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In response to an earlier workshop covering the pros and cons of quantification below the LLOQ (BLQ) the author reviews the topics discussed from the bioanalytical standpoint. Important considerations for estimating concentrations below the LLOQ include: method signal-to-noise, baseline shape and condition, close lying interference peaks (especially for protein methods), matrix effect, adsorption and stability of the analyte at low concentrations and carryover. These methodological issues are discussed as possible contributors to inaccuracy in BLQ estimations, and appropriate cautions are provided via examples. A proposed method for the evaluation of BLQ estimations utilizes extended incurred sample reanalysis analysis where BLQ samples or spiked simulated samples are analyzed with quality controls and standards in addition to those in the original study. Generally, BLQ estimations are discouraged, with the recommendation that any extrapolations should be done in close collaboration between the pharmacokinetic (PK) and bioanalytical scientists in consultation with the regulatory agency.

A refinement of protein LC–MS bioanalysis is to use predigestion immunoaffinity capture to extract the drug from matrix prior to digestion. Because of their increased sensitivity, such hybrid assays have been successfully validated and applied to a number of clinical studies; however, they can also be subject to potential interferences from antidrug antibodies, circulating ligands or other matrix components specific to patient populations and/or dosed subjects. The purpose of this paper is to describe validation experiments that measure immunocapture efficiency, digestion efficiency, matrix effect and selectivity/specificity that can be used during method optimization and validation to test the resistance of the method to these potential interferences. The designs and benefits of these experiments are discussed in this report using an actual assay case study.

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.

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.

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

This paper was developed with the support of the International Consortium for Innovation and Quality in Pharmaceutical Development (IQ). IQ is a not-for-profit organization of pharmaceutical and biotechnology companies with a mission of advancing science-based and scientifically driven standards and regulations for pharmaceutical and biotechnology products worldwide. Within the IQ, various working groups (WG) have been formed, where the microsampling WG is committed to providing a scientific forum for the advancement of both wet and dry microsampling techniques within the pharmaceutical industry. This first output from the microsampling WG is to summarize and reflect on the current knowledge and opinions on DBS sampling, to stimulate discussion, and to encourage future creative applications of DBS sampling. Dried blood spot (DBS) sampling has established itself as an innovative sampling technique where wet blood is spotted onto absorbent paper or other paper materials and allowed to dry (1–4). DBS offers several potential benefits inherent to the technique, namely a low blood volume, simplified blood sample collection (5), and convenient sample storage and transfer. In certain applications, DBS sampling has been shown to stabilize certain analytes or metabolites without the addition of chemical modifiers (6–9). DBS has been routinely applied for decades in neonatal screening for phenylketonuria and other congenital metabolic disorders (10). The utility of DBS sampling has also been demonstrated for therapeutic drug monitoring (11) and for epidemiological studies (e.g., HIV and HBV detection/monitoring) (12) due to the practical advantages along with simplified sample collection and handling procedures. Finally, DBS can also be used for quantitative biomarker (PD) assessment from blood, where appropriate. However, the technique is relatively new to the pharmaceutical industry and to government regulators overseeing new drug applications. Nevertheless, over the past 5 to 7 years, the technique has been extensively evaluated for quantifying drug exposure in nonclinical and/or clinical studies in various stages of drug discovery and development. The ease to collect, transfer, store, and process small volumes of blood samples has generated considerable interest in providing utility in volume-limited situations (e.g., small rodent, human pediatric studies) for toxicokinetic (TK), pharmacokinetic (PK), or pharmacodynamic (PD) sampling. Discovery and nonclinical studies Rodent animal models are typically employed in these studies. The reduced blood volumes required for DBS can enable serial bleeding and, consequently, elimination of satellite animal groups and reduction of compound use. The ability to eliminate the satellite animal groups enables the assessment of exposure and toxic effects within the same animal. Studies involving expensive animal models (i.e., transgenic mice, knock-out mice, humanized mice, etc.) further highlight a persuasive scientific and economic case for DBS sampling since a complete pharmacokinetic profile can be obtained from a single study animal without the need for extra rodents merely for generating exposure data. These are perfectly in line with the principles of the 3Rs: reduction, refinement, and replacement of humane animal research (13–15). With greater emphasis from the regulatory authorities to study new drugs for infants, neonates, and pediatric populations, the requirement to conduct associated nonclinical juvenile rodent toxicity studies serves as an ideal scenario where the advantage of low blood volume in DBS sampling is undeniable. Although the advantages of DBS heavily favor rodent studies, it can also be used to refine non-rodent studies.

Levulinic acid (LA), a competitive inhibitor of δ-aminolevulinic acid (ALA) dehydratase (EC 4.2.1.24), has been used extensively in the study of ALA formation during greening. When [1-14C]LA is administered to etiolated barley (Hordeum vulgare L. var. Larker) shoots in darkness, 14CO2 is evolved. This process is accelerated when such tissues are incubated with 2 millimolar ALA or placed under continuous illumination. Label from the C-1 of LA becomes incorporated into organic acids, amino acids, sugars, lipids, and proteins during a 4-hour incubation in darkness or in the light. This metabolism is discussed in relation to the use of LA as a tool in the study of chlorophyll synthesis in higher plants.

Primary leaves of 7- to 9-day-old etiolated seedlings of Phaseolus vulgaris L. var. Red Kidney infiltrated in darkness with aqueous solutions of α, α′-dipyridyl, o-phenanthroline, pyridine-2-aldoxime, pyridine-2-aldehyde, 8-hydroxyquinoline, or picolinic acid synthesize large amounts of magnesium protoporphyrin monomethyl ester and lesser amounts of magnesium protoporphyrin, protoporphyrin, and protochlorophyllide. Pigment formation proceeds in a linear manner for up to 21 hours after vacuum infiltration with 10 mm α, α′-dipyridyl. Etiolated tissues of Zea mays L., Cucumis sativus L., and Pisum sativum L. respond in the same way to dipyridyl treatment. Compounds active in eliciting this response are aromatic heterocyclic nitrogenous bases which also act as bidentate chelators and form extremely stable complexes with iron; other metal ion chelators, such as ethylenediaminetetraacetic acid, salicylaldoxime, and sodium diethyldithiocarbamate, do not elicit any pigment synthesis. The ferrous, ferric, cobaltous, and zinc chelates of α, α′-dipyridyl are similarly ineffective. If levulinic acid is supplied to etiolated bean leaves together with α, α′-dipyridyl, porphyrin production is inhibited and δ-aminolevulinic acid accumulates in the tissue. Synthesis of porphyrins proceeds in the presence of 450 micrograms per milliliter chloramphenicol or 50 micrograms per milliliter cycloheximide with only partial diminution. We propose that heme or an iron-protein complex blocks the action of the enzyme(s) governing the synthesis of δ-aminolevulinic acid in etiolated leaves in the dark and that iron chelators antagonize this inhibition, leading to the biosynthesis of δ-aminolevulinic acid and porphyrins.

The effect of light on the levels of activity of six enzymes which are light-modulated in higher plants was examined in the photosynthetic procaryot Anacystis nidulans. Ribulose-5-phosphate kinase (EC 2.7.1.19) was found to be light-activated in vivo and dithiothreitol-activated in vitro while glucose-6-phosphate dehydrogenase (EC 1.1.1.49) was light-inactivated and dithiothreitol-inactivated. The enzymes fructose-1,6-diphosphate phosphatase (EC 3.1.3.11), sedoheptulose-1,7-diphosphate phosphatase, NAD- and NADP-linked glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12; EC 1.2.1.13) were not affected by light treatment of the intact algae, but sedoheptulose-diphosphate phosphatase and the glyceraldehyde-3-phosphate dehydrogenases were dithiothreitol-activated in crude extracts. Light apparently controls the activity of the reductive and oxidative pentose phosphate pathway in this photosynthetic procaryot as in higher plants, through a process which probably involves reductive modulation of enzyme activity.

The in vivo oxidation of the C(4) and C(5) of 5-aminolevulinic acid (ALA) to CO(2) has been studied in etiolated barley (Hordeum vulgare L. var. Larker) leaves in darkness. The rate of (14)CO(2) evolution from leaves fed [4-(14)C]ALA is strongly inhibited by aminooxyacetate, anaerobiosis, and malonate. The rate of (14)CO(2) evolution from leaves fed [5-(14)C]ALA is also inhibited by these treatments but to a lesser extent. These results suggest that (a) one step in ALA catabolism is a transamination reaction and (b) the C(4) is oxidized to CO(2) via the tricarboxylic acid cycle to a greater extent than is the C(5).

The capacity of a particulate pea (Pisum sativum L.) leaf chloroplast system for light-modulation of enzyme activity is diminished by brief exposure to sodium sulfite and, when intact seedlings are exposed to atmosphric SO

When [2,4- 14 C]porphobilinogen (PBG) or [2 (aminomethyl),5- 14 C]PBG is administered to etiolated barley ( Hordeum vulgare L. var. Larker) leaves in darkness, label becomes incorporated into CO 2 , organic and amino acids, sugars, lipids, and proteins during a 4-hour incubation. Less than 1% of the label, however, is incorporated into porphyrins. The rate of 14 CO 2 evolution from leaves fed [2,4- 14 C]PBG is strongly inhibited by anaerobiosis but is unaffected by aminooxyacetic acid, while the rate of 14 CO 2 evolution from [2(aminomethyl),5- 14 C]PBG is strongly inhibited by aminooxyacetic acid but is not affected by anaerobiosis. These results suggest that: (a) exogenous PBG is taken up and metabolized by etiolated barley leaves; (b) PBG is not metabolized exclusively to porphyrins but can be converted to a variety of intermediary metabolites; (c) this metabolism involves reactions which are partially dependent upon O 2 and pyridoxal phosphate.

Light inactivation of glucose-6-phosphate dehydrogenase within the pea (Pisum sativum L.) leaf chloroplast has a narrow pH optimum between 7.2 and 7.4 and is NADP-sensitive. The pH optimum for dark activation is slightly lower. Inactivation apparently results in a simple decrease in maximal velocity of the chloroplastic and cytoplasmic forms of the enzyme with no concomitant change in pH optimum or K(m) (glucose 6-phosphate).