Anne-Françoise Aubry

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

ABSTRACTThis 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.

Full-scan high resolution accurate mass spectrometry (HRMS) in regulated bioanalysis: LC–HRMS for the quantitation of prednisone and prednisolone in human plasma

A liquid chromatography-full scan high resolution accurate mass spectrometry (LC-HRMS) method for quantifying prednisone and prednisolone in human plasma using a quadrupole time-of-flight mass spectrometer (Q-TOF) was developed. Plasma samples were extracted using a liquid-liquid extraction procedure. Full scan data were acquired in the TOF only mode and extracted ion chromatograms were generated post-acquisition with the exact masses of the analytes. The calibration range was 5-2500 ng/mL, with a Lower Limit of Quantitation (LLOQ) of 5 ng/mL. The assay accuracy was between 98.4% and 106.3%. The between-run (inter-day) and within-run (intra-day) precision were within 1.7% and 2.9%, respectively. The matrix effect was between 0.98 and 1.10 for the six different lots of human plasma evaluated. Pooled incurred samples were analyzed by the method and the results matched those obtained from an LC-MS/MS method. In addition, qualitative information on phospholipids, and other endogenous components were also extracted from the full-scan data acquired.

Selecting the Correct Weighting Factors for Linear and Quadratic Calibration Curves with Least-Squares Regression Algorithm in Bioanalytical LC-MS/MS Assays and Impacts of Using Incorrect Weighting Factors on Curve Stability, Data Quality, and Assay Performance

A simple procedure for selecting the correct weighting factors for linear and quadratic calibration curves with least-squares regression algorithm in bioanalytical LC-MS/MS assays is reported. The correct weighting factor is determined by the relationship between the standard deviation of instrument responses (σ) and the concentrations (x). The weighting factor of 1, 1/x, or 1/x(2) should be selected if, over the entire concentration range, σ is a constant, σ(2) is proportional to x, or σ is proportional to x, respectively. For the first time, we demonstrated with detailed scientific reasoning, solid historical data, and convincing justification that 1/x(2) should always be used as the weighting factor for all bioanalytical LC-MS/MS assays. The impacts of using incorrect weighting factors on curve stability, data quality, and assay performance were thoroughly investigated. It was found that the most stable curve could be obtained when the correct weighting factor was used, whereas other curves using incorrect weighting factors were unstable. It was also found that there was a very insignificant impact on the concentrations reported with calibration curves using incorrect weighting factors as the concentrations were always reported with the passing curves which actually overlapped with or were very close to the curves using the correct weighting factor. However, the use of incorrect weighting factors did impact the assay performance significantly. Finally, the difference between the weighting factors of 1/x(2) and 1/y(2) was discussed. All of the findings can be generalized and applied into other quantitative analysis techniques using calibration curves with weighted least-squares regression algorithm.

Simple and efficient digestion of a monoclonal antibody in serum using pellet digestion: comparison with traditional digestion methods in LC–MS/MS bioanalysis

BACKGROUND Fast, efficient and reproducible digestion is critical for LC-MS/MS quantitative bioanalysis of therapeutic proteins. Traditional digestion methods require a pretreatment, such as sequential denaturation, reduction and alkylation, which are very time-consuming. RESULTS Pellet digestion, which does not require the serial pretreatments of denaturation, reduction and alkylation, was evaluated using a test monoclonal antibody (mAb) with 16 disulfide bonds, and compared with traditional digestion methods. For the test mAb, pellet digestion provided much better digestion efficiency compared with direct digestion, and provided similar or better digestion efficiency compared with digestion-with-pretreatment. In particular, for two peptides with very low digestion efficiency under direct digestion, pellet digestion improved the digestion yield by approximately 30-fold, which was similar to or better than what digestion-with-pretreatment offered. This method was then successfully applied to an LC-MS/MS assay of the test mAb in monkey serum. CONCLUSION Pellet digestion will be a very useful technique for high-throughput and reliable LC-MS/MS bioanalysis of mAbs and other large proteins, including ones with multiple disulfide bonds.

Multiplexed LC-MS/MS method for the simultaneous quantitation of three novel hepatitis C antivirals, daclatasvir, asunaprevir, and beclabuvir in human plasma

Dual or triple combination regimens of novel hepatitis C direct-acting antivirals (DAA, daclatasvir, asunaprevir, or beclabuvir) provide high sustained virological response rates and reduced frequency of resistance compared to clinical monotherapy. To support pharmacokinetic (PK) assessments in clinical studies, a multiplexed liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantitation of daclatasvir, asunaprevir, beclabuvir (BMS-791325) and its active metabolite (BMS-794712) in human plasma was developed and validated. Human plasma samples were extracted with methyl-t-butyl ether followed by an LC-MS/MS analysis, which was conducted in a multiple reaction monitoring (MRM) mode. The lower limits of quantitation (LLOQ) were 1 ng/mL for daclatasvir, asunaprevir, and BMS-794712, and 2 ng/mL for beclabuvir. Intra-run precision (≤4.5% CV), inter-run precision (≤2.9% CV), and accuracy (±5.3% deviation) based on different concentration levels (low, geometric mean, mid and high) of the quality control samples (QCs) provided evidence of the methods accuracy and precision. Selectivity and matrix effect on LC-MS/MS detection, stability in plasma, and potential interference of coadministered drugs (ribavirin and interferon) were all evaluated and the results were acceptable. Method reproducibility was demonstrated by the reanalysis of a portion of study samples. The cross-validation results for QCs demonstrated the equivalency between this method and two single-analyte methods which were previously validated for quantitation of daclatasvir in human plasma. This approach of using a multiplexed LC-MS/MS method for the simultaneous quantitation of three DAAs is time- and cost-effective, and can maintain good data quality in sample analysis.

Quantitative bioanalysis of antibody-conjugated payload in monkey plasma using a hybrid immuno-capture LC-MS/MS approach: Assay development, validation, and a case study.

Antibody drug conjugates (ADCs) are complex molecules composed of two pharmacologically distinct components, the cytotoxic payload and the antibody. The measurement of the payload molecules that are attached to the antibody in vivo is important for the evaluation of the safety and efficacy of ADCs, and can also provide distinct information compared to the antibody-related analytes. However, analyzing the antibody-conjugated payload is challenging and in some cases may not be feasible. The in vivo change in drug antibody ratio (DAR), due to deconjugation, biotransformation or other clearance phenomena, generates unique and additional challenges for ADC analysis in biological samples. Here, we report a novel hybrid approach with immuno-capture of the ADC, payload cleavage by specific enzyme, and LC-MS/MS of the cleaved payload to quantitatively measure the concentration of payload molecules still attached to the antibody via linker in plasma. The ADC reference material used for the calibration curve is not likely to be identical to the ADC measured in study samples due to the change in DAR distribution over the PK time course. The assay clearly demonstrated that there was no bias in the measurement of antibody-conjugated payload for ADC with varying DAR, which thus allowed accurate quantification even when the DAR distribution dynamically changes in vivo. This hybrid assay was fully validated based on a combination of requirements for both chromatographic and ligand binding methods, and was successfully applied to support a GLP safety study in monkeys.

Systematic evaluation of the root cause of non-linearity in liquid chromatography/tandem mass spectrometry bioanalytical assays and strategy to predict and extend the linear standard curve range

RATIONALE The linear range of a liquid chromatography/tandem mass spectrometry (LC/MS/MS) bioanalytical assay is typically about three orders of magnitude. A broader standard curve range is favored since it can significantly reduce the time, labor and potential errors related to sample dilution - one of the bottlenecks in sample analysis. Using quadratic regression to fit the standard curve can, to a certain degree, extend the dynamic range. However, the use of a quadratic regression is controversial, particularly in regulated bioanalysis. METHODS A number of compounds, with different physicochemical properties and ionization efficiencies, were evaluated to understand the cause of the non-linear behavior of the standard curve. RESULTS The standard curve behavior is primarily associated with the absolute analyte response but not the analyte concentration, the properties of the analyte, or the nature of the matrix when a stable-isotope-labeled internal standard (SIL-IS) is used. For all the test compounds, a non-linear curve was observed when signals exceeded a certain response, which depends on the detector used in the mass spectrometer. With typical API4000 instruments used for the experiments, this critical response level was determined to be ~1 E+6 counts per second (cps) and it was successfully used to predict the linear ranges for the test compounds. By simultaneously monitoring two selective reaction monitoring (SRM) channels of different intensity and using SIL-IS, a linear range of five orders of magnitude was achieved. CONCLUSIONS In this work, the root cause of the non-linear behavior of the standard curve when using a SIL-IS was investigated and identified. Based on the findings, an improved multiple SRM channels approach was proposed and successfully applied to obtain a linear dynamic range of five orders of magnitude for one test compound. This approach may work particularly well for LC/MS/MS bioanalytical assay of dried blood spot (DBS) samples, for which a direct dilution is cumbersome.

High-resolution MS in regulated bioanalysis: where are we now and where do we go from here?

While triple quadrupole MS remains the workhorse of bionanalytical laboratories, LC coupled with high-resolution MS (LC-HRMS) is making headway in drug discovery. LC-HRMS is well suited for quantitative bioanalysis with the inherent advantage of post-acquisition data mining, which is not possible with triple quadrupole systems operated in SRM mode. LC-HRMS can, thus, accomplish the core task of a bioanalytical laboratory--accurate determination of a targeted analyte--with the added bonus of being able to monitor other compounds of interest either at the time of sample analysis, or as an afterthought, after sample analysis, with no additional effort in sample preparation, chromatographic optimization or sample reinjection. Despite these advantages, LC-HRMS has not been broadly adopted in regulated bioanalytical laboratories. The slow progress in embracing the technology may be due, in part, to difficulties in replacing an entire fleet of triple quadrupole MS. Additional reasons are that data mining is of less benefit in development, especially late-stage, than in discovery and that the technical and regulatory challenges associated with the change of platform are perceived to be significant. In addition, the current platform of LC-HRMS introduced by instrument companies has not been tailored to the core responsibility of the bioanalytical community. In marketing current LC-HRMS systems, there is a tendency to combine the needs of the bioanalytical community with those of the drug metabolism community, despite their inherent differences. As a result, the current HRMS systems available lack some basic features desired for bioanalysis, but include features that are not important for bioanalysis making the systems unnecessarily complex and expensive. A simple, cost effective, ideal HRMS system for a bioanalytical laboratory would provide HRMS with high resolving power (the higher the better), no MS/MS capability, and with software suitable for quantitative analysis and appropriate for use in regulated laboratories. Under this scenario, one can foresee a future where part of the regulated bioanalytical work will be accomplished using LC-HRMS, reserving triple quadrupole-based LC-MS/MS for assays that require exquisite sensitivity.

Systematic investigation of orthogonal SPE sample preparation for the LC–MS/MS bioanalysis of a monoclonal antibody after pellet digestion

BACKGROUND Increasing assay sensitivity is critical for promoting the application of LC-MS/MS quantitative bioanalysis of therapeutic proteins. A sample processing method that can selectively remove the abundant background peptides in the serum tryptic digests and retain the target peptides can greatly improve the assay sensitivity. RESULTS Mixed-mode strong-cation exchange SPE was systematically investigated as an orthogonal sample separation technique to reversed-phase UHPLC for the analysis of a test monoclonal antibody, BMS-986012, in monkey serum after pellet digestion. Strong cation exchange SPE efficiently removed most of the background peptides and reduced the matrix effect and background level in the monitored mass transition channels. As a result, improved sensitivity was observed for the surrogate peptides VVSV and SLIY. CONCLUSION This orthogonal approach provides a simple and easy-to-develop sample preparation method that can selectively remove most background peptides and extract the target peptides, therefore, improving the LC-MS/MS assay sensitivity.

Innovative Use of LC-MS/MS for Simultaneous Quantitation of Neutralizing Antibody, Residual Drug, and Human Immunoglobulin G in Immunogenicity Assay Development

Immunogenicity testing for antidrug antibodies (ADA) faces challenges when high levels of the drug are present in clinical patient samples. In addition, most functional cell-based assays designed to characterize the neutralizing ability of ADA are vulnerable to interference from endogenous serum components. Bead extraction and acid dissociation (BEAD) has been successfully applied to extract ADA from serum samples prior to conduction of cell-based assays. However, in the BEAD, certain amounts of the drug and endogenous serum components (so-called residual drug and serum components) from serum samples are carried over to final BEAD eluates due to formation of protein complexes with ADA or nonspecific binding with the beads. Using current enzyme-linked immunosorbent assay (ELISA)-based ligand-binding assays, it is difficult to evaluate the residual drug, which is complexed with excessive amounts of ADA and endogenous serum components in the BEAD eluates. Here, we describe an innovative application of LC-MS/MS for simultaneous detection of the residual human monoclonal antibody drug and endogenous human IgG and the neutralizing antibody positive-control (NAb-PC) in the BEAD eluates. In this study, the low levels of the residual drug and human IgG in the BEAD eluates indicate that the BEAD efficiently removed the high-concentration drug and serum components from the serum samples. Meanwhile, the NAb-PC recovery (∼42%) in the BEAD provided an acceptable detection limit for the cell-based assay. This novel application of LC-MS/MS to immunogenicity assay development demonstrates the advantages of LC-MS/MS in selectivity and multiplexing, which provides direct and fast measurements of multiple components for immunogenicity assay development.