Recommendations for validation of LC-MS/MS bioanalytical methods for protein biotherapeutics white paper p highlights

Recommendations for validation of LC-MS/MS bioanalytical methods for protein biotherapeutics white paper p highlights g Rand Jenkins on behalf of the AAPS Bioanalytical Focus Group Protein Bioanalysis by MS Committee (PBMSC) & the PBMS Method Validation Consortium EBF 8 th Open Symposium 18 November 2015

Overview 1 Bioanalytical science and regulatory landscape 2 PrD-LC-MS BA approach 3 Highlights of the WP recommendations 4 Technical concerns 5 Areas needing clarification/expansion i 6 Final thoughtsht

Bioanalytical Landscape why the need for WPs? Technology Biotherapeutics are increasingly popular and rapidly evolving Molecular constructs are becoming more complex Analytical instruments and techniques continue to advance New scientific questions must be answered

Bioanalytical Landscape why the need for WPs? Technology Biotherapeutics are increasingly popular and rapidly evolving Molecular constructs are becoming more complex Analytical instruments and techniques continue to advance New scientific questions must be answered Regulatory Regulatory guidelines evolve very slowly Traditional focus (i.e., SM by LC-MS and LM by LBA) may or may not be appropriate for new applications of science & technology Regulators don t have enough experience with new techniques Studies are conducted globally and submitted to different regulators Multiple BMV guidelines coming out from different regions/countries Bioanalysts are uncertain

When do we need a fully validated assay? The general rule that can be applied is that if the data generated will support regulatory action, such as assessing safety and/or efficacy, or supporting labeled-dosing instructions or patient treatment, then the data must be reliable and the analytical assays should be fully validated Booth B. When do you need a validated assay? Bioanalysis 3(24), 2729 30 (2011).

When do we need a fully validated assay? The results of animal toxicokinetic studies and of clinical trials, including bioequivalence studies are used to make critical decisions supporting the safety and efficacy of a medicinal drug substance or product. It is therefore paramount that the applied bioanalytical methods used are well characterised, fully validated and documented to a satisfactory standard in order to yield reliable results. Guideline on bioanalytical method validation. European Medicines Agency, Committee for Medicinal Products for Human Use, London, UK (2011). For pivotal studies that require regulatory action for approval or labeling, such as BE or PK studies, the bioanalytical methods should be fully validated. For exploratory methods used for the sponsor s internal decision i making, less validation may be sufficient i Guidance for Industry: Bioanalytical Method Validation - Draft Guidance. U.S. Department of Health and Human Services, FDA, Rockville, MD, USA (2013)

Editor ial For reprint orders, please contact reprints@future-science.com LC MS/MS of large molecules in a regulated bioanalytical environment which acceptance criteria to apply? The current thinking from the EBF Topic Team is to start with a conservative approach when defining acceptance criteria and not to propose acceptance criteria that are still too demanding for the technology/analytical approach Keywords: acceptance criteria European Bioanalysis Forum LC MS peptide p protein regulated bioanalysis Analysis of large molecules has become the talk of t he day in the bioanalyt ical communit y. T he increasing importance of peptides and proteins as therapeutic agents, together with the enormous possibilities offered by new MS-based technology, has opened a new world for the bioanalytical scientist. The European Bioanalysis Forum (EBF) has been following these new developments closely and havededicated atopicteam (TT) to di scuss and share experiences on t he bioanalysi s of large molecules with LC MS-based technologies. In this Editorial, the EBF wants to share their perspect ive on how to i ntegrate LC MS of peptides and proteinsin regulated bioanalysis. With larger molecules, that is, peptides, proteins and oligonucleotides, becoming increasi ngly i mport ant as t herapeut ic agent s i n t he future, the scientific community is building latter methods detect molecules based on binding affinity and 3D conformational structure, they may not be able to distinguish between a protein and itsmetabolites. MS-based methods do have that ability and will be able to generate more accurate data on unchanged peptide/ protein concentrations, compared with LBA, i n cases where met abol i sm hampers accurat e data from LBA. MS methods normally give total drug concentrations. For LBA methods, this may depend on the type of assay, and they can give either total or free drug concentration. Depending on the project in development, the crossreactivity of LBAs towards analyte-like like compounds (potentially active metabolites) can be an advantage to better describe the PD. However, in case we need to document the accurate PK behavior of the compound, M S-based Magnus Knutsson Ferring Pharmaceuticals A/S, Copenhagen, Denmark Ronald Schmidt Sanofi-Aventis Deutschland GmbH, Frankfurt, Germany Philip Timmerman Author for correspondence: Bioanalysis Department, Janssen R&D NV, Turnhoutseweg 30, Beerse, Belgium Tel.: +32 1460 3581 E-mail: ptimmerm@its.jnj.com Knutsson M, Schmidt R, Timmerman P. LC MS/MS of large molecules in a regulated bioanalytical environment which acceptance criteria to apply? Bioanalysis 5(18), 2211 2214 (2013).

Protein LC-MS/MS Bioanalysis General Strategy Protein/Peptide in Biomatrix Sample MW < ~10 kda MW > ~10 kda Direct Measurement (Intact Analyte) Indirect Measurement (Proteolytic Peptides) Extraction/Enrichment (Chemical (e.g. PPT, SPE) or Affinity Capture) Extraction/Enrichment (Chemical (e.g. PPT) or Affinity Capture) LC MS/MS Proteolytic Digestion LC MS/MS Clean up (SPE or Peptide AC) 9

PrD-LC-MS/MS Method Approaches

Recombinant human IgM antibody example rhigm MW 900 kda AC, denature, reduce & alkylate, digest with trypsin LLIYDITK (surrogate peptide) Czajkowskya, D. M. and Shao, Z. (2000) Proc. Natl. Acad. Sci. USA 106, 14960 14965.

WP highlights

accuracy & & precision i

European Bioanalysis Forum Editorial the EBF propose the following when it comes to acceptance criteria for large molecules using LC MS/MS: for smaller intact tanalytes (e.g. peptides and oligonucleotides) l it is normally recommended to apply 4 6 15 acceptance criteria. For larger analytes, especially if a hybrid LC MS/MS approach is employed, 4 6 20 acceptance criteria are recommended. Knutsson M, Schmidt R, Timmerman P. LC MS/MS of large molecules in a regulated bioanalytical environment which acceptance criteria to apply? Bioanalysis 5(18), 2211 2214 (2013).

Validation Parameters Parameter Protein LBA Small Molecule LC MS/MS Calibration curve regression function Non linear with 4 or 5 parameter logistic. Anchor points may be used Linear preferred, non linear with justification Protein LC MS/MS via Surrogate Peptide (Recommendation) Linear recommended; non linear models dlmay be acceptable with some affinity capture methods Lower Limit of Quantification (RE, CV) 25% 20% 25% Calibration standards 20% (except LLOQ 15% (except LLOQ) 20% (except LLOQ) (RE, CV) and ULOQ) Accuracy & precision (RE, CV) Within 20% (LLOQ/ULOQ QCs within 25%) Min. 6 runs Within 15% (LLOQ QC within 20%) Min. 3 runs Within 20% (LLOQ QC within 25%) Min. 3 runs 15

Validation Parameters Parameter Protein LBA Small Molecule LC MS/MS Selectivity/specificity Non specific matrixrelated interferences: using individual matrix lots; include hemolyzed, lipemic, disease population, as appropriate it 10 lots; LLOQ: 80% of fortified lots Accuracy within 25% 6 lots; blanks: <20% of LLOQ or <5% of IS LLOQ: 80% of fortified lots Accuracy within 20% Protein LC MS/MS via Surrogate Peptide (Recommendation) 6 10 lots; blanks: <20% of LLOQ or <5% of IS LLOQ: 80% of fortified lots Accuracy within 25% Specific interferences: using LLOQ (and ULOQ for LBAs) QC samples Fortified with available lbl ADA, soluble target, catabolites, or con meds (LM only?). Accuracy within 25% Fortified with available lbl metabolites Fortified with available ADA, soluble target, or con meds, as catabolites or conmeds, appropriate. as appropriate. Accuracy within 20% Accuracy within 25% 16

Validation Parameters Parameter Protein LBA Small Molecule LC MS/MS Protein LC MS/MS via Surrogate Peptide (Recommendation) Dilutional integrity/linearity X (20%) X (15%) X (20%) Parallelism X (30%) endogenous endogenous Recovery NA X X Digestion efficiency NA NA maybe Matrix effect NA X (15%) X (20%) Carry over Gyrolab X (<20%) X (<20%) Stock stability? X (5 7%) X (10%) Extract stability NA X (15%) X (20%) Matrix stability X (20%) X (15%) X (20%) Critical reagents X NA enzymes & AC ISR (at least 67%) X (30%) X (20%) X (30%) 17

Hybrid BMV approach PrD-LC-MS/MS BA General consensus Combine appropriate small and large molecule elements Accuracy & precision Apply 4-6-20 (LM) criteria; LLOQ (25%), but S/N still 5:1 Selectivity/specificity Apply A l SM approach for matrix blanks, zero standards, d and LLOQ spikes For AC-based methods, increase matrix lots (including disease-state) and evaluate potential binding interferences similar to LM approach Recovery Should be sufficient & reproducible overall

concerns

Reference standards quality and COAs PrD-LC-MS/MS BA Technical concerns

Contrary to conventional molecules, a pure reference material that can serve as a calibration standard is either difficult or sometimes impossible to obtain for this class of (protein) compounds. Therefore extreme care should be taken in order to ensure that the reference material used in the different analytical calibration processes is representative of the material used in clinical i l trials, including clinical i l pharmacokinetics. i GUIDELINE ON THE CLINICAL INVESTIGATION OF THE PHARMACOKINETICS OF THERAPEUTIC PROTEINS. EMEA, Committee for Medicinal Products for Human Use (CHMP), London, UK (2007) Doc. Ref. CHMP/EWP/89249/2004.

Reference standards quality and COAs Enrichment efficiency y( (degree of purification ) reproducibility Digestion completeness and consistency enzymes as critical reagents PrD-LC-MS/MS BA Technical concerns Recovery reproducibility focus on overall (combine with MF experiment?)

Procedure for evaluation of processing recovery and matrix effect Jiang H et al, Fully Validated LC-MS/MS Assay for the Simultaneous Quantitation of Coadministered Therapeutic Antibodies in Cynomolgus Monkey Serum, Anal. Chem., 2013, 85 (20), 9859 9867

Monitoring peptides selection and evaluation (criteria?) PrD-LC-MS/MS BA Technical concerns

PrD-LC-MS/MS BA Monitoring peptides One or more secondary proteotypic peptides chosen and analyzed, along with the surrogate peptide, for monitoring purposes A notable change in peptide response ratio(s) trending over a PK or TK profile may be indicative of a biotransformation issue Monitoring peptides must have the following properties Unique to the analyte protein Sufficiently free from matrix interference Adequately stabile and sensitive by SRM Located in a subunit or other key part of the biotherapeutic Not used for quantification Response ratios evaluated for consistency but generally without Response ratios evaluated for consistency, but generally without specific acceptance criteria (case-by-case)

Monitoring peptides selection and evaluation (criteria?) PrD-LC-MS/MS BA Technical concerns Internal standards SIL-protein, ext d SIL-peptide, p SIL-peptide, p analogs? overall suitability? some method steps may be uncontrolled

Affinity capture-based approaches

Monitoring peptides selection and evaluation (criteria?) PrD-LC-MS/MS BA Technical concerns Internal standards SIL-protein, ext d SIL-peptide, p SIL-peptide, p analogs? overall suitability? some method steps may be uncontrolled Affinity capture (LB) considerations critical reagent worries? selectivity/specificity i ifi i evaluation curve non-linearity (use quadratic or 4/5-PL fits?)

Protein LC-MS/MS BA Matrix stability 37 C C (whole blood/collection) 20-25 C (ambient) 0-4 C (ice) -20 C (freezer) -80 C C (cryo)

Stability assumption No loss of response = analyte stable Loss of response = analyte unstable Maybe, or maybe not.

Monitoring peptides selection and evaluation (criteria?) PrD-LC-MS/MS BA Technical concerns Internal standards SIL-protein, ext d SIL-peptide, p SIL-peptide, p analogs? overall suitability? some method steps often uncontrolled Affinity capture (LB) considerations critical reagent worries? selectivity/specificity i ifi i evaluation curve non-linearity (use quadratic or 4/5-PL fits?) Stability false negative result if selected peptides unaffected? false positive result if NSB or other issue?

Protein LC-MS BA Areas for clarification/expansion Hybrid constructs MDBs (multidomain biologics) Intact protein quantification (LC-HRAMS) Catabolite analysis ADAs? What else?

Feedback from regulatory authorities Regulators have very limited experience with PrD-LC-MS/MS data in submissions Regulators have been present during many discussions of WP recommendations at various meetings/workshops 8th Workshop on Recent Issues in Bioanalysis (WRIB) 2014 AAPS-NBC Workshop on Method Development, Validation, and Troubleshooting of Ligand-Binding Assays in the Regulated Environment, 2014 6th Japan Bioanalysis i Forum Symposium: challenge of regulated bioanalysis, i Feb 2015 AAPS Annual Conference, Oct 2015 EBF Open Symposium, Nov 2015 Regulators have recommended that proposed BA strategies be discussed with them early on so they can understand the methodology and be able to ask questions ahead of time

Protein LC-MS BA Final thoughts PrD-LC-MS technology is not addressed in BMV guidances Several industry ygroups have considered and proposed recommended BMV practices Consensus to combine SM and LM elements There are still areas for clarification/expansion Apply good science and justify when appropriate

Acknowledgements AAPS white paper co-authors: Rand Jenkins, PPD; Jeffrey X. Duggan & John Yu, Boehringer-Ingelheim Pharmaceuticals; Anne-Françoise Aubry, Jianing Zeng, & Yan J. Zhang, Bristol Myers Squibb; Jean W. Lee, BioQualQuan; Laura Cojocaru, Tandem Labs; Dawn Dufield, Pfizer; Fabio Garofolo, Algorithme Pharma; Surinder Kaur & Keyang Xu, Genentech; Gary A. Schultz, Quintiles; Ziping Yang, Novartis; and Faye Vazvaei, Roche Pre-submission reviewers: Bradley Ackermann & Michael Berna, Eli Lilly and Company; Lakshmi Amaravadi & Lauren Stevenson, Biogen Idec; Mark Arnold & Binodh DeSilva, Bristol Myers Squibb; Suzanne Brignoli & An Song, Genentech; Margarete Brudny-Kloeppel, Bayer Pharma AG; Jimmy Flarakos, Novartis; Eric Fluhler & Lindsay King, Pfizer; Stephen Lowes; Matt Szapacs & Eric Yang, GlaxoSmithKline

Acknowledgements Special thanks to: Jeff Duggan Anne-Françoise Aubry Jianing Zeng * Faye Vazvaei EBF program committee

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