Applications of Mass Spectrometry for Biotherapeutic Characterization Case Studies of Disulfide Characterization and Separation free Modes of Analysis Steven L. Cockrill Amgen Colorado Analytical Sciences
Agenda Introduction Structural Characterization Case Study 1: Elucidation of IgG2 Disulfide Connectivity Separation-free Modes of Analysis Case Study 2: Sequence Confirmation Case Study 3: High-throughput Glycan Analysis Conclusions Acknowledgements
Case Study 1: Elucidation of IgG2 Disulfide Linkages
Background Classical structure of human IgGs (including IgG2) first reported in 1960s and 1970s: Frangione et al., Nature 1969 Milstein et al., Biochem. J. 1971 Recent landmark work by Amgen colleagues revealed unexpected isoforms resulting from disulfide variants: Wypych et al., J. Biol. Chem., 2008 Dillon et al., J. Biol. Chem., 2008 1 1 2 Light Chain 4 5 Heavy Chain 6 7 11 12 CH1 VH CH2 CH3 27 27 However, specific connectivity between novel Fab and hinge disulfide linkages still unknown 11 13 17 23 11 13 17 23 7 11 12 6 5 4 1 2 1 CL VL Fab Hinge:Hinge Peptide Fc
Separation of Disulfide Isoforms Achieved by RP HPLC under Non reducing Conditions Methodology first reported by Amgen colleagues: Dillon et al., J. Chromatogr. A. 2006 2500 RP-1 RP-2 RP-3 RP-4 RP-5 2000 Absorbance at 215 nm 1500 1000 500 Fraction Observed Mass (Da) Purity of Enriched Fraction (%) Proportion Distribution (%) RP-1 147,357 86.3 43.1 RP-2 147,359 58.3 8.1 RP-3 147,360 82.8 26.2 RP-4 147,361 94.2 13.6 RP-5 147,368 97.1 9.1 0 5 10 15 20 25 30 Time (min)
Non reduced Peptides in each RP Fraction Fraction RP-1, compared to Unfractionated (RS) and Blank F,G RP-1a RP-1c RP-1b RP-1d RP-1e Peptide Observed Mass (Da) Theoretical Mass (Da) Absorbance at 215 nm RP-1f c f RP-1g d e RP-1 b g a h i RS Lys-C-related Blank 89 90 91 92 93 94 95 96 97 98 99 100 RP-1a 26,160.85 26,160.46 RP-1b 25,547.15 25,546.75 RP-1c 25,935.57 25,935.18 RP-1d 25,321.88 25,321.47 RP-1e 25,709.24 24,794.32 25,709.89 24,793.89 RP-1f 25,096.58 25,096.18 RP-1g 24,568.39 25,096.27 24,568.60 25,096.18 Time (min) RP-1 ~25 kda
Non reduced Peptides in each RP Fraction Fraction RP-2, compared to Unfractionated (RS) and Blank F,G Absorbance at 215 nm RP-2a a RP-2b b Carry-over from RP-1 c f d e g h i RP-2 RS Peptide Observed Mass (Da) Theoretical Mass (Da) RP-2a 12,773.50 12,773.38 RP-2b 12,547.61 12,548.09 Lys-C-related Blank 89 90 91 92 93 94 95 96 97 98 99 100 Time (min) RP-2 ~12 kda
Non reduced Peptides in each RP Fraction Fraction RP-3, compared to Unfractionated (RS) and Blank RP-3a Carry-over from RP-2 RP-3d F,G RP-3c RP-3e Peptide Observed Mass (Da) Theoretical Mass (Da) Absorbance at 215 nm RP-3b b a Lys-C-related c d e f g h i RP-3 RS RP-3a 10,095.99 10,096.13 RP-3b 9,568.29 9,568.55 RP-3c 15,450.70 15,450.63 RP-3d 15,225.48 15,225.34 RP-3e 14,999.83 15,000.05 Blank 89 90 91 92 93 94 95 96 97 98 99 100 Time (min) RP-3a same as Peptide G Other RP-3 ~15 kda
Non reduced Peptides in each RP Fraction Fraction RP-4, compared to Unfractionated (RS) and Blank RP-4a, RP-4b Absorbance at 215 nm RP-4c F,G a b Lys-C-related c d e f g h i RP-4 RS Peptide Observed Mass (Da) Theoretical Mass (Da) RP-4a 5,129.09 5,129.21 RP-4b 10,096.05 10,096.13 RP-4c 9,568.60 9,568.55 Blank 89 90 91 92 93 94 95 96 97 98 99 100 Time (min) RP-4a, 4b same as expected peptides RP-4 is classical structure
Non reduced Peptides in each RP Fraction Fraction RP-5, compared to Unfractionated (RS) and Blank RP-5a, RP-5b Absorbance at 215 nm RP-5c F,G a b Lys-C-related c d e f g h i RP-5 RS Peptide Observed Mass (Da) Theoretical Mass (Da) RP-5a 5,129.09 5,129.21 RP-5b 10,095.98 10,096.13 RP-5c 9,568.36 9,568.55 Blank 89 90 91 92 93 94 95 96 97 98 99 100 Time (min) RP-5a, 5b same as expected peptides RP-5 is classical structure
Status Check, and Path Forward Summary of non-reduced Lys-C peptide maps of RP fractions: RP-1, RP-2, RP-3 represent unexpected variants RP-4, RP-5 show only expected peptides, consistent with classical structure RP-1 is ~25 kda variant RP-2 is ~12 kda variant RP-3 is ~15 kda variant 6 6 6 7 7 7 12 11 11 12 11 12 All three variants (RP-1, RP-2, RP-3) are distributed among peptides in crown region of non-reduced peptide map How are these peptides arranged through disulfide linkages?
New Approach to Elucidate Linkages Between Closely spaced Cys Residues Significant analytical challenge to determine linkages between specific residues when Cys are closely-spaced, or worse, adjacent Historical approaches include limited reduction/alkylation, cyanylation/cleavage, or presence of fortuitous cleavage site for proteolysis IgG2 hinge peptide (H11) not ideally suited to these methods: No cleavage possible C C V E C P P C P A P P V A G P S V F L F P P K Cleavage unlikely (steric hindrance) New approach developed interfacing manual coupling and cleavage cycles of Edman chemistry with LC-MS analysis
We Developed a New Methodology to Elucidate Complex Disulfide Connectivity Manual execution of phenylisothiocyanate (PITC) coupling and cleavage via Edman chemistry: Case study using RP-3 (~15 kda variant) PITC TFA Leaving H6a H 2 N- Group G MW = 1,815 Da P S V F P L A P C S R S T S E L12 H 2 N- S H 2 N- C H 2 N- C C V E C P P C P A P P V A G P S V F L F P P K P K C V E C P P C P A P P V A G P S V F L F P P K Residual Group MW = 5,330 Da Disulfide-linked peptide from Glu-C Digest of peptide c (~15 kda) MW = 7,574 8,519 Da F N R G E C Leaving Group MW = 962 Da -NH 2 -NH 2 -NH 2 H11 H11-12
Different Connectivity Distinguished by Masses of Leaving and Residual Groups G P S V F P L A P C S R S T S E C C V E C P P C P A P P V A G P S V F L F P P K P K Leaving Group MW = 474 Da S C F N R G E C C V E C P P C P A P P V A G P S V F L F P P K Residual Group MW = 7,631 Da G P S V F P L A P C S R S T S E C C V E C P P C P A P P V A G P S V F L F P P K P K Leaving Group MW = 962 Da C S F N R G E C C V E C P P C P A P P V A G P S V F L F P P K Residual Group MW = 7,144 Da G P S V F P L A P C S R S T S E Leaving Groups MW = 1,815 Da MW = 962 Da S F N R C C G E C C V E C P P C P A P P V A G P S V F L F P P K P K C V E C P P C P A P P V A G P S V F L F P P K Residual Group MW = 5,330 Da
Evaluation of Leaving Groups for RP 3 Analysis of leaving groups confirmed presence of two species (L12* and H6a*) after first cycle Leaving groups alone do not necessarily provide definitive proof of structure Analysis of residual groups required to confirm structure EIC Signal Intensity P S V F P L A P C S R S T S E H6a* C C F N R G E C L12* 0 10 20 30 40 50 60 Retention Time (min)
Evaluation of Residual Groups for RP 3 IgG2-A/B 3 * C V E C P P C P A P P V A G P S V F L F P P K P K C V E C P P C P A P P V A G P S V F L F P P K IgG2-A/B 3 * EIC Signal Intensity P S V F P L A P C S R S T S E C C V E C P P C P A P P V A G P S V F L F P P K P K C C V E C P P C P A P P V A G P S V F L F P P K IgG2-A/B 4 * 0 10 20 30 40 50 60 Retention Time (min) F N R G E C IgG2-A/B 4 * Note leaving group associated Data confirms presence of with IgG2-A/B 4 not observed two IgG2-A/B subvariants (poor retention on column)
Connectivity of IgG2 Disulfide Isoforms RP-2 is related to IgG2-B but RP-3 contains is IgG2-A/B single copy of hinge and and contains Fab peptides two subisoforms RP-1 is IgG2-B and contains two subisoforms Successful development and application of novel methodology for elucidation of connectivity of recently reported IgG2 disulfide variants Zhang and Cockrill. 2009, Anal. Chem. 81:7314-7320 Zhang, Harder, Connelly, Maheu, and Cockrill. 2010, Anal. Chem. 82:1090-1099
Case Study 2: Separation free Sequence Confirmation
Protein Sequence Confirmation is a Regulatory Expectation, per ICH Guidelines ICH Guideline Q6B, Appendix 6: BLA Section 3.2.S.3.1: Elucidation of Structure Confirm the position of every amino acid residue within the protein sequence More extensive than sequence coverage A B C D Mass = 1000 Da
Traditional Paradigm: LC MS/MS Lys C Peptide Map T: ITM S + c ESI F ull ms [300.00-2000.00] 100 95 90 85 80 75 LH28 GFYPSDIAVEWESNGQPENNYK Mass = 2543 Da 1272.98 LH28 m/z 1273 (2+) ion isolated automatically via software and subjected to subsequent fragmentation 70 65 1199.78 Relative Abundance 60 55 50 45 40 35 30 25 20 15 10 5 0 1155.71 764.42 1111.67 653.38 565.40 785.48 1023.59 1697.58 382.79 1327.78 1595.72 1780.66 1909.19 400 600 800 1000 1200 1400 1600 1800 2000 m/z
LC MS/MS Sequence Confirmation: LH28 Scans: 1 Decent coverage, but still missing >30% confirmation!!!
Shortcomings of LC MS/MS Approach Disadvantages of LC-MS/MS MS data acquisition limited by chromatographic timescale Co-elution of multiple peptides Software control T: ITM S + c ES I Full ms [300.00-2000.00] 100 Relative Abundance 95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 1199.78 Software missed this co-eluting peptide LH28 m/z 1273 (2+) 1199.78 1272.98 < 10 sec peak width 15 10 5 0 1155.71 764.42 1111.67 653.38 1023.59 1697.58 565.40 785.48 382.79 1327.78 1595.72 1780.66 1909.19 400 600 800 1000 1200 1400 1600 1800 2000 m/z
New Paradigm: Lys C Nano Infusion MS/MS Peptide Map T : IT M S + p NSI E F u ll ms [200.00-2000.00] 100 938.96 95 90 85 80 75 70 661.84 839.40 1064.52 Manually isolate LH28 m/z 1273 (2+); subject to subsequent fragmentation 65 Relative Abundance 60 55 50 45 40 419.68 581.80 782.12 710.04 1162.56 1311.60 1365.68 35 30 25 20 560.28 1273.04 1502.72 15 10 5 280.68 327.72 435.72 488.28 1412.28 1680.24 1548.12 1731.92 1873.80 1912.16 0 200 400 600 800 1000 1200 1400 1600 1800 2000 m/z
NSI MS/MS Sequence Confirmation: LH28 Scans: 50 100% confirmation with no separation!!! (30 sec) Application provides significant cost and time savings, with improved data quality in support of regulatory filings
Case Study 3: High throughput Glycan Analysis
Evaluation of Glycan Profiles is a pain point for the Biotherapeutic Industry Regulatory expectations are becoming more stringent with respect to demonstrating process control Clone selection, cell culture conditions, and raw material inputs all influence a biotherapeutic s glycan population Current methods/technologies are time and resource-intensive Industry is consistently looking for new methods for rapid high throughput screening that are fully automatable
Contemporary Glycan Mapping Methods RP-MS method ~180 minutes per sample (100 samples = 11.8 days) Column and 2-AB labeling kit cost RRRP-MS and HILIC-MS methods ~60 minutes per sample (100 samples = 4.2 days) Column and 2-AB labeling kit cost UPLC-RP-MS and CZE methods ~15 minutes per sample (100 samples = 25 hours) Column or capillary and 2-AB or 2-APTS labeling kit cost Challenge of CZE-MS for characterization Agilent Lab-on-a-chip ~10 minutes per sample (100 samples = 16.6 hours) Requires specific instrumentation, difficult characterization In each case, separation speed is limiting factor for throughput
Stable Isotopic MS Glycan Mapping Workflow Sample Control PNGaseF release 2 hr @ 80 C Fluorescent labeling ( 12 [C 6 ]AA) 1 hr @ 80 C PNGaseF release 2 hr @ 80 C Fluorescent labeling ( 13 [C 6 ]AA) 1 hr @ 80 C Combine Sample and Control Normal Phase PhyTip TM Phynexus MEA or Tecan 30 min / 96 samples Carbopack B PhyTip TM Phynexus MEA or Tecan 30 min / 96 samples 137.14 Da 143.09 Da Mass Spectrometry Nano-ESI MS, MALDI MS 50 msec / sample Prien, Prater, Qin, and Cockrill. 2010, Anal. Chem. 82:1498-1508
Assay Performance Shows Accuracy and Linearity for NSI and MALDI Sources RI: 100:99.2 RI: 50.7:100 RI: 19.7:100 RI: 11.1:100 Prien, Prater, Qin, and Cockrill. 2010, Anal. Chem. 82:1498-1508
Application of MS Glycan Profiling Rapid Monitoring Effects of Process Change 50 HPLC 2-AB 50 MS-Based Stable Isotope 2-AA %Difference ((SampleX-RS)/RS x 100%) 40 30 20 10 0-10 -20-30 Process 1 Process 2 Process 3 % Difference ((SampleX-RS)/RS*100) 40 30 20 10 0-10 -20-30 Process 1 Process 2 Process 3-40 -40-50 4.0 days for results 4.0 hours for results -50 Processing time for 96 samples UPLC Agilent Lab-on-a-chip Stable isotopic profiling PNGase F release 2 hr N/A (performed on chip) 2 hr Application provides significant cost and time savings allowing for efficiencies in process characterization Derivatization 1 hr N/A (no derivatization) 1 hr Clean up 1 hr N/A (no derivatization) 1 hr Data collection 24 hr (15 min / sample) 16 hr (10 min / sample) < 1 min (0.5 sec / sample ) Total time 28 hr 16 hr ~4 hr
Acknowledgements Thank you! Questions? IgG2 Disulfide Connectivity Bing Zhang Adam Harder Heather Connelly Lorna Maheu Separation-free Modes of Analysis Adam Harder Justin Prien Brad Prater Qiang Qin