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

Transcription

1 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

2 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

3 Introduction IGG1 3

4 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

5 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

6 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

7 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: Atwell S, et al. (1997) Stable heterodimers from remodeling the domain interface of a homodimer using a phage display library. J. Mol. Biol. 270: desired mainproduct possible sideproduct 7

8 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

9 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

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

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

12 Results: bispecific MAB of a research clone 12

13 Results: bispecific MAB of a research clone 13

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

15 Results: bispecific MAB of a research clone 15

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

17 Results: bispecific MAB of a research clone 17

18 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

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

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 20

21 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

22 Results: bispecific MAB of a research clone RT: 4,17-9, ,83 TIC of native digest NL: 7,90E7 TIC F: ms MS f ,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 f ,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 f NL: 7,36E2 m/z= 507,18-507,21 MS f ,5 5,0 5,5 6,0 6,5 7,0 7,5 8,0 8,5 9,0 Time (min) 22

23 Results: bispecific MAB of a research clone RT: 4,17-9, ,83 TIC of native digest NL: 7,90E7 TIC F: ms MS f ,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 f ,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 f NL: 7,36E2 m/z= 507,18-507,21 MS f ,5 5,0 5,5 6,0 6,5 7,0 7,5 8,0 8,5 9,0 Time (min) 23

24 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

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

26 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

27 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

28 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/ n.n. n.n. C230/ 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 C n.n. n.n. C n.n. n.n. C198 99,49 0,47 0,04 C325 C371 C429 28

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

30 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

31 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 C C429

32 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

33 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

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

35 Doing now what patients need next 35

36 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

37 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