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.KEY WORDS: affinity capture mass spectrometry; industry white paper; method validation; protein LC-MS/MS quantification; regulated bioanalysis.

Primary leaves of 7-to 9-day-old etiolated

An LC-MS/MS method was developed to quantify an antisense oligonucleotide against Raf-1 expression (rafAON) in monkey and mouse plasma and in mouse tissue homogenates from animals dosed with a liposome-entrapped rafAON easy-to-use formulation (LErafAON-ETU) intended for use as an antineoplastic agent. RafAON was extracted from mouse and monkey plasma using solid-phase extraction. Tissues were homogenized and sample cleanup was achieved by protein precipitation. RafAON and the internal standard (IS) were separated on a Hamilton PRP-1 column and quantified by tandem mass spectrometry using an electrospray source in negative ion mode. The total run time was 4.0 min. The peak areas of two rafAON transitions were summed and plotted against the peak area of an IS transition to generate the standard curve. In monkey plasma the linear range was 50-10,000 ng/mL, and in mouse plasma it was 25-5000 ng/mL. The lower limit of quantification was 500 ng/mL (10 microg/g tissue) in heart, kidney, liver, lung and spleen homogenates, and the standard curve was linear up to 10,000 ng/mL. Accuracy, precision and stability were evaluated and found to be acceptable in all three matrices. The assay was used to support pharmacokinetics and tissue distribution studies of LErafAON-ETU in mice and monkeys.

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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.