to 8 hours with organic solvent DMSO at about 12-18% v/v;
e) Tangential flow filtration – II;
f) Hydrophobic interaction chromatography (HIC) wherein, HIC is operated in flow through mode with phosphate and sodium chloride (NaCl) as a buffer system;
g) Tangential flow filtration – III; and
h) 0.2 µm filtration.
Another embodiment of the present invention is to provide a method for purification of Trastuzumab Emtansine, wherein the method comprises hydrophobic interaction chromatography operated on flow through mode comprising steps of:
a) Equilibration with phosphate and sodium chloride buffer system
b) Loading of Trastuzumab Emtansine
c) Collection of purified Trastuzumab Emtansine in flow through mode, 10 mAU onwards (ascending; 2 mm path length chromatography system) to 3-5 CV of post load wash
d) Post load wash with equilibration buffer
e) Regeneration with water
f) Sanitization with sodium hydroxide
Another embodiment of the present invention is to provide a method for preparing an antibody maytansinoid conjugate comprising the steps of:
contacting a linker with a maytansinoid to form a first mixture comprising the linker and the maytansinoid, then contacting the first mixture with an antibody, in a solution having about 10-20% v/v of organic solvent DMSO at pH of about 6 to about 8, for about 2 to 10 hours to provide a second mixture comprising (i) the antibody maytansinoid conjugate, wherein the antibody is chemically coupled through the linker to the maytansinoid, (ii) free maytansinoid, and (iii) reaction by-products.
Another embodiment of the present invention is to provide a method for preparing Trastuzumab Emtansine comprising the steps of:
contacting a SMCC linker with a DM1 to form a first mixture comprising the SMCC and the DM1, then contacting the first mixture with an Trastuzumab, in a solution having about 10-20% v/v of organic solvent DMSO at pH of about 6 to about 8, for about 2 to 10 hours to provide a second mixture comprising (i) the Trastuzumab Emtansine, wherein the Trastuzumab is chemically coupled through the SMCC to DM1, (ii) free DM1, and (iii) reaction by-products.
Another embodiment of the present invention is to provide a method for preparing an antibody drug conjugate comprising the steps of:
(a) contacting a drug with a bifunctional crosslinking reagent to covalently attach a linker to the drug and thereby prepare a first mixture comprising drug having linkers bound thereto,
(b) optionally, subjecting the first mixture to tangential flow filtration, selective precipitation, adsorptive filtration, or an adsorptive chromatography resin and thereby prepare a purified first mixture of cell-binding agents having linkers bound thereto,
(c) conjugating an antibody to the drug having linkers bound thereto in the purified first mixture by reacting the drug having linkers bound thereto with an antibody in a solution having about 10-20% v/v of organic solvent DMSO at pH of about 6 to about 8, for about 2 to 10 hours to prepare a second mixture comprising (i) antibody chemically coupled through the linker to the drug, (ii) free drug, and (iii) reaction by-products, and
(d) subjecting the second mixture to a tangential flow filtration, selective precipitation, adsorptive filtration, or an adsorptive chromatography resin to purify the antibody chemically coupled through the linkers to the drug from the other components of the second mixture and thereby obtain final purified antibody drug conjugate.
In another embodiment of the present invention organic solvent DMSO in the conjugation reaction is used at concentration preferably about 10-18 % v/v, preferably about 12-15 % v/v more preferably about 10-12 % v/v.
The embodiments of the present invention are further described using specific examples herein after. The examples are provided for better understanding of certain embodiments of the invention and not, in any manner, to limit the scope thereof. Possible modifications and equivalents apparent to those skilled in the art using the teachings of the present description and the general art in the field of the invention shall also from the part of this specification and are intended to be included within the scope of it.
EXAMPLES:
Example 1: Conjugation process
TDM1 was produced by performing conjugation reaction at 5L reaction scale, where Trastuzumab was conjugated with SMCC-DM1 (linker-drug/ payload) in a single step reaction in the presence of an organic solvent, DMSO. This reaction was performed at room temperature by controlling various parameters such as pH, DMSO concentration, protein concentration, and drug: antibody molar ratio. All reaction components were split in two parts, A and B, and passed through a static mixer to ensure homogenous mixing. Static mixer was operated at a constant Reynolds’s number (2000-5000). Conjugation reaction occurred on Lysine residues on Trastuzumab which act as sites for binding of SMCC bifunctional linker. The reaction resulted in a mixture with highly heterogeneous conjugated species distribution from D0, indicating no or zero drug conjugated to mAb, to D12, indicating up to twelve drugs conjugated to mAb. In order for TDM1 to be high in potency and exhibit least cytotoxicity, a drug to antibody ratio (DAR) of 3.2-3.6 was desired.
The process parameters for TDM1 conjugation are as follows:
Reaction pH = 7.2 (+ 0.3), Reaction time = 2 to 10 h, DMSO %= 10-20 % v/v, SMCC-DM1: mAb ratio = 1: 8.7 & Target mAb reaction concentration = 0.5 - 5 g/L, Static mixer internal diameter= 0.001 to 0.050 m, Ratio of component A: component B= 1: 5.667.
Example 2: Drug conjugation at 18L scale
In another example TDM1 was produced by performing conjugation reaction at 18L reaction scale, where Trastuzumab was conjugated with SMCC-DM1 (linker-drug/ payload) in a single step reaction in the presence of an organic solvent, DMSO. Static mixer was operated at a constant Reynolds’s number (2000-5000). For TDM1 to be high in potency and exhibit least cytotoxicity, a drug to antibody ratio (DAR) of 3.2-3.6 was desired.
Example 3: Drug conjugation at 50L scale and purification process
In another example TDM1 was produced by performing conjugation reaction at 50L reaction scale, where Trastuzumab was conjugated with SMCC-DM1 (linker-drug/ payload) in a single step reaction in the presence of an organic solvent, DMSO. Static mixer was operated at a constant Reynolds’s number (2000-5000). For TDM1 to be high in potency and exhibit least cytotoxicity, a drug to antibody ratio (DAR) of 3.2-3.6 was desired.
Following conjugation reaction, the reaction mixture was processed through tangential flow filtration (TFF) to remove unreacted components and exchange it into the buffer matrix for the next step hydrophobic interaction chromatography (HIC).
HIC was performed in flow through (FT) mode contrary to conventional Bind & elute mode to attain desired DAR distribution of conjugated species (Figure-6). HIC was optimized to be operated in a buffer system containing phosphate and sodium chloride (NaCl). The conjugation reaction output, post TFF, was conditioned by adding NaCl which now act as HIC input. As the input was loaded onto the column, packed with resin, the higher DAR species were observed to bind to the resin while the relatively lower DAR species flow through the column and were collected. Following loading of the input, a post load wash was given with the equilibration buffer and collected with the product. Post collection of the desired product, the higher order DAR species bound to the resin were stripped off by passing water through the column and collected separately, thereby regenerating the resin.
The process parameters of HIC in FT mode are provided below:
Operating pH: 5.0 – 8.0, Operating conductivity= 5 - 50 mS/cm, Buffer: phosphate and NaCl.
With this invention, due to the binding of higher DAR species, the species distribution obtained in the flow through was closer to the desired one and overall DAR was also found to be in the desired range.
Table 1: Results of conjugation reaction and HIC output at 5L, 18L, 50L & 200L scales
Sample DAR By UV D0 D1 D2 D3 D4 D5 D6 D7 D8 D9 D10 D11 D12
Conjugation reaction output scale (5L) 5.44 2.1 6.9 12.6 16 15.5 13 10 7.5 5.2 4 3.2 2.5 1.6
HIC Output Scale (5 L) 3.49 3.5 10.9 19.2 21.7 18.1 10.8 5.1 2.7 1.7 1.7 1.9 1.6 1
Conjugation reaction output scale (18L) 4.98 3.4 10.3 16.3 17.7 16 11.9 8.3 5.7 3.7 2.8 2.2 1.7 0.00
HIC Output Scale (18 L) 3.26 5.6 14.6 21.2 20.8 16 9.4 4.9 2.8 1.8 1.6 1.5 0.00 0.00
Conjugation reaction output scale (50L) 3.64 3.7 10.7 17.4 18.9 16.4 11.8 7.8 5.1 3.1 2.2 1.7 1.2 0.00
HIC Output Scale (50 L) 3.31 6 15.7 23.9 22 14.9 7.5 3.6 2.1 1.5 1.3 1.4 0.00 0.00
Conjugation reaction output scale (200L) 3.76 4.4 11.5 18.5 19.3 16.3 11.2 7.2 4.8 3.1 2 1.6 0.00 0.00
HIC Output Scale (200 L) 2.74 5.6 14 21.8 21.5 16.6 9.2 4.8 2.6 1.3 1.2 1.3 0.00 0.00

The process presented in this embodiment have significant advantages over previously disclosed methods in the following:
• Robust and Controlled Conjugation Reaction at Any Scale: The current process ensures robustness and precise control during the conjugation reaction, regardless of the scale of production. This reliability is crucial for consistent product quality and reproducibility.
• Ease of Chromatography Unit Operation in Flowthrough Mode: Unlike traditional chromatography methods, which may involve multiple steps and complex elution profiles, this process operates in a flowthrough mode.
• The simplified chromatography unit operation streamlines the manufacturing process, reducing time and complexity.
• Significant Reduction in Analysis Time: The new process incorporates streamlined analytical methods, resulting in faster analysis times. Reduced analysis time accelerates product development and allows for quicker decision-making during production.
• Very High Yielding Process Reducing COGs (Cost of Goods): The improved process yields a higher quantity of the desired product per unit of starting material. Increased yield directly impacts cost-effectiveness by minimizing raw material usage and waste.
In summary, the modified process offers substantial advantages over previous methods, enhancing efficiency, cost-effectiveness, and overall product quality.

,CLAIMS:

We claim,

1. A method for purification of an antibody-drug-conjugate, wherein the method comprises steps of:
a) Antibody DS thawing;
b) Carboxypeptidase B (CpB) treatment;
c) Tangential flow filtration – I;
d) Conjugation reaction;
e) Tangential flow filtration – II;
f) Hydrophobic interaction chromatography (HIC);
g) Tangential flow filtration – III; and
h) 0.2 µm filtration.
2. A method for purification of an antibody drug conjugate, wherein the method comprises hydrophobic interaction chromatography operated in flow through mode.

3. The method for purification of a Trastuzumab emtansine according to claim 2, wherein the said chromatography operated in flow through mode comprising steps of:
a) Equilibration with phosphate and sodium chloride buffer system
b) Loading of Trastuzumab Emtansine
c) Collection of purified Trastuzumab Emtansine in flow through mode, 10 mAU onwards (ascending; 2 mm path length chromatography system) to 3-5 CV of post load wash
d) Post load wash with equilibration buffer
e) Regeneration with water
f) Sanitization with sodium hydroxide
4. The method for purification of ADC according to claim 1, wherein the method comprises steps of:
a) Trastuzumab DS thawing;
b) Carboxypeptidase B (CpB) treatment;
c) Tangential flow filtration – I;
d) Conjugation reaction wherein, the conjugation reaction is carried out at pH about 6 to 8 for about 2 to 10 hours with organic solvent DMSO at about 10-20% v/v.
e) Tangential flow filtration – II;
f) Hydrophobic interaction chromatography (HIC) wherein, HIC is operated in flow through mode with phosphate and sodium chloride (NaCl) as a buffer system;
g) Tangential flow filtration – III; and
h) 0.2 µm filtration.
5. A method for preparing a Trastuzumab emtansine comprising the steps of: contacting a SMCC linker with a DM1 to form a first mixture comprising the SMCC and the DM1, then contacting the first mixture with an Trastuzumab, in a solution having about 10-20% v/v of organic solvent DMSO at pH of about 6 to about 8, for about 2 to 10 hours to provide a second mixture comprising (i) Trastuzumab emtansine, wherein the Trastuzumab is chemically coupled through the SMCC to DM1, (ii) free DM1, and (iii) reaction by-products.
6. The method for preparing an antibody drug conjugate according to claim 5, where in the method comprises the steps of:
(a) contacting a drug with a bifunctional crosslinking reagent to covalently attach a linker to the drug and thereby prepare a first mixture comprising drug having linkers bound thereto,
(b) optionally, subjecting the first mixture to tangential flow filtration, selective precipitation, adsorptive filtration, or an adsorptive chromatography resin and thereby prepare a purified first mixture of cell-binding agents having linkers bound thereto,
(c) conjugating an antibody to the drug having linkers bound thereto in the purified first mixture by reacting the drug having linkers bound thereto with an antibody in a solution having about 10-20% v/v of organic solvent DMSO at pH of about 6 to about 8, for about 2 to 10 hours to prepare a second mixture comprising (i) antibody chemically coupled through the linker to the drug, (ii) free drug, and (iii) reaction by-products, and
(d) subjecting the second mixture to a tangential flow filtration, selective precipitation, adsorptive filtration, or an adsorptive chromatography resin to purify the antibody chemically coupled through the linkers to the drug from the other components of the second mixture and thereby obtain final purified antibody drug conjugate.