Analytical Characterization of Biotherapeutics: Looking to the Future with Biosimilars

Transcription

1 Analytical Characterization of Biotherapeutics: Looking to the Future with Biosimilars Jared Auclair, Ph.D. Northeastern University Department of Chemistry and Chemical Biology Barnett Institute of Chemical and Biological Analysis Biopharmaceutical Analysis Training Laboratory

2 History of Northeastern s Burlington Campus Kostas Hall Elliott Hall military housing entrance to campus Currently: Innovation Campus US Army Nike Missile Base 9/4/59 Acquired by Northeastern from US in 1963

3 BATL : Focuses on Biotherapeutic Training and Analysis Forging New Global Collaborations 1-Day Intensive Courses: Introduction to Protein Analysis Protein Structure Analytics in Developing Biopharmaceuticals From Discovery ~20 economiesto Market through Analytical eyes 2-Day Short Courses: Intact Protein Analysis Glycoprotein and Glycan Analysis Recombinant Ab and Biosimilars ~60 economies

4 DNA double-helix 1950s Recombinant DNA technology 1970s Insulin approved early 1980s Zarxio approved in March 2015 (US) History of Biotherapeutics

5 What is a biotherapeutic? Major MajorClasses Classes Major Classes As defined by the national cancer institute: From living organisms Recombinant DNA 3. Vaccines 4. Gene Therapy A type of treatment that uses substances made from living organisms to treat disease. These substances may occur naturally in the body or may be made in the laboratory. Stem cell therapy mab hgh Blood transfusion erythropoetin Organ transplant

6 Importance of Protein (Antibody) Drugs Product Type 2016 Revenue (USD bn) 2010 Revenue (USD bn) Humira Biologic Avastin Biologic Rituxan Biologic Enbrel Biologic Crestor Small molecule Seretide/Advair Respiratory/device Remicade Biologic Herceptin Biologic Revlimid Small molecule Lantus Biologic

7 Biopharmaceutical Development Process Original biologics (innovator drugs) coming off patent Leads to creation of generic* drugs (biosimilars) More complex than creating a small molecule generic *Biosimilars are NOT generics; as they are made in living organisms and are unique molecules.

8 Biosimilars active drug substance (e.g. recombinant protein) made by a living organism or derived from a living organism by means of recombinant DNA or controlled gene expression methods often times to mimic a drug on the market Post-translation modifications Production/isolation/purification may result in chemical modifications According to the FDA: Ø that the biological product is highly similar to the reference product (innovator) notwithstanding minor differences in clinicallyinactive components; and Ø there are no clinically meaningful differences between the biological product and the reference product in terms of the safety, purity, and potency of the product.

9 Common misperception when creating a biosimilar. Graphic by J. Tella (NEU)

10 Highly similar analytical data reduces the risk of clinical Differences More Analytical data required Intact Mass Analysis Peptide Map Glycan Analysis Steven Kozlowski, OPS/CDER/US FDA, WCBP 2013

11 Analytical Workflow for Biotherapeutics Totality of the Evidence Intact Mass Analysis Peptide Mapping Released Glycans Top Down Analysis Bottom Up Analysis Retention Time Fluorescence Mass Top Down Analysis (RP)-UPLC-MS (RP)-UPLC-MS/MS HILIC-UPLC-MS

12 Analytical Workflow for Biotherapeutics Totality of the Evidence Intact Mass Analysis Peptide Mapping Released Glycans Top Down Analysis Bottom Up Analysis Retention Time Fluorescence Mass Top Down Analysis UPLC-MS (RP)-UPLC-MS/MS HILIC-UPLC-MS

13 PTMs of SOD1 identified from human nervous tissue Δ+119 Da I went on to characterize the most predominant modification (Auclair, 2013a) Human spinal cord

14 (Auclair, 2013a) Protective cysteinylation: most prevalent PTM

15 Protein S-thiolation (cysteinylation) studied using Intact Mass Proteomics Traditional proteomics techniques (e.g. reduction) remove S-thiolation. Bruker 9.4T FTICR Waters Q-Tof

16 Anaerobic Purification Reduces the Amount of S-thiolation Intensity [arb. Units} S-thiolation (Auclair, 2014). m/z Molecular Mass (Da)

17 Analytical Workflow for Biotherapeutics Totality of the Evidence Intact Mass Analysis Peptide Mapping Released Glycans Top Down Analysis Bottom Up Analysis Retention Time Fluorescence Mass Top Down Analysis RP-UPLC-MS (RP)-UPLC-MS/MS HILIC-UPLC-MS

18 Liquid Chromatography Separation of a Monoclonal Antibody 5-95 % B (100% acetonitrile with 0.1 % formic acid) over 60 minutes

19 Intact Analysis: mab MS

20 Intact Mass Shows Glycosylation Delta ~ 160 Da

21 Heterogeneous Mixture: Dissociating mab Intact Ab Half Ab Heavy chain Light chain

22 Liquid Chromatography Highlights Dissociating and Aggregating Antibodies Dissociating antibody chains These peaks will increase as the Ab dissociates

23 Analytical Workflow for Biotherapeutics Totality of the Evidence Intact Mass Analysis Peptide Mapping Released Glycans Top Down Analysis Bottom Up Analysis Retention Time Fluorescence Mass Top Down Analysis (RP)-UPLC-MS RP-UPLC-MS/MS HILIC-UPLC-MS

24 HPLC Immuno-affinity Isolation of clusterin Sample injection 2 protein depletion Clusterin anti-body Unbound proteins + column column salts and other particulates Pump Anti-body conjugated on porous bead C4 reverse phase Desalting column Purified Clusterin sample collector Unbound proteins +salts and Other particulates 250 mvolts Clusterin IgG and albumin depleted proteins Minutes Gbormittah et al, JPR (2015)

25 Glycopeptides Identification Glycosylation site Trypsin MH+ (Da) GluC MH+ (Da) Site occupancy status N-86 KKEDALN/DETR KKEDALN/DETRE Fully glycosylated KKKEDALN/DETR KKEDALN/DETRESE Fully glycosylated KKKEDALN/DETRE Fully glycosylated KKKEDALN/DETRESE Fully glycosylated N-103 ELPGVCNETMMALWEECK PCLK N/A N-145 QLEEFLNQSSPFYFWMNGD RIDSLLE FLNQSSPFYFWMNGDRID SLLE N/A N-291 EIRHN/DSTGCLR IRHN/DSTGCLRMKD Fully glycosylated HN/DSTGCLR Fully glycosylated N-317 EILSVDCSTNNPSQAK ILSVDCSTNNPSQAKLRRE Not glycosylated N-354 MLN/DTSSLLEQLNE Partially glycosylated N-374 LAN/DLTQGEDQYYLR Fully glycosylated Gbormittah et al, JPR (2015)

26 Cysteine Scrambling: Traditional proteomics is misleading in identification of the site of modification Need Top Down Proteomics (Auclair, 2013a) cysteine scrambling

27 Analytical Workflow for Biotherapeutics Totality of the Evidence Intact Mass Analysis Peptide Mapping Released Glycans Top Down Analysis Bottom Up Analysis Retention Time Fluorescence Mass Top Down Analysis (RP)-UPLC-MS Top Down MS/MS HILIC-UPLC-MS

28 Top Down Analysis Avoids Cysteine Scrambling: Cysteine 111 is the site of modification Mass of SOD1 C-terminus y139 (Auclair, 2013a)

29 Thus, best practice includes both a Top Down and Bottom-up Analysis

30 Top Down and Bottom Up Analysis are Complimentary: Human Growth Hormone Sequence map of hgh showing peptides and modifications identified by bottom-up and top-down proteomics.

31 Analytical Workflow for Biotherapeutics Totality of the Evidence Intact Mass Analysis Peptide Mapping Released Glycans Top Down Analysis Bottom Up Analysis Retention Time Fluorescence Mass Top Down Analysis (RP)-UPLC-MS (RP)-UPLC-MS/MS HILIC-UPLC-MS

32 NIBRT Glycobase Gradient (right bottom) Retention time calibrated using a dextran ladder (right top) Glycans are released with PNGase F Labeled with 2-AB fluorophore Analyzed using HILIC column and MS Released Glycan Analysis: HILIC-LC-MS

33 Released Glycan Analysis from mab Extracted ion chromatogram for glycan mass Fluorescent and Mass Spec Confirmation of released Glycans

34 Analytical Workflow for Biotherapeutics Totality of the Evidence SUMMARY Intact Mass Analysis Peptide Mapping Released Glycans Totality of the Evidence Throughout the Life Cycle Top Down Analysis Bottom Up Analysis Retention Time Fluorescence Mass (including post approval analysis; fingerprinting) Top Down Analysis (RP)-UPLC-MS (RP)-UPLC-MS/MS HILIC-UPLC-MS

35 Acknowledgements Northeastern Jeff Agar William Hancock Brandeis Greg Petsko Dagmar Ringe Umass Medical School Robert Brown Daryl Bosco Kristin Boggio Melissa Rotunno Waters Kelly Johnson Barbara Sullivan