Challenges in Quantification of Protein Therapeutic Catabolites - New Analytical Tools

Transcription

1 Challenges in Quantification of Protein Therapeutic Catabolites - New Analytical Tools Eliza Fung, Ph.D. Analytical & Bioanalytical Development Bristol-Myers Squibb 2014 AAPS Annual Meeting November 5,

2 Quantification of Small Molecule Therapeutics Metabolites Biotransformation of small molecule therapeutics (SMT, with molecular weights < 1000) is catalyzed by one or more of drug-metabolizing enzymes (e.g., Cytochrome P450) The metabolites of the SMT can be quantified with welladapted analytical technologies, e.g., LC-MS/MS Does the same apply to protein therapeutics??? 2

3 Protein Therapeutics Class of Protein Therapeutics Monoclonal Antibodies, including Fab (Fragment antigen-binding) Examples Erbitux, Lucentis, Yervoy, Opdivo Antibody-drug conjugates (ADC) Kadcyla, Adcetris Bispecific biologics catumaxomab Large peptides Hydrocarbon stapled a- helical peptide Protein therapeutics comprise different classes of proteins with various levels of complexity and heterogeneity, and different physiochemical properties. In addition, changes can occur at any point during manufacturing, prior to patient administration, in vivo after administration, during PK sample collection and analysis 3

4 Biotransformation of Protein Therapeutics De-pegylation (Pegylated protein) Proteolysis, glycosylation Deamidation and isomerization (e.g., Asn to Asp, Asp to Iso-Asp) Oxidation (e.g., Methionine) 4

5 Literature Examples Pathway Proteolysis Deamidation (Asn) Oxidation Examples GLP-1: N-terminal truncation, resulting in loss of biological activities Deamidation of Asn in human growth hormone releasing factor, resulting in fold reduction in potency IFN a2a, on methionine, resulting in enhanced immunogenicity; Insulin, on cysteine, resulting in aggregate formation References: 1. Bongers J. et al, Int. J. Peptide Protein Res. 39, 1992, Torosantucci R. et al, Pharm. Res. 31, 2014, Ryff J. C., J. Interferon Cytokine Res. 17 Suppl 1, 1997, S Hochuli E., J. Interferon Cytokine Res. 17 Suppl 1, 1997, S Constantino H.R., et al, Pharm. Res. 11, 1994, Xiao Q., et al, Biochemistry 40, 2001,

6 Current Gold Standard in Quantifying Protein Therapeutics in Biological Matrices Ligand-Binding Assays (LBA) Signal Detection Analyte Examples of Assay Platforms: ELISA MSD (Meso-Scale Discovery) Gyrolab Forte-Bio Luminex Capture by antidody Solid Surface 6

7 Challenges in Quantifying Protein Therapeutics Metabolites / Catabolites in Biological Matrices 1. LBA relies on interactions between analytes and the capture reagents, thus it may or may not be possible to distinguish between the drug and its metabolites / catabolites, especially when the metabolic site is not part of the binding epitope Binding with capture antibody Binding with capture antibody More selective detection techniques needed to distinguish them 7

8 Challenges in Quantifying Protein Therapeutics Metabolites / Catabolites in Biological Matrices 2. Metabolites / Catabolites may have very low concentrations 3. Presence of other endogenous proteins in serum / plasma samples, potentially at much higher concentrations Think of a needle in a haystack 8

9 Challenges in Quantifying Protein Therapeutics Metabolites / Catabolites in Biological Matrices 2. Metabolites / Catabolites may have very low concentrations 3. Presence of other endogenous proteins in serum / plasma samples, potentially at much higher concentrations Think of a needle in a haystack Highly sensitive and selective detection techniques needed 9

10 Emerging Technologies in Quantifying Protein Therapeutics LC-MS has been the method of choice in quantifying SMT and in proteomics, and it has been used to quantify protein therapeutics Mass spectrometers detect analytes by their mass-tocharge ratios. Tandem mass spectrometers (MS/MS) and full-scan high resolution mass spectrometers (HRMS) have been used, with MS/MS being more sensitive in detection LC - MS analyte Sample other compounds 10

11 Advantages of LC-MS/MS in the Quantitation of Protein Catabolites LC-MS/MS is very selective, able to differentiate analytes with different masses, even with a difference of one oxygen atom (of mass of ~16 Da) Assay selectivity can be further improved by combining with immunocapture, to isolate the analytes of interest before LC- MS/MS analysis 11

12 General Methodology for Protein Quantification by LC-MS/MS Proteins (pegylated or non-pegylated) are digested by enzymes, e.g., trypsin; or hydrolyzed by chemical means, e.g., limited acid hydrolysis, to produces multiple peptides, usually in the MW range of Da. These unique peptides serve as surrogates of the protein These surrogate peptides can be analyzed by MS easily. Usually, one peptide is selected for quantitation Enzymatic digestion or limited acid hydrolysis 12

13 Case Study #1: C-terminal Clipping of a Fusion Protein 13

14 Case Study #1 - Background pab Capture Domain 1 Linker Domain 2 Intact pab Capture C-terminal Binding site C-terminal clipping Domain 1 Linker Domain 2 Clipped C-terminal region is susceptible to proteolysis, resulting in a loss of activity First generation capture reagent (polyclonal Ab, pab capture) could not distinguish between intact protein and its clipped version Exploratory LC-MS/MS assay was developed to measure both C- terminal intact and total (intact + clipped) protein concentrations 14

15 Challenge #1 - Generating Suitable Surrogate Peptides Domain 1 Linker Domain 2 Intact Domain 1 Linker Domain 2 Trpysin cleavage site Clipped Total (clipped + intact) protein concentration: Trypsin digestion generates multiple surrogate peptides, unique to the fusion protein, across different parts of the protein. A total of 3 surrogate peptides, chosen across Domain 1, Linker and Domain 2, away from the C-terminal, were quantified. All intact and clipped versions of the protein are quantified 15

16 Challenge #1 - Generating Suitable Surrogate Peptides (continued) Domain 1 Linker Domain 2 Asp---Pro Intact Domain 1 Linker Domain 2 Trpysin cleavage site Clipped Intact protein concentration: Trypsin digestion does not generate a suitable surrogate peptide encompassing intact C-terminus An acid-labile Asp-Pro amide bond close to C-terminus. Incubating the protein in dilute formic acid at elevated temperature breaks the amide bond and generates a surrogate peptide encompassing the C-terminus 16

17 Challenge #2 - Isolating the Fusion Protein and its Metabolites / Catabolites Capture mab Intact Domain 1 Linker Domain 2 Capture mab Clipped Domain 1 Linker Domain 2 The protein binds strongly to serum albumin, making surrogate peptide generation and subsequent MS detection very difficult, if not impossible Applied immunocapture for sample cleanup A capture mab, targeting Domain 1 region was used to capture all intact and clipped versions of proteins 17

18 Methodology Serum samples extracted by immunocapture Trypsin digestion Acid hydrolysis 3 surrogate peptides by LC-MS/MS total protein concentration 1 surrogate peptide by LC-MS/MS intact protein concentration 18

19 Intensity, cps Analyte Area / IS Area Representative Standard Curve and LLOQ XIC of +MRM (10 pairs): / Da Max cps. LLOQ = 200 ng/ml %DEV < ±2.6% Between run %CV < 11.5% Within run %CV < 12.8% Total 3 runs "Linear" Regression ("1 / (x * x)" weighting): y = x (r = ) XIC of +MRM (10 pairs): / Da Max cps Blank with IS Time, min e4 4.0e4 6.0e4 8.0e4 1.0e5 Analyte Conc. / IS Conc. 19

20 Concentration (ng/ml) Results dosing dosing LBA Concentration C-terminal peptide (intact) YYY Domain PKE1 peptide (total) YLY N-terminal FGF21-1 Peptide (total) ALK Center FGF21-2 Peptide (total) Hour Monkey study with 10 mpk dose The LC-MS/MS assay provided the concentration of the intact protein in circulation and facilitated the rapid decision making for the program 20

21 Case Study #2: Isomerization of Aspartic Acid in Complementarity Determining Region (CDR) 21

22 Deamidation & Isomerization Hydrolysis Hydrolysis Non-enzymatic [OH - ]-dependent 22 Deamidation of Asn in mabs can be a major route of degradation (shelf life; in vivo) Deamidation in the PENNY peptide in Fc of IgG1 demonstrated at high ph; alternate site at low ph Deamidation in the CDR of mabs (NG/NS) have been reported to cause loss of activity May raise concerns regarding efficacy and immunogenicity

23 Isomerization of Aspartic Acid in CDR Isomerization upon storage: Clear relationship between amount of isomerized antibody and age of drug product (~4.2% increase in isomerization per year) Effect on Potency: Increase in Iso-Asp variant correlated with decrease in potency What happens in vivo? Lower concentration than in drug substance and drug product, need more sensitive technique 23

24 I n t e n s i t y, c p s Representative Chromatogram of Asp and Iso-Asp Surrogate Peptides XIC of +MRM (1 pair): / Da from Sample 2 (Iso-Asp_100_0.2 mg/ml) of liug4_ _isoasp_set1.wiff (Turbo Spray) Max. 3.6e4 cps. 3.6e4 3.4e4 3.2e4 3.0e4 2.8e4 2.6e4 2.4e4 2.2e4 2.0e4 Iso-Asp Asp Iso-Asp and Asp need to be chromatographically separated because they have the same mass 1.8e4 1.6e4 1.4e4 1.2e4 1.0e Interference Time, min Courtesy of Jonathan Haulenbeek & Guowen Liu

25 Comparing the Ratios of Iso-Asp/Asp in the Drug and ex vivo samples Drug spiked in monkey Serum (mg/ml) Average Peak area ratio Iso-Asp/Asp %Iso-Asp Average Peak area ratio Iso-Asp/Asp %Iso-Asp Timepoints, Pooled samples A B C D E F Note: Different lots of DS/DP were used in the spike samples and pooled samples 25 Courtesy of Jonathan Haulenbeek & Guowen Liu

26 Case Study 2 - Summary Observations: The feasibility of using LC-MS/MS method to measure the ratio of Asp / iso Asp in vivo has been demonstrated Synthesized isomerized peptide standard to confirm its stability during digestion Additional Work: Additional stability work to ensure analyte stability during sample processing and storage Animal studies to evaluate impact of isomerization on in vivo efficacy and potential immunogenicity 26

27 Concluding Remarks Emerging hybrid technologies such as LC-MS/MS, in combination with immunocapture are new tools to quantify the metabolites / catabolites of protein therapeutics These new tools are meant to provide complementary data to the ligand binding assays Combining data from these complementary techniques provides a better insight into the metabolism of the protein therapeutics, and its impact on their efficacy, safety, PK/PD relationship, immunogenicity 27

28 Acknowledgement Mark Arnold Anne Aubry Binodh Desilva Jonathan Haulenbeek Alexander Kozhich Guowen Liu Paul Morin Steven Piccoli Renuka Pillutla Frank Zambito Jianing Zeng 28

29 BACK-UP 29

30 References J. Bongers, E.P. Heimer, T. Lambros, Y.E. Pan, R.M. Campell, A.M. Felix, Int. J. Peptide Protein Res. 39, 1992, R. Torosantucci, C. Schoneich, W. Jiskoot, Pharm. Res. 31, 2014, J.C. Ryff, J. Interferon Cytokine Res. 17 Suppl 1, 1997, S29-33 E. Hochuli, J. Interferon Cytokine Res. 17 Suppl 1, 1997, S15-21 H.R. Constantino, R. Langer, A.M. Klibanow, Pharm. Res. 11, 1994, Q. Xiao, J. Giguere, M. Parisien, W. Jeng, S.A. St-Pierre, P.L. Brubaker, M.B. Wheeler, Biochemistry 40, 2001,

31 Intens. [a.u.] Intact C-terminal Fragment Peptide from Acid Digestion [M+H] m/z 31 Courtesy of Jonathan Haulenbeek