Recommendations for Validation of LC-MS/MS Bioanalytical Methods for Protein Biotherapeutics
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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.
KEY WORDSaffinity capture mass spectrometry industry white paper method validation protein LC-MS/MS quantification regulated bioanalysis
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.
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.
Antibody-drug conjugate; ADCs consist of a monoclonal antibody covalently conjugated with one or more drug molecules via a chemical linker.
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.
Specific form of affinity capture that uses an immunological reagent, such as an antibody. Immunopurification or immunoprecipitation (IP) and immunoenrichment (IE) are synonymous terms.
Technology or instrument combining liquid chromatographic separation with tandem mass spectrometric detection
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.
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.
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.
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.
Proteins used for treating diseases. A similar, but broader term, Biotherapeutics, applies to all types of biomolecules that can be used for treating diseases.
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.
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 (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
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.
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: 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.
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.
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: 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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