Jürgen Fichtl, Development Characterization Pharma Technical Development, Penzberg, Germany Mass Spec 2013, September 23-26, Boston

Analytical Strategies for Assessment of Disulfide Linkages in Biopharmaceuticals Jürgen Fichtl, Development Characterization Pharma Technical Development, Penzberg, Germany Mass Spec 2013, September 23-26, Boston

Introduction Example 1: bispecific MAB Method description Research Clone Results of a research clone Results of the best producing clone Example 2: MAB with open SH Determination of free thiols Comparison with RP HPLC Determination of incorrect disulfide pairing Conclusions 2

Introduction IGG1 3

Introduction What disulfid modifications may occur? open sulfhydryl groups mainly in VL and CH3 ( Lacy ER et al 2008) Basic conditions may cause the formation of a non reducable thioether between HC - LC (J. Smith et al 2005) long term storage / posttranslational modification - may cause the formation of trisulfide within all inter chain bridges (S. Gu et al 2010) Heat stress conditions -may cause scrambling of C370 $ C96 (Yi Wang et al 2011) - fragmentation of hinge region (J. Vlasak et al 2007) Important role on function and stability IGG1 4

Introduction Analytical approaches Free thiols: - Reversed Phase Chromatography - Derivatization of free thiols with fluorimetric detection - Tryptic peptide map after derivatization of free thiols sum of all free thiols site-specific percentage of open disulfide bonds Incorrect disulfide pairing: - ESI-MS - Tryptic peptide map LCMS comparison before/after reduction poor sensitivity for minor species 5

Introduction Example 1: bispecific MAB Method description Research Clone Results of a research clone Results of the best producing clone Example 2: MAB with open SH Determination of free thiols Comparison with RP HPLC Determination of incorrect disulfide pairing Conclusions 6

Example 1: bispecific MAB Bispecific antibodies: Merge of two antibodies Ridgway JB, et al. (1996) 'Knobs-into-holes' engineering of antibody CH3 domains for heavy chain heterodimerization. Protein Eng 9:617-621 Atwell S, et al. (1997) Stable heterodimers from remodeling the domain interface of a homodimer using a phage display library. J. Mol. Biol. 270:26-35. desired mainproduct possible sideproduct 7

Sample preparation for LCMS peptide map Native tryptic digest (50µg antibody; c>2mg/ml) Alkylation of free SH: Buffer exchange: Digest (combination of LysC and Trypsin): - 1. Step: Addition of MAB : LysC 70:1 MAB : Trypsin 25:1-2.Step: Addition of MAB : Trypsin 25:1 Termination of digestion incubation 2h / 37 C incubation 16h / 37 C 8

Data evaluation LC MSMS on Thermo LTQ Orbitrap Top5 LCMSMS method (FT R=30.000; fragmentation in iontrap): Identification of SS-bridged peptides Dbond Algorithm (Seonhwa Choi et al 2009) : - java based software tool which screens all MSMS data to find b-/y- ions of SS bridged peptides - search based on local storage - workflow similar to mascot search (parameter files, modifications, fasta-file, excel export) Quantification by XIC evaluation relative determination of false SS-bridged peptides 9

Results: bispecific MAB of a research clone regular tryptic peptide (rel amount: 8%) 10

Results: bispecific MAB of a research clone regular tryptic peptide (rel amount: 8%) 11

Results: bispecific MAB of a research clone 12

Results: bispecific MAB of a research clone 13

Results: bispecific MAB of a research clone Int 37% 9% 25% 18% m/z 14

Results: bispecific MAB of a research clone 15

Results: bispecific MAB of a research clone Int 37% 9% 25% 18% m/z 16

Results: bispecific MAB of a research clone 17

Results: bispecific MAB of a research clone Quantification by SS bridge HC $ HC Description ratio HC1 366 HC2 343 HC1_$_HC2 88,0 HC2 343 HC2 343 HC2-Dimer 11,4 HC1 366 HC1 366 HC1_Dimer 0,6 18

Results: bispecific MAB of a research clone Int 37% 9% 25% 18% m/z 19

Results: bispecific MAB of a research clone Quantification by each cysteine SS Bond Description ratio LC1_Cys 213 LC1 213 HC1 232 LC1_$_HC1 27,7 LC1 213 HC2 214 LC1_$_HC2 49,6 LC1 213 LC1 213 LC1-Dimer 22,6 LC1 213 LC2 226 LC1_$_LC2 0,1 20

Results: bispecific MAB of a research clone Quantification by each cysteine SS Bond Description ratio LC1_Cys 213 LC1 213 HC1 232 LC1_$_HC1 27,7 LC1 213 HC2 214 LC1_$_HC2 49,6 LC1 213 LC1 213 LC1-Dimer 22,6 LC1 213 LC2 226 LC1_$_LC2 0,1 21

Results: bispecific MAB of a research clone RT: 4,17-9,37 100 80 60 6,83 TIC of native digest NL: 7,90E7 TIC F: ms MS f12100211 40 20 0 100 80 60 40 4,27 5,56 6,15 8,37 6,57 4,49 5,82 5,26 6,41 7,44 4,85 5,14 7,11 7,55 7,99 8,11 8,64 8,74 9,16 7,08 XIC of SS-peptide NL: 2,80E4 m/z= 507,18-507,21 F: ms MS f12100211 20 0 100 80 60 40 20 0 100 80 60 4,85 4,28 5,98 6,58 4,33 7,44 5,09 4,48 5,28 5,70 4,55 4,86 6,46 8,11 8,27 5,38 6,40 7,18 7,57 7,99 8,45 8,79 9,05 9,21 4,77 6,54 6,85 7,08 7,37 TIC of reduced digest XIC of SS-peptide NL: 2,69E7 TIC MS f12101211 NL: 7,36E2 m/z= 507,18-507,21 MS f12101211 40 20 0 4,5 5,0 5,5 6,0 6,5 7,0 7,5 8,0 8,5 9,0 Time (min) 22

Results: bispecific MAB of a research clone RT: 4,17-9,37 100 80 60 6,83 TIC of native digest NL: 7,90E7 TIC F: ms MS f12100211 40 20 0 100 80 60 40 4,27 5,56 6,15 8,37 6,57 4,49 5,82 5,26 6,41 7,44 4,85 5,14 7,11 7,55 7,99 8,11 8,64 8,74 9,16 7,08 XIC of SS-peptide NL: 2,80E4 m/z= 507,18-507,21 F: ms MS f12100211 20 0 100 80 60 40 20 0 100 80 60 4,85 4,28 5,98 6,58 4,33 7,44 5,09 4,48 5,28 5,70 4,55 4,86 6,46 8,11 8,27 5,38 6,40 7,18 7,57 7,99 8,45 8,79 9,05 9,21 4,77 6,54 6,85 7,08 7,37 TIC of reduced digest NL: 2,69E7 TIC MS f12101211 NL: 7,36E2 m/z= 507,18-507,21 MS f12101211 40 20 0 4,5 5,0 5,5 6,0 6,5 7,0 7,5 8,0 8,5 9,0 Time (min) 23

Introduction Example 1: bispecific MAB Method description Research Clone Results of a research clone Results of the best producing clone Example 2: MAB with open SH Determination of free thiols Comparison with RP HPLC Determination of incorrect disulfide pairing Conclusions 24

Results: Bispecific MAB of best producing clone - No incorrect disulfide linkages detected! - Disulfide linkages covered except for: - - 2 peptides > 7000 Da (bad CID fragmentation data) - - 1 peptide with two SS linkages - - 1 peptide due to incomplete digest incomplete digest 25

Introduction Example 1: bispecific MAB Method description Research Clone Results of a research clone Results of the best producing clone Example 2: MAB with open SH Determination of free thiols Comparison with RP HPLC Determination of incorrect disulfide pairing Conclusions 26

Sample preparation In-house optimized reduced tryptic pepmap by derivatization with NEM/NEM-d5 0µg antibody; c>2mg/ml) Derivatization free SH with NEM Buffer Exchange to remove NEM Label concentrate by centrifugation Reduction of linked disulfides Derivatization of ne novo formed thiols with NEM-d5: Buffer Exchange to remove NEM-d5 Label Tryptic Digest Termination of Digestion 27

Example 2: Determination of free thiols C265 C230 C233 Peptid N NEM-d5 [%] N NEM [%] N SH [%] Peptid N NEM-d5 [%] N NEM [%] N SH [%] C22 65,59 34,41 n.n. C96 64,27 35,73 n.n. C149 H12 97,04 2,73 0,23 C204 99,65 n.n. 0,35 C224 99,42 0,58 n.n. C218 n.n. n.n. n.n. C230/233 100 n.n. n.n. C230/233 100 n.n. n.n. C265 94,83 4,9 0,27 C325 n.n. n.n. n.n. C371 95,43 4,39 0,18 C429 96,6 3,28 0,12 C23 96,78 3,14 0,08 C92 100 n.n. n.n. C138 100 n.n. n.n. C198 99,49 0,47 0,04 C325 C371 C429 28

Example 2: comparison with RP HPLC Ratio of free thiol LCMS ca 36% 41% 46% 13% calculated probability based on LCMS data 29

Example 2: Determination of incorrect disulfide pairing NEM-Tag C230 C233 SS Bond Description Ratio ph 7 C22 NEM HC_$_NEM 31.5 C22 C96 HC_$_HC 56.5 C22 C198 HC_$_LC 1.4 C22 C265 HC_$_LC 3.9 C22 minor species 3.0 C265 C325 C371 C429 30

Example 2: Determination of incorrect disulfide pairing NEM-Tag SS Bond Description Ratio ph 7 C96 NEM HC_$_NEM 47.4 C96 C22 HC_$_HC 48.7 C230 C233 C96 C265 HC_$_LC 1.6 C96 C371 HC_$_LC 1.0 C96 minor species 1.3 C265 C325 C371 31 C429

Introduction Example 1: bispecific MAB Method description Research Clone Results of a research clone Results of the best producing clone Example 2: MAB with open SH Determination of free thiols Comparison with RP HPLC Determination of incorrect disulfide pairing Conclusions 32

Conclusion Disulfide mismatching Native Tryptic Pepmap Evaluation by DBond - highly sensitive method to identify minor species of incorrect disulfide pairing - complex evaluation not practical for high troughput applications - induced scrambling during basic tryptic digest ESI-MS - high troughput application - not sensitive to minor species Free thiols - RP HPLC results correlate with tryptic peptide map with NEM/ NEM-d5 derivatization - tryptic peptide map with NEM/ NEM-d5 derivatization appropriate for identification of low ratios of free thiols

Acknoledgements Eunok Paek (University of Soeul; Dbond Algortithm) Nadja Alt Dietmar Reusch Nadine Flöser Manuel Schott Ingo Linder Andreas Adler

Doing now what patients need next 35

Example 2: Determination of Incorrect Disulfide Pairing Influence of Digest conditions C22 Label Area ph8,5 Area ph7 Area ph6 [%] [%] [%] HC_C96 30,1 56,5 52,5 HC_C265 12,1 3,9 0,8 LC_C198 14,2 1,4 0,2 HC_C148 13,4 0,9 0,1 LC_C138 9,8 0,8 0,1 HC_C325 2,6 0,6 0,1 HC_C371 5,5 0,3 0,1 LC_C23 5,3 0,2 0,0 HC_C22 1,1 0,2 0,0 HC_C224 1,3 0,1 0,0 HC_C429 1,0 0,0 0,0 LC_C92 1,0 0,0 0,0 HC_C204 0,3 0,0 0,0 NEM 2,1 35,1 46,1 C96 Label Area ph8,5 Area ph7 Area ph6 [%] [%] [%] HC_C22 36,8 48,7 34,0 HC_C265 7,3 1,6 0,6 HC_C371 10,9 1,0 0,2 LC_C198 15,7 0,4 0,1 LC_C138 4,0 0,4 0,0 HC_C325 2,6 0,3 0,0 HC_C148 6,8 0,2 0,1 HC_C429 7,4 0,0 0,0 HC_C224 1,4 0,0 0,0 HC_C96 0,6 0,0 0,0 HC_C204 0,4 0,0 0,0 NEM 6,3 47,4 64,9 36

ph 6.0 ph 7.0 ph 8.5 ph 6.0 ph 7.0 ph 8.5 ph 7.0 Compare of digest conditions MAB A native MAB B native MAB C native MAB D reduced kda kda 200 200 160 97 160 97 66 66 55 55 36 31 36 31 21 21 14 14 6 6 2.5 2.5 37