Faye Vazvaei

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.

Bioanalytical method validation considerations for LC–MS/MS assays of therapeutic proteins

This paper highlights the recommendations of a group of industry scientists in validating regulated bioanalytical LC-MS/MS methods for protein therapeutics in a 2015 AAPSJ White Paper. This group recommends that most of the same precision and accuracy validation criteria used for ligand-binding assays (LBAs) be applied to LC-MS/MS-based assays where proteins are quantified using the LC-MS/MS signal from a surrogate peptide after proteolytic digestion (PrD-LCMS methods). PrD-LCMS methods are generally more complex than small molecule LC-MS/MS assays and may often include LBA procedures, leading to the recommendation for a combination of chromatographic and LBA validation strategies and appropriate acceptance criteria. Several key aspects of this bioanalytical approach that are discussed in the White Paper are treated here in additional detail. These topics include selectivity/specificity, matrix effect, digestion efficiency, stability and critical reagent considerations.

Method transfer, partial validation, and cross validation: recommendations for best practices and harmonization from the global bioanalysis consortium harmonization team.

This paper presents the recommendations of the Global Bioanalytical Consortium Harmonization Team on method transfer, partial validation, and cross validation. These aspects of bioanalytical method validation, while important, have received little detailed attention in recent years. The team has attempted to define, separate, and describe these related activities, and present practical guidance in how to apply these techniques.

Repeat Analysis and Incurred Sample Reanalysis: Recommendation for Best Practices and Harmonization from the Global Bioanalysis Consortium Harmonization Team

The A7 harmonization team (A7 HT), a part of the Global Bioanalysis Consortium (GBC), focused on reviewing best practices for repeat analysis and incurred sample reanalysis (ISR) as applied during regulated bioanalysis. With international representation from Europe, Latin America, North America, and the Asia Pacific region, the team first collated common practices and guidance recommendations and assessed their suitability from both a scientific and logistical perspective. Subsequently, team members developed best practice recommendations and refined them through discussions and presentations with industry experts at scientific meetings. This review summarizes the team findings and best practice recommendations. The few topics where no consensus could be reached are also discussed. The A7 HT recommendations, together with those from the other GBC teams, provide the basis for future international harmonization of regulated bioanalytical practices.

Validation of LC-MS/MS bioanalytical methods for protein therapeutics.

Background There are more than 100 protein and peptide drugs currently approved by the US FDA and now over 100 more are currently in active development by pharmaceutical companies worldwide [1]. The quantification of protein therapeutics has traditionally relied upon ligand-binding assays (LBAs). Such assays can be very sensitive and selective if properly developed using appropriate reagents, but are nevertheless susceptible to interferences due to circulating ligands, antidrug antibodies (ADAs), cross reactions and various forms of non-specific binding. LC– MS/MS analysis has emerged as an effective quantitative tool for protein bioanalysis. Of particular interest is LC–MS/MS analysis of therapeutic proteins via quantification of unique surrogate peptides that are produced by enzymatic digestion, a technique that is being widely used [1–4]. For simplicity, we will call this type of analysis protein digestion LC–MS/MS (PrD-LC–MS). PrD-LC– MS can be used as an alternative and supplemental approach to LBAs, and this has been extensively discussed in recent years [1–4]. Such LC–MS/MS methods, where the protein biotherapeutic is digested directly in the biomatrix prior to LC–MS/MS, can provide bioanalytical data with low interference from ADAs and other circulating ligands [5]. These methods can yield more representative quantification of total drug, but they may have limited sensitivity. Where higher sensitivity has been required, analyte enrichment or affinity capture concentration steps can be added either before [6] or after [7] proteolytic digestion. These enrichment methods can improve sensitivity by eliminating background signals either from the biomatrix itself or from the digest prior to peptide LC–MS/MS. As the methodology for PrD-LC–MS matures, there will be an increased need to use these assays in a regulated environment, both for good laboratory practice (GLP) toxicology studies and for regulated sample bioanalysis in clinical trials. Neither the recent EMA guidance [8], nor the FDA draft Bioanalytical Method Validation (BMV) guidance [9] has specifically discussed the validation of PrD-LC–MS or its application to regulated bioanalysis. Several questions need to be addressed for any regulated PrD-LC–MS assay validation:

Investigating the effect of autoinduction in cynomolgus monkeys of a novel anticancer MDM2 antagonist, idasanutlin, and relevance to humans

Abstract 1. Idasanutlin (RG7388) is a potent p53-MDM2 antagonist currently in clinical development for treatment of cancer. The purpose of the present studies was to investigate the cause of marked decrease in plasma exposure after repeated oral administration of RG7388 in monkeys and whether the autoinduction observed in monkeys is relevant to humans. 2. In monkey liver and intestinal microsomes collected after repeated oral administration of RG7388 to monkeys, significantly increased activities of homologue CYP3A8 were observed (ex vivo). Investigation using a physiologically based pharmacokinetic (PBPK) model suggested that the loss of exposure was primarily due to induction of metabolism in the gut of monkeys. 3. Studies in monkey and human primary hepatocytes showed that CYP3A induction by RG7388 only occurred in monkey hepatocytes but not in human hepatocytes, which suggests the observed CYP3A induction is monkey specific. 4. The human PK data obtained from the first cohorts confirmed the lack of relevant induction as predicted by the human hepatocytes and the PBPK modelling based on no induction in humans.

Sensitive, High-Throughput, and Robust Trapping-Micro-LC-MS Strategy for the Quantification of Biomarkers and Antibody Biotherapeutics

For LC-MS-based targeted quantification of biotherapeutics and biomarkers in clinical and pharmaceutical environments, high sensitivity, high throughput, and excellent robustness are all essential but remain challenging. For example, though nano-LC-MS has been employed to enhance analytical sensitivity, it falls short because of its low loading capacity, poor throughput, and low operational robustness. Furthermore, high chemical noise in protein bioanalysis typically limits the sensitivity. Here we describe a novel trapping-micro-LC-MS (T-μLC-MS) strategy for targeted protein bioanalysis, which achieves high sensitivity with exceptional robustness and high throughput. A rapid, high-capacity trapping of biological samples is followed by μLC-MS analysis; dynamic sample trapping and cleanup are performed using pH, column chemistry, and fluid mechanics separate from the μLC-MS analysis, enabling orthogonality, which contributes to the reduction of chemical noise and thus results in improved sensitivity. Typically, the selective-trapping and -delivery approach strategically removes >85% of the matrix peptides and detrimental components, markedly enhancing sensitivity, throughput, and operational robustness, and narrow-window-isolation selected-reaction monitoring further improves the signal-to-noise ratio. In addition, unique LC-hardware setups and flow approaches eliminate gradient shock and achieve effective peak compression, enabling highly sensitive analyses of plasma or tissue samples without band broadening. In this study, the quantification of 10 biotherapeutics and biomarkers in plasma and tissues was employed for method development. As observed, a significant sensitivity gain (up to 25-fold) compared with that of conventional LC-MS was achieved, although the average run time was only 8 min/sample. No appreciable peak deterioration or loss of sensitivity was observed after >1500 injections of tissue and plasma samples. The developed method enabled, for the first time, ultrasensitive LC-MS quantification of low levels of a monoclonal antibody and antigen in a tumor and cardiac troponin I in plasma after brief cardiac ischemia. This strategy is valuable when highly sensitive protein quantification in large sample sets is required, as is often the case in typical biomarker validation and pharmaceutical investigations of antibody therapeutics.

Effect of Hepatic Impairment on the Pharmacokinetics of Alectinib

Alectinib is approved and recommended as the preferred first‐line treatment for patients with anaplastic lymphoma kinase (ALK)‐positive non–small cell lung cancer. The effect of hepatic impairment on the pharmacokinetics (PK) of alectinib was assessed with physiologically based PK modeling prospectively and in a clinical study. An open‐label study (NCT02621047) investigated a single 300‐mg dose of alectinib in moderate (n = 8) and severe (n = 8) hepatic impairment (Child‐Pugh B/C), and healthy subjects (n = 12) matched for age, sex, and body weight. Physiologically based PK modeling was conducted prospectively to inform the clinical study design and support the use of a lower dose and extended PK sampling in the study. PK parameters were calculated for alectinib, its major similarly active metabolite, M4, and the combined exposure of alectinib and M4. Unbound concentrations were assessed at 6 and 12 hours postdose. Administration of alectinib to subjects with hepatic impairment increased the area under the plasma concentration–time curve from time 0 to infinity of the combined exposure of alectinib and M4 to 136% (90% confidence interval [CI], 94.7‐196) and 176% (90%CI 98.4‐315), for moderate and severe hepatic impairment, respectively, relative to matched healthy subjects. Unbound concentrations for alectinib and M4 did not appear substantially different between hepatic‐impaired and healthy subjects. Moderate hepatic impairment had only a modest, not clinically significant effect on alectinib exposure, while the higher exposure observed in severe hepatic impairment supports a dose adjustment in this population.

Regulated Bioanalysis: Documentation and Reports

The early Crystal City Bioanalytical workshops did not discuss the requirements for bioanalytical documentation and reports. The major bioanalytical guidance from FDA and EMA provided only broad outlines for the requirements in bioanalytical documentation and reports. The bioanalytical practitioners were therefore left to decide what to document in the reports and what to maintain in the archives for inspection. This has led to bioanalytical reports of various shapes and sizes. Parallel to the development of the bioanalytical guidance, ICH developed an electronic Common Technical Document (eCTD) guidance for preparing regulatory reports. Recently, FDA issued a mandatory guidance for submission of data and reports electronically for certain submissions. These latter changes have also been influencing how the bioanalytical data and reports would be submitted in the future. This chapter provides an overview of the bioanalytical documentation and reports for bioanalytical studies intended for regulatory submission.