Unbiased in-depth characterization of CEX fractions from a stressed mab by MS. Matthias Berg, Novartis Pharma, BTDM
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1 Unbiased in-depth characterization of CEX fractions from a stressed mab by MS Matthias Berg, Novartis Pharma, BTDM
2 Characterization of CEX fractions Biopharmaceuticals like IgGs show a certain degree of microheterogeneity, e.g. post translational modifications These modifications are introduced during protein expression, purification, and storage They can reduce the potency, change stability, or increase immunogenicity and must therefore be characterized during drug development CEX is widely used to monitor charge heterogeneity and separate protein variants In this study a stressed recombinant IgG1 was analyzed by CEX and fractions were collected for further characterization 2 CASSS Mass Spec 2015 Characterization of CEX fractions by MS Matthias Berg
3 CEX-UV profile (collected fractions indicated) mau 1800 Main peak fraction AF1 AF2 AF3 BF1 BF Elution Volume (ml) 3 CASSS Mass Spec 2015 Characterization of CEX fractions by MS Matthias Berg
4 Characterization of collected CEX fractions by MS Intact LC-MS analysis: identify degradation products (clipping, half mab, free LC or HC), PTMs Reduced/alkylated LC-MS analysis: identify clipping and PTMs PepMap analysis after red/carboxy and LysC digest: identify PTMs like deamidation or oxidation, and confirm findings from intact and reduced analyses 4 CASSS Mass Spec 2015 Characterization of CEX fractions by MS Matthias Berg
5 MS data evaluation Intact and reduced/alkylated LC-MS: use time-resolved deconvolution instead of averaged spectra for better visualization and assisted data interpretation using Genedata Expressionist PepMap: identify differences between fractions based on quantitative data from charge states using Genedata Expressionist. Characterize only observed differences 5 CASSS Mass Spec 2015 Characterization of CEX fractions by MS Matthias Berg
6 Overlay of UV chromatograms from all CEX fractions (intact) AF1 AF2 AF3 Main peak BF1 BF2 6 CASSS Mass Spec 2015 Characterization of CEX fractions by MS Matthias Berg
7 Time-resolved deconvolution of the main peak (RT 19 to 21 min) Possible Peak Assignment 1 bg0, bg0-f 2 2bG0 3 bg0, bg1 4 2bG1 5 1bG1, 1bG2 Retention time (min) Mass (Da) 7 CASSS Mass Spec 2015 Characterization of CEX fractions by MS Matthias Berg
8 Time-resolved deconvolution of all fractions (RT 19 to 21 min) Retention time (min) Retention time (min) Frac AF1 AF2 AF3 Main peak BF1 BF2 Possible Assignment 2bG0 2bG0 2bG0 2bG0 2bG0, pe 2bG0, - C-Term Gly Mass (Da) Mass (Da) Mass (Da) 8 CASSS Mass Spec 2015 Characterization of CEX fractions by MS Matthias Berg
9 Time-resolved deconvolution of the main peak (relative quantitation of glycoforms) AF1 AF2 AF3 Main Peak 9 CASSS Mass Spec 2015 Characterization of CEX fractions by MS Matthias Berg
10 Deconvoluted area around 19 min (intact, analyzed as received) AF1 AF2 AF3 Main peak BF1 BF2 AF3 AF2 AF1 AF1 AF2 AF3 Retention Time (min) Mass (Da) LC HC LC HC LC HC 10 CASSS Mass Spec 2015 Characterization of CEX fractions by MS Matthias Berg
11 Summary for intact analyses of CEX fractions Basic fractions contain mainly N- and C-Term modified HC Glycosylation profile changes Different fragments of the intact mab are observed: Fab, Fab-DK and Fab-DKTH (more abundant in acidic fractions) Different clipping fragments of LC and HC are observed: - in the HC between the loops V H and C H 1 - in the LC between the loops V L and C L 11 CASSS Mass Spec 2015 Characterization of CEX fractions by MS Matthias Berg
12 Overlay of UV chromatograms from all CEX fractions (reduced/alkylated) AF1 AF2 AF3 Main peak BF1 BF2 LC HC 12 CASSS Mass Spec 2015 Characterization of CEX fractions by MS Matthias Berg
13 Time-resolved deconvolution of the LC peak from the main peak fraction (RT 23 min) Retention Time (min) LC LC, oxidized Mass (Da) LC, glycated 13 CASSS Mass Spec 2015 Characterization of CEX fractions by MS Matthias Berg
14 Time-resolved deconvolution of the LC peak from all fractions (RT 23 min) AF1 AF2 AF3 Main peak BF1 BF2 Retention Time (min) Mass (Da) LC LC, oxidized LC, glycated 14 CASSS Mass Spec 2015 Characterization of CEX fractions by MS Matthias Berg
15 Relative quantification of the light chain species AF1 AF2 AF3 Main BF5 BF6 LC 6.0 Relative quantification of the glycation level LC, glycation 0.0 AF1 AF2 AF3 Main peak BF1 BF2 15 CASSS Mass Spec 2015 Characterization of CEX fractions by MS Matthias Berg
16 Time-resolved deconvolution of the HC peak from all fractions (RT 30 min) AF1 AF2 AF3 Main peak BF1 BF2 Retention Time (min) Retention Time (min) HC, 0K, bg0 HC, 0K, bg0-f HC, 0K, bg1 HC, 0K, bg2 Mass (Da) HC, 0K, bg0, pe HC, 0K, bg1, pe HC, bg0, 0GK HC, bg1, 0GK 16 CASSS Mass Spec 2015 Characterization of CEX fractions by MS Matthias Berg
17 Time-resolved versus averaged spectra deconvolution AF1 AF2 AF3 Main peak BF1 BF2 Main peak BF1 BF2 Retention Time (min) HC, 0K, bg0 HC, 0K, bg0-f HC, 0K, bg1 Mass (Da) HC, 0K, bg0, pe HC, bg1, 0K, pe HC, bg0, 0GK HC, bg1, 0GK 17 CASSS Mass Spec 2015 Characterization of CEX fractions by MS Matthias Berg
18 Summary for reduced/alkyated analyses of CEX fractions Glycation levels increase in acidic fractions Basic fractions contain mainly pe (BF1), and a truncated HC C-Term (BF2) Glycosylation profile changes from more bg2 (acidic) to more bg0-f (main) Clipping found in intact analysis could be confirmed 18 CASSS Mass Spec 2015 Characterization of CEX fractions by MS Matthias Berg
19 Overlay of UV chromatograms of red/carboxy Lys-C digested CEX fractions AF1 AF2 AF3 Main peak BF1 BF2 19 CASSS Mass Spec 2015 Characterization of CEX fractions by MS Matthias Berg
20 How to find the differences between the different CEX fractions Retention Time (min) >2000 clusters corresponding to a single charge state were detected and quantified 20 CASSS Mass Spec 2015 Characterization of CEX fractions by MS Matthias Berg m/z
21 How to find the differences between the different CEX fractions Create a new group only with the selected clusters, which are more intense 21 CASSS Mass Spec 2015 Characterization of CEX fractions by MS Matthias Berg
22 Modifications found for the heavy chain 22 CASSS Mass Spec 2015 Characterization of CEX fractions by MS Matthias Berg
23 Verifying the assignments, e.g. deamidation Retention Time (min) m/z 23 CASSS Mass Spec 2015 Characterization of CEX fractions by MS Matthias Berg
24 MS/MS data of deamidated peptide: XXXXXXINSQGK Pre-peak Mod +1Da Unmod Post-peak Mod +1Da Unmod/Pre-peak XXXXXXINSQGK Post-peak y 419.2m/z XXXXXXINSEGK y 420.2m/z 24 CASSS Mass Spec 2015 Characterization of CEX fractions by MS Matthias Berg
25 MS/MS data of deamidated peptide: XXXXXXINSQGK Pre-peak Mod +1Da Unmod Post-peak Mod +1Da Unmod XXXXXXINSQGK Pre-peak y 533.3m/z XXXXXXIDSQGK y 534.2m/z Post-peak XXXXXXINSEGK y 534.2m/z 25 CASSS Mass Spec 2015 Characterization of CEX fractions by MS Matthias Berg
26 Peptide H17-18 TKPREEQYNSTYRVVSVLTVLHQDWLNGK, without glycosylation, deamidated AF1 AF2 AF3 Fr1 Fr2 Retention Time (min) Fr3 Main, 4 Main peak BF1 BF2 Fr5 Fr6 Retention Time (min) m/z 26 CASSS Mass Spec 2015 Characterization of CEX fractions by MS Matthias Berg
27 Comparison of relative quantification of the different glycoforms on the heavy chain 70.0 PepMap 70.0 Reduced AF1 AF AF1 AF AF3 Main peak 30.0 AF3 Main peak bg0-f bg0 bg1 bg2 0.0 bg0-f bg0 bg1 bg2 27 CASSS Mass Spec 2015 Characterization of CEX fractions by MS Matthias Berg
28 Summary for PepMap analyses of CEX fractions Identification of charged variants based on quantitative data from charge state cluster Exact localization of certain modifications like deamidation, glycation Glycosylation profile changes from more bg1 (acidic) to more bg0 (main) Clipping found in intact and reduced analysis could be confirmed 28 CASSS Mass Spec 2015 Characterization of CEX fractions by MS Matthias Berg
29 Summary AF1 AF2 AF3 Main BF1 BF2 Acidic Main Basic Glycation Glycosylation profile pe in BF1 0GK in BF2 Deamidation Clipping The evaluation approach using time-resolved deconvolution for intact and reduced data and display in a 2D heat map allowed: - recognition of small mass differences with certainty - better data interpretation facilitated by pattern recognition - direct quantification of modifications (glycation, pe) Identification of quantitative differences between the fractions on charge state level allowed: - unbiased view of the data - faster evaluation and confirmation - identification of the unexpected 29 CASSS Mass Spec 2015 Characterization of CEX fractions by MS Matthias Berg
30 Acknowledgement LPAD-MS group: Blandine Denefeld Francois Griaud Marie-Claude Djidja Manuel Lang Heloise Cornuel Markus Blümel LPAD-PCA group: Wolfram Kern Robin Mende Dirk Chelius Genedata: Peter Haberl 30 CASSS Mass Spec 2015 Characterization of CEX fractions by MS Matthias Berg
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