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Recommendations for Validation of LC-MS/MS Bioanalytical Methods for Protein Biotherapeutics

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ABSTRACT

This paper represents the consensus views of a cross-section of companies and organizations from the USA and Canada regarding the validation and application of liquid chromatography tandem mass spectrometry (LC-MS/MS) methods for bioanalysis of protein biotherapeutics in regulated studies. It was prepared under the auspices of the AAPS Bioanalytical Focus Group’s Protein LC-MS Bioanalysis Subteam and is intended to serve as a guide to drive harmonization of best practices within the bioanalytical community and provide regulators with an overview of current industry thinking on applying LC-MS/MS technology for protein bioanalysis. For simplicity, the scope was limited to the most common current approach in which the protein is indirectly quantified using LC-MS/MS measurement of one or more of its surrogate peptide(s) produced by proteolytic digestion. Within this context, we considered a range of sample preparation approaches from simple in-matrix protein denaturation and digestion to complex procedures involving affinity capture enrichment. Consideration was given to the method validation experiments normally associated with traditional LC-MS/MS and ligand-binding assays. Our collective experience, thus far, is that LC-MS/MS methods for protein bioanalysis require different development and validation considerations than those used for small molecules. The method development and validation plans need to be tailored to the particular assay format being established, taking into account a number of important factors: the intended use of the assay, the test species or study population, the characteristics of the protein biotherapeutic and its similarity to endogenous proteins, potential interferences, as well as the nature, quality, and availability of reference and internal standard materials.

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ACKNOWLEDGMENTS

The authors wish to acknowledge the contributions of the many colleagues, within and outside their respective companies, who have provided valuable input with their reviews of the draft manuscript. We would also like to thank the AAPS organization and the Bioanalytical Focus Group for their support throughout this initiative. Additionally, we would like to recognize the Ligand-Binding Assay and Bioanalytical Focus Groups for their preliminary reviews of the draft manuscript. Their comments are greatly appreciated. Among all those that have provided valuable feedback, we would specifically like to acknowledge the following reviewers: Bradley L. Ackermann, Eli Lilly and Company; Lakshmi Amaravadi, Biogen Idec; Mark E. Arnold, Bristol-Myers Squibb Company; Michael J. Berna, Eli Lilly and Company; Suzanne Brignoli, Genentech; Margarete Brudny-Kloeppel, Bayer Pharma AG; Binodh DeSilva, Bristol-Myers Squibb Company; Jimmy Flarakos, Novartis Institutes for Biomedical Research; Eric Fluhler, Pfizer Inc.; Lindsay E. King, Pfizer Inc.; Stephen Lowes, Quintiles Bioanalytical and ADME Labs; An Song, Genentech; Lauren F. Stevenson, Biogen Idec; Matt Szapacs, GlaxoSmithKline; and Eric Yang, GlaxoSmithKline.

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Authors and Affiliations

  1. Chromatographic Sciences, PPD Bioanalytical Laboratories, 2244 Dabney Road, Richmond, Virginia, 23230, USA

    Rand Jenkins

  2. Drug Metabolism and Pharmacokinetics, Boehringer-Ingelheim Pharmaceuticals, Inc., Ridgefield, Connecticut, USA

    Jeffrey X. Duggan & John Yu

  3. Analytical and Bioanalytical Development, Bristol-Myers Squibb, Route 206 and Province Line Road, Princeton, New Jersey, 08543, USA

    Anne-Françoise Aubry, Jianing Zeng & Yan J. Zhang

  4. BioQualQuan, LLC, 370 Spring Park Road, Camarillo, California, 93012, USA

    Jean W. Lee

  5. Bioanalytical Division, Tandem Labs, 115 Silvia Street, West Trenton, New Jersey, 08628, USA

    Laura Cojocaru

  6. Pharmacokinetics Dynamics and Metabolism Department, Pfizer Worldwide Research and Development, Andover, Massachusetts, 01810, USA

    Dawn Dufield

  7. Bioanalytical Services, Algorithme Pharma, 575, Armand-Frappier Blvd, Laval, Quebec, H7V 4B3, Canada

    Fabio Garofolo

  8. BioAnalytical Sciences, Genentech, 1 DNA Way, South San Francisco, California, 94080, USA

    Surinder Kaur & Keyang Xu

  9. Quintiles Bioanalytical and ADME Labs, 19 Brown Road, Ithaca, New York, 14850, USA

    Gary A. Schultz

  10. Drug Metabolism and Pharmacokinetics, Novartis Institutes of Biomedical Research, One Health Plaza, 435/3131, East Hanover, New Jersey, 07936, USA

    Ziping Yang

  11. Roche Pharma Research and Early Development, Pharmaceutical Sciences, Global DMPK and Bioanalytical R&D, Roche Innovation Center New York, 430 East 29th Street, New York, New York, 10016, USA

    Faye Vazvaei

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  1. Rand Jenkins
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  2. Jeffrey X. Duggan
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  4. Jianing Zeng
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Corresponding author

Correspondence to Faye Vazvaei.

GLOSSARY

ADA

Anti-drug antibodies (also called ATAs, anti-therapeutic antibodies); ADAs are antibodies that are produced by the immune response (animal or human) against a dosed biotherapeutic.

ADC

Antibody-drug conjugate; ADCs consist of a monoclonal antibody covalently conjugated with one or more drug molecules via a chemical linker.

Affinity Capture

The purification and/or enrichment of an analyte (protein or peptide) from the sample (matrix or digest) onto a solid support (bead, column, membrane, or plate) via an immobilized reagent (e.g., antibody or target) having a high binding affinity.

Immunoaffinity Capture

Specific form of affinity capture that uses an immunological reagent, such as an antibody. Immunopurification or immunoprecipitation (IP) and immunoenrichment (IE) are synonymous terms.

LC-MS/MS

Technology or instrument combining liquid chromatographic separation with tandem mass spectrometric detection

LBA

Ligand-binding assay refers to any analytical method, in which quantification is based on macromolecular interactions. The complex formed between a ligand and a binding molecule (either of which may be the analyte) is typically detected using enzyme-linked colorimetric, electrochemiluminescence or fluorescence methods. Immunoassay is one form of ligand-binding assay in which antibodies are used as binding reagents.

Matrix Effect

The effect (i.e., suppression or enhancement) of chromatographically co-eluting residual matrix components of a biological sample on the ionization signal (typically ESI) of the detected analyte in the mass spectrometer.

Monitoring Peptide

A peptide that is unique to the target protein analyte and selected to monitor to confirm or assess its structural integrity and evaluate assay robustness.

Parallelism

Assessment of dilutional linearity using incurred samples, intended to demonstrate that the analyte concentration vs. response relationship observed for incurred samples is sufficiently similar to that measured for non-dosed standard and QC samples prepared in pooled matrix.

Protein Therapeutic

Proteins used for treating diseases. A similar, but broader term, Biotherapeutics, applies to all types of biomolecules that can be used for treating diseases.

Proteolytic Digestion

Procedure by which proteins are digested, primarily using enzymes such as trypsin, at cleavage points specifically related to their amino acid sequence, thereby reproducibly producing predictable peptide fragments. Chemical proteolysis with acid or cyanogen bromide is also sometimes used.

Selectivity/Specificity

The ability of the bioanalytical method to measure and differentiate the analytes in the presence of components that may be expected to be present. These could include metabolites, impurities, degradants, or matrix components (11).

Selected Reaction Monitoring

Selected reaction monitoring (SRM) refers to a tandem mass spectrometric method in which a precursor ion of a particular mass-to-charge ratio is selected in the first stage of a tandem mass spectrometer and a product ion produced by fragmentation of the precursor ion is selected in the second mass spectrometer stage for detection

SISCAPA®

Stable isotope standards and capture by anti-peptide antibodies, a technique for simultaneous and specific affinity capture of a signature peptide and its SIL-peptide IS from a protein digest sample.

Signature Peptide

General term for a specially selected peptide that is unique to the target protein analyte and can be used for quantification (i.e., as a surrogate) of the target protein or for other qualitative analytical purposes.

SIL-IS, SIL-Protein IS, and SIL-Peptide IS

SIL-IS: stable isotope-labeled internal standard; a compound that has the same chemical structure as the analyte, except that some of atoms of the molecule are replaced with the corresponding stable isotopes, such as, 13C, 15N, 2H, and 18O. They can be used in quantitative LC-MS/MS assays to normalize the response of an analyte for variations in sample preparation, injection volume, matrix effects, and other instrumental conditions.

SIL-Protein IS: stable isotope-labeled protein internal standard, typically made by incorporating stable isotope containing amino acids into the protein during recombinant synthesis.

SIL-Peptide IS: stable-isotope-labeled peptide internal standard, typically made by incorporating stable isotope containing amino acids into the surrogate peptide during chemical synthesis.

Soluble Target

An endogenous receptor or its soluble fragment that can be found in the circulation or excretion, to which a biotherapeutic drug is specifically designed to bind.

Surrogate Peptide

A peptide that is specific to the target protein analyte, in the context of the assay and the intended sample matrix, and quantified in lieu of the target protein analyte in a LC-MS/MS assay

Overall Recovery, Process Recovery, and Digestion Efficiency

Overall recovery: the combination of recoveries through all processes of an LC-MS/MS protein bioanalysis method, including pre-digestion, proteolytic digestion, and post-digestion treatments. It is typically calculated from the ratio of the detector responses obtained from matrix samples spiked with intact protein analyte prior to any processing, against blank matrix sample extracts spiked after final processing with a stoichiometric amount of surrogate peptide.

Process recovery: the recovery determined for each individual process step.

Digestion efficiency: the completeness or recovery of the digestion process. This is usually expressed as the molar equivalent ratio of surrogate peptide recovered from the final bioanalytical process to that of the nominal molar concentration of the biotherapeutic spiked into the QC sample pre-digestion.

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Jenkins, R., Duggan, J.X., Aubry, AF. et al. Recommendations for Validation of LC-MS/MS Bioanalytical Methods for Protein Biotherapeutics. AAPS J 17, 1–16 (2015). https://doi.org/10.1208/s12248-014-9685-5

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