Recommendations for validation of LC-MS/MS bioanalytical ... · PrD-LC-MS/MS BAMS/MS BA Monitoring peptides • One or more secondary proteotypic peptides chosen and analyzed, along

Recommendations for validation ofLC-MS/MS bioanalytical methods for protein

biotherapeutics—white paper highlightsp p p g g

Rand Jenkinson behalf of the AAPS Bioanalytical Focus GroupProtein Bioanalysis by MS Committee (PBMSC)

& the PBMS Method Validation Consortium

EBF 8th 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

A di l ifi ti / i5 Areas needing clarification/expansion

6 Fi l th ht6 Final thoughts

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

RegulatoryRegulatory• Regulatory guidelines evolve very slowly• Traditional focus (i e SM by LC-MS and LM by LBA) may or may notTraditional 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 techniquesRegulators 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/countriesp g g g

Bioanalysts are uncertain

When do we need a fully validated assay?

“The general rule that can be applied is that if the dataThe 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 instructionssafety 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”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 bioanalyticalproduct. 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.”y y

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 i t l d i i ki l lid ti b ffi i t”internal decision making, less validation may be sufficient”

Guidance for Industry: Bioanalytical Method Validation - Draft Guidance. U.S. Department of Health and Human Services FDA Rockville MD USA (2013)Department of Health and Human Services, FDA, Rockville, MD, USA (2013)

Ed it o r ia l

For reprint orders, please contact [email protected]

LC–MS/MS of large molecules in a regulated bioanalytical environment – which acceptance

p , p p @f

bioanalytical environment – which acceptance criteria to apply?

Keywords: acceptance criteria European Bioanalysis Forum LC–MS peptide protein regulated bioanalysis

“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…”

y p p y p p p g y

Analysis of large molecules has become the talk of the day in the bioanalytical community. The increasing importance of peptides and proteins as therapeutic agents, together with the enormous

latter methods detect molecules based on bind-ing affinity and 3D conformational structure, they may not be able to distinguish between a protein and itsmetabolites. MS-based methods

Magnus KnutssonFerring Pharmaceuticals A/S, Copenhagen, Denmark

Ronald Schmidt therapeutic agents, together with the enormous possibilities offered by new MS-based technol-ogy, has opened a new world for the bioanalyti-cal scientist. The European Bioanalysis Forum (EBF) has been following these new developments closelyand havededicated aTopicTeam (TT) to

protein and its metabolites. MS based methods do have that ability and will be able to gener-ate more accurate data on unchanged peptide/protein concentrations, compared with LBA, in cases where metabolism hampers accurate data from LBA MS methods normally give

Ronald Schmidt Sanofi-Aventis Deutschland GmbH, Frankfurt, Germany

closelyand have dedicated aTopicTeam (TT) to discuss and share experiences on the bioanalysis of large molecules with LC–MS-based technologies. In this Editorial, the EBF wants to share their per-spective on how to i ntegrate LC–MS of peptides and proteinsin regulated bioanalysis

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

Philip TimmermanAuthor for correspondence: Bioanalysis Department Janssen and proteins in regulated bioanalysis.

With larger molecules, that is, peptides, pro-teins and oligonucleotides, becoming increas-ingly important as therapeutic agents in the future, the scientific community is building

crossreactivity of LBAs towards analyte-like compounds (potentially active metabolites) can be an advantage to better describe the PD. However, in case we need to document the accu-rate PK behavior of the compound, MS-based

Bioanalysis Department, Janssen R&D NV, Turnhoutseweg 30, Beerse, Belgium Tel.: +32 1460 3581 E-mail: [email protected]

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/ y gy

Protein/Peptide in Biomatrix Sample

MW < ~10 kDa MW > ~10 kDa

Indirect Measurement(Proteolytic Peptides)

Direct Measurement (Intact Analyte) (Proteolytic Peptides)(Intact Analyte)

Extraction/Enrichment Extraction/Enrichment/(Chemical (e.g. PPT, SPE)

or Affinity Capture)

/(Chemical (e.g. PPT)or Affinity Capture)

ProteolyticDigestion

LC‐MS/MSClean up (SPE or

LC‐MS/MS

DigestionPeptide AC)

9

PrD-LC-MS/MS Method Approaches

Recombinant human IgM antibody exampleg y p

rhIgMMW 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&

accuracy&&i i&i iprecisionprecision

European Bioanalysis Forum EditorialEuropean Bioanalysis Forum Editorial

“ …the EBF propose the following when it comes toacceptance criteria for large molecules using LC–MS/MS: for

ll i t t l t ( tid d li l tid ) itsmaller intact analytes (e.g. peptides and oligonucleotides) itis normally recommended to apply 4–6–15 acceptancecriteria For larger analytes especially if a hybrid LC MS/MScriteria. For larger analytes, especially if a hybrid LC–MS/MSapproach is employed, 4–6–20 acceptance criteria arerecommended ”recommended.

Knutsson M, Schmidt R, Timmerman P. LC–MS/MS of large molecules in a regulated bioanalytical environment which acceptance criteria to apply?regulated bioanalytical environment – which acceptance criteria to apply? Bioanalysis 5(18), 2211–2214 (2013).

Validation ParametersParameter Protein LBA Small Molecule

LC‐MS/MSProtein LC‐MS/MSvia Surrogate Peptide(Recommendation)

Calibration curvei f ti

Non‐linear with4 5 t

Linear preferred,li ith

Linear recommended; li d lregression function 4 or 5 parameter

logistic. Anchor points may be used

non‐linear with justification

non‐linear models may be acceptable with some affinity‐capture methods

Lower Limit of Quantification

25% 20% 25%Quantification(RE, CV)

Calibration standards 20% (except LLOQ 15% (except LLOQ) 20% (except LLOQ)Calibration standards(RE, CV)

20% (except LLOQ and ULOQ)

15% (except LLOQ) 20% (except LLOQ)

Accuracy & precision(RE, CV)

Within 20%(LLOQ/ULOQ QCs within 25%)

Within 15%(LLOQ QC within 20%)

Within 20%(LLOQ QC within 25%)Min. 3 runs)

Min. 6 runs)

Min. 3 runs15



Selectivity/specificityNon specific matrix

10 lots; LLOQ:80% of fortified lots

6 lots; blanks:<20% of LLOQ

6‐10 lots; blanks:<20% of LLOQNon‐specific matrix‐

related interferences: using individual matrix lots; include

80% of fortified lots Accuracy within 25%

<20% of LLOQor <5% of ISLLOQ:80% of fortified lots

<20% of LLOQor <5% of ISLLOQ:80% of fortified lotslots; include

hemolyzed, lipemic, disease population, as

i t



appropriate

Specific interferences: ( d

Fortified with l bl

Fortified with l bl b l

Fortified with available l blusing LLOQ (and ULOQ

for LBAs) QC samplesavailable ADA, soluble target, catabolites, or con‐

available metabolitesor con‐meds, as appropriate.

ADA, soluble target, catabolites or con‐meds, as appropriate.

meds (LM only?).Accuracy within 25%

Accuracy within 20% Accuracy within 25%

16



Dilutionalintegrity/linearity ParallelismR

X (20%)X (30%)NA

X (15%)endogenousX

X (20%)endogenousXRecovery

Digestion efficiencyMatrix effect

NANANA

XNAX (15%)

XmaybeX (20%)Matrix effect

Carry overStock stability

NAGyrolab?

X (15%)X (<20%)X (5‐7%)

X (20%)X (<20%)X (10%)

Extract stabilityMatrix stability

NAX (20%)

X (15%)X (15%)

X (20%)X (20%)

Critical reagentsISR (at least 67%)

XX (30%)

NAX (20%)

enzymes & ACX (30%)

17

PrD-LC-MS/MS BAPrD-LC-MS/MS BAGeneral consensus

• Hybrid BMV approach−Combine appropriate small and large molecule elementsCombine appropriate small and large molecule elements

• Accuracy & precisionApply 4 6 20 (LM) criteria; LLOQ (25%) but S/N still ≥ 5:1−Apply 4-6-20 (LM) criteria; LLOQ (25%), but S/N still ≥ 5:1

• Selectivity/specificityA l SM h f t i bl k t d d d−Apply SM approach for matrix blanks, zero standards, and LLOQ spikes

−For AC-based methods increase matrix lots (includingFor AC-based methods, increase matrix lots (including disease-state) and evaluate potential binding interferences similar to LM approach

• Recovery−Should be sufficient & reproducible overallp

concernsconcernsconcernsconcerns

PrD-LC-MS/MS BAPrD LC MS/MS BATechnical concerns

• Reference standards– quality and COAs

“Contrary to conventional molecules, a pure referencematerial that can serve as a calibration standard is eithermaterial that can serve as a calibration standard is either difficult or sometimes impossible to obtain for this class of (protein) compounds(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 i li i l i l i l di li i l h ki i ”in clinical trials, including clinical pharmacokinetics.”

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 (degree of “purification”) y ( g p )– reproducibility

• Digestiong– completeness and consistency– enzymes as “critical reagents”

• 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 CoadministeredTherapeutic Antibodies in Cynomolgus Monkey Serum, Anal. Chem., 2013, 85 (20), 9859–9867


• Monitoring peptides– selection and evaluation (criteria?)

PrD-LC-MS/MS BAPrD-LC-MS/MS BAMonitoring 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− 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)



• Internal standards– SIL-protein, ext’d SIL-peptide, SIL-peptide, analogs?p p p p p g– overall suitability?– some method steps may be “uncontrolled”

Affinity capture-based approaches



• Internal standards– SIL-protein, ext’d SIL-peptide, SIL-peptide, analogs?p p p p p g– overall suitability?– some method steps may be “uncontrolled”

• Affinity capture (LB) considerations– critical reagent worries?

l i i / ifi i l i– selectivity/specificity evaluation– curve non-linearity (use quadratic or 4/5-PL fits?)

Protein LC-MS/MS BAMatrix stability

37 °C (whole blood/collection)37 C (whole blood/collection)20-25 °C (ambient)0-4 °C (ice)( )

-20 °C (freezer)

-80 °C (cryo)-80 C (cryo)

Stability assumptiony p

No loss of response = analyte stableLoss of response = analyte unstableLoss of response = analyte unstable

M b b tMaybe, or maybe not.



• Internal standards– SIL-protein, ext’d SIL-peptide, SIL-peptide, analogs?p p p p p g– overall suitability?– some method steps often “uncontrolled”

• Affinity capture (LB) considerations– critical reagent worries?

l i i / ifi i l i– selectivity/specificity 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?– false positive result if NSB or other issue?

Protein LC-MS BAAreas for clarification/expansion

• Hybrid constructs MDBs (multidomain biologics)• Hybrid constructs—MDBs (multidomain biologics)• Intact protein quantification (LC-HRAMS)• Catabolite analysis

ADA ?• 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 J Bi l i F S i h ll f l t d bi l i F b 2015 6th Japan Bioanalysis Forum Symposium: challenge of regulated bioanalysis, Feb 2015

AAPS Annual Conference, Oct 2015

EBF O S i N 2015 EBF Open Symposium, Nov 2015

• Regulators have recommended that proposed BA strategiesbe discussed with them early on so they can understand thebe discussed with them early on so they can understand the methodology and be able to ask questions ahead of time

Protein LC-MS BAFinal thoughts

• PrD-LC-MS technology is not addressed in BMV guidancesBMV guidances

• Several industry groups have considered and y g pproposed recommended BMV practices

• Consensus to combine SM and LM elements• 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:• AAPS white paper co-authors:Rand Jenkins, PPD; Jeffrey X. Duggan & John Yu, Boehringer-IngelheimPharmaceuticals; Anne-Françoise Aubry, Jianing Zeng, & Yan J. Zhang,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; SurinderKaur & Keyang Xu, Genentech; Gary A. Schultz, Quintiles; Ziping Yang,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; MargareteBrudny-Kloeppel, Bayer Pharma AG; Jimmy Flarakos, Novartis; Eric Fluhler & Lindsay King, Pfizer; Stephen Lowes; Matt Szapacs & Eric Yang, GlaxoSmithKline

Acknowledgements

• Special thanks to:• Special thanks to:

Jeff Duggan

Anne-Françoise Aubry

Jianing ZengJianing Zeng

* Faye Vazvaei

EBF program committee

D h h h l dD h h h l dDo not go where the path may lead, Do not go where the path may lead, go instead where there is no pathgo instead where there is no pathand leave a trailand leave a trail

——Ralph Waldo Emerson Ralph Waldo Emerson

Changegis inexorable

LargeSmall TO Large

Documents

Recommendations for validation of LC-MS/MS bioanalytical ... · PrD-LC-MS/MS BAMS/MS BA Monitoring peptides • One or more secondary proteotypic peptides chosen and analyzed, along