Use of a PVDF membrane to purify cell-binding agent cytotoxic agent conjugates

Abstract

The invention provides processes for preparing purified cell-binding agent cytotoxic agent conjugates comprising subjecting a mixture comprising a cell-binding agent cytotoxic agent conjugate and one or more impurities to a polyvinyl difluoride (PVDF) membrane to remove at least a portion of the impurities from the mixture, thereby providing a purified cell-binding agent cytotoxic agent conjugate.

1 - 5 . (canceled) 
     
     
         6 . A process for preparing a purified antibody-maytansinoid conjugate, comprising:
 (a) contacting an antibody with a maytansinoid to form a first mixture comprising the antibody and the maytansinoid, then contacting the first mixture with a bifunctional crosslinking reagent comprising a linker, in a solution having a pH of about 6 to about 8, to provide a second mixture comprising the antibody-maytansinoid conjugate comprising the antibody chemically coupled through the linker to the maytansinoid and one or more impurities;   (b) subjecting the second mixture to a polyvinyl difluoride (PVDF) membrane to remove at least a portion of the impurities, thereby providing a purified second mixture of the antibody-maytansinoid conjugate; and   (c) subjecting the purified second mixture after step (b) to tangential flow filtration, selective precipitation, non-adsorptive chromatography, adsorptive filtration, adsorptive chromatography, or a combination thereof, to further purify the antibody-maytansinoid conjugate from the impurities and thereby prepare a purified third mixture of the antibody-maytansinoid conjugate, wherein the purified third mixture comprises a reduced amount of the impurities as compared to the purified second mixture.   
     
     
         7 . The process of  claim 6 , wherein step (b) is sequentially repeated two, three, or four times prior to step (c). 
     
     
         8 . The process of  claim 6 , wherein adsorptive chromatography is utilized in step (c). 
     
     
         9 . The process of  claim 6 , wherein the adsorptive chromatography is selected from hydroxyapatite chromatography, hydrophobic charge induction chromatography (HCIC), hydrophobic interaction chromatography (HIC), ion exchange chromatography, mixed mode ion exchange chromatography, immobilized metal affinity chromatography (IMAC), dye ligand chromatography, affinity chromatography, reversed phase chromatography, and combinations thereof. 
     
     
         10 . The process of  claim 9 , wherein the adsorptive chromatography is ion exchange chromatography. 
     
     
         11 . The process of  claim 10 , wherein the ion exchange chromatography is ceramic hydroxyapatite (CHT) chromatography. 
     
     
         12 . The process of  claim 6 , wherein tangential flow filtration is utilized in step (c). 
     
     
         13 . The process of  claim 6 , wherein the contacting in step (a) is effected by providing the antibody in a reaction vessel, adding the maytansinoid to the reaction vessel to form the first mixture comprising the antibody and the maytansinoid, and then adding the bifunctional crosslinking reagent to the first mixture. 
     
     
         14 . The process of  claim 6 , further comprising holding the mixture between steps a-b or steps b-c to release the unstably bound linkers from the antibody. 
     
     
         15 . The process of  claim 14 , wherein the mixture is held for about 20 hours at a temperature of about 2° C. to about 8° C. 
     
     
         16 . The process of  claim 6 , further comprising quenching the second mixture between steps (a)-(b) to quench any unreacted maytansinoid and/or unreacted bifunctional crosslinking reagent. 
     
     
         17 . The process of  claim 16 , wherein the mixture is quenched by contacting the second mixture with a quenching reagent that reacts with the free maytansinoid. 
     
     
         18 . The process of  claim 17 , wherein the quenching reagent is selected from 4 maleimidobutyric acid, 3-maleimidopropionic acid, N-ethylmaleimide, iodoacetamide, and iodoacetamidopropionic acid. 
     
     
         19 . The process of  claim 6 , wherein the process further comprises subjecting the mixture to an ion exchange chromatography membrane between steps (a) and (b). 
     
     
         20 . The process of  claim 6 , wherein the process further comprises subjecting the mixture to an ion exchange chromatography membrane between steps (b) and (c). 
     
     
         21 - 22 . (canceled) 
     
     
         23 . The process of  claim 6 , wherein the maytansinoid comprises a thiol group. 
     
     
         24 - 34 . (canceled) 
     
     
         35 . The process of  claim 23 , wherein the maytansinoid is N 2′ -deacetyl-N 2′ -(3-mercapto-1-oxopropyl)-maytansine (DM1) or N 2′ -deacetyl-N 2′ -(4-methyl-4-mercapto-1-oxopentyl)-maytansine (DM4). 
     
     
         36 - 51 . (canceled) 
     
     
         52 . The process of  claim 6 , wherein the one or more impurities are selected from maytansinoid dimers, aggregates of the antibody-maytansinoid conjugate, free maytansinoid, unconjugated linker, and mixtures thereof. 
     
     
         53 . The process of  claim 52 , wherein the mixture comprises maytansinoid dimers as an impurity, and some portion of the maytansinoid dimers is removed from the mixture to provide the purified antibody-maytansinoid conjugate. 
     
     
         54 . The process of  claim 53 , wherein the maytansinoid dimer comprises DM1-DM1. 
     
     
         55 . The process of  claim 53 , wherein the maytansinoid dimer comprises DM1-MCC-DM1. 
     
     
         56 . The process of  claim 53 , wherein the maytansinoid dimer comprises DM1-DM1 and DM1-MCC-DM1. 
     
     
         57 - 58 . (canceled) 
     
     
         59 . The process of  claim 53 , wherein maytansinoid dimer comprises DM1-DM1 and DM1-SPP-DM1. 
     
     
         60 . The process of  claim 53 , wherein the maytansinoid dimer comprises DM1-SPP-DM1. 
     
     
         61 . The process of  claim 6 , wherein the pH of the mixture is about 7 to about 8. 
     
     
         62 . The process of  claim 6 , wherein the pH of the mixture is about 6.5 to about 7.5. 
     
     
         63 . The process of  claim 6 , wherein the pH of the mixture is about 87.5. 
     
     
         64 - 75 . (canceled) 
     
     
         76 . The process of  claim 6 , wherein the bifunctional crosslinking reagent is an acid labile linker, a disulfide containing linker, a photolabile linker, a peptidase labile linker, or an esterase labile linker. 
     
     
         77 . The process of  claim 6 , wherein the bifunctional crosslinking reagent is a disulfide-containing cleavable linker. 
     
     
         78 . The process of  claim 6 , wherein the bifunctional crosslinking reagent is a non-cleavable linker. 
     
     
         79 . The process of  claim 6 , wherein the bifunctional crosslinking reagent comprises an N-succinimidyl ester moiety, an N-sulfosuccinimidyl ester moiety, a maleimido-based moiety, or a haloacetyl-based moiety. 
     
     
         80 . The process of  claim 77 , wherein the bifunctional crosslinking reagent is selected from N succinimidyl 3-(2-pyridyldithio)propionate (SPDP), N-succinimidyl 4-(2-pyridyldithio)butanoate (SPDB), N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP), and N-succinimidyl-4-(2-pyridyldithio)2-sulfo butanoate (sulfo-SPDB). 
     
     
         81 . The process of  claim 78 , wherein the bifunctional crosslinking reagent is selected from N-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate (SMCC), N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxy-(6-amidocaproate) (LC-SMCC), κ-maleimidoundecanoic acid N-succinimidyl ester (KMUA), γ-maleimidobutyric acid N-succinimidyl ester (GMBS), β-maleimidopropyloxy-succinimidyl ester (BMPS), ε-maleimidocaproic acid N-hydroxysuccinimide ester (EMCS), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), N-(α-maleimidoacetoxy)-succinimide ester (AMAS), succinimidyl-6-(β-maleimidopropionamido)hexanoate (SMPH), N-succinimidyl 4-(p-maleimidophenyl)-butyrate (SMPB), and N-(p-maleimidophenyl)isocyanate (PMPI), sulfo-Mal, PEG4-Mal and CX1-1. 
     
     
         82 - 83 . (canceled) 
     
     
         84 . The process of  claim 6 , wherein the antibody is a monoclonal antibody. 
     
     
         85 . The process of  claim 6 , wherein the antibody is a humanized monoclonal antibody. 
     
     
         86 . The process of  claim 84 , wherein the antibody is selected from huB4, huC242, trastuzumab, bivatuzumab, sibrotuzumab, huDS6, rituximab, anti-CD33 antibody, anti-CD27L antibody, anti-Her2 antibody, anti-EGFR antibody, anti-EGFRvIII antibody, Cripto, anti-CD138 antibody, anti-CD38 antibody, anti-EphA2 antibody, integrin targeting antibody, anti-CD37 antibody, anti-folate receptor antibody, anti-Her3 antibody, B-B4 antibody and anti-IGFIR antibody. 
     
     
         87 - 98 . (canceled)