16 GCC Closed Forum: ICH M10 implementation; NGS, qPCR/dPCR, flow cytometry validation; tissue biomarkers; IS response; immunogenicity harmonization; bioanalytical industry status.

The 16th GCC Closed Forum was held in Orlando, FL, USA, on 23 June 2023. Representatives from international bioanalytical Contract Research Organizations were in attendance in order to discuss scientific and regulatory issues specific to bioanalysis. The issues discussed at the meeting included: IS response, flow cytometry, changes to the bioanalytical industry, NGS assays, biomarker assay for tissues, dPCR validation, immunogenicity harmonization and ICH M10 implementation.

Recommendations for the content and management of Certificates of Analysis for reference standards from the GCC for bioanalysis.

The 13th Global CRO Council (GCC) closed forum for bioanalysis was held in New Orleans, LA, USA on 5 April 2019. This GCC meeting was organized to discuss the contents of the 2019 ICH M10 Bioanalytical Method Validation Draft Guideline published in February 2019 and consolidate the feedback of the GCC members. While ICH M10 will cover requirements for reference standards, one of the biggest challenges facing the CRO community is the lack of consistency and completeness of Certificates of Analysis for reference standards used in regulated bioanalysis.

GCC Consolidated Feedback to ICH on the 2019 ICH M10 Bioanalytical Method Validation Draft Guideline.

The 13 GCC Closed Forum for Bioanalysis was held in New Orleans, Louisiana, USA on April 5, 2019. This GCC meeting was organized to discuss the contents of the 2019 ICH M10 Bioanalytical Method Validation Draft Guideline published in February 2019 and consolidate the feedback of the GCC members. In attendance were 63 senior-level participants from eight countries representing 44 bioanalytical CRO companies/sites.

Translational Pharmacokinetic/Pharmacodynamic Characterization and Target-Mediated Drug Disposition Modeling of an Anti-Tissue Factor Pathway Inhibitor Antibody, PF-06741086.

Tissue factor pathway inhibitor (TFPI) exhibits multiple isoforms, which are known to present in multiple locations such as plasma, endothelium, and platelets. TFPI is an endogenous negative modulator of the coagulation pathway, and therefore, neutralization of TFPI function can potentially increase coagulation activity. A human monoclonal antibody, PF-06741086, which interacts with all isoforms of TFPI is currently being tested in clinic for treating hemophilia patients with and without inhibitors.

2014 White Paper on recent issues in bioanalysis: a full immersion in bioanalysis (Part 2 - hybrid LBA/LCMS, ELN & regulatory agencies' input).

The 2014 8th Workshop on Recent Issues in Bioanalysis (8th WRIB), a 5-day full immersion in the evolving field of bioanalysis, took place in Universal City, California, USA. Close to 500 professionals from pharmaceutical and biopharmaceutical companies, contract research organizations and regulatory agencies worldwide convened to share, review, discuss and agree on approaches to address current issues of interest in bioanalysis. The topics covered included both small and large molecules, and involved LCMS, hybrid LBA/LCMS, LBA approaches and immunogenicity.

Recommendations for validation of LC-MS/MS bioanalytical methods for protein biotherapeutics.

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.

Minimalistic sample preparation strategies for LC-MS quantification of large molecule biopharmaceuticals: a case study highlighting alpha-1 antitrypsin protein.

Background: Large molecule biotherapeutics pose a distinctive bioanalytical challenge for LC-MS assay development, particularly when optimizing sample enrichment steps. Alpha-1 antitrypsin (AAT) is used as an example for highlighting large-molecule assay-development strategies.

Results: Two sensitive and selective LC-MS/MS-based quantification assays were developed.

2013 White Paper on recent issues in bioanalysis: 'hybrid'--the best of LBA and LCMS.

The 2013 7th Workshop on Recent Issues in Bioanalysis was held in Long Beach, California, USA, where close to 500 professionals from pharmaceutical and biopharmaceutical companies, CROs and regulatory agencies convened to discuss current topics of interest in bioanalysis. These 'hot' topics, which covered both small and large molecules, were the starting point for fruitful exchanges of knowledge, and sharing of ideas among speakers, panelists and attendees. The discussions led to specific recommendations pertinent to bioanalytical science.

Leukotrienes, but not angiotensin II, are involved in the renal effects elicited by the prolonged cyclooxygenase-2 inhibition when sodium intake is low.

It is known that cyclooxygenase-2 (COX-2) inhibition elicits significant renal hemodynamics alterations when sodium intake is low. However, the mechanisms involved in these renal changes are not well known. Our objective was to evaluate the role of angiotensin II and 5-lipooxygenase-derived metabolites in the renal effects induced by prolonged COX-2 inhibition when sodium intake is low.

Multiplex transcriptional analysis of paraffin-embedded liver needle biopsy from patients with liver fibrosis.

Background: The possibility of extracting RNA and measuring RNA expression from paraffin sections can allow extensive investigations on stored paraffin samples obtained from diseased livers and could help with studies of the natural history of liver fibrosis and inflammation, and in particular, correlate basic mechanisms to clinical outcomes.

Results: To address this issue, a pilot study of multiplex gene expression using branched-chain DNA technology was conducted to directly measure mRNA expression in formalin-fixed paraffin-embedded needle biopsy samples of human liver. Twenty-five genes were selected for evaluation based on evidence obtained from human fibrotic liver, a rat BDL model and in vitro cultures of immortalized human hepatic stellate cells.

Online immunoaffinity LC/MS/MS. A general method to increase sensitivity and specificity: How do you do it and what do you need?

There is an increased emphasis on hyphenated techniques such as immunoaffinity LC/MS/MS (IA-LC/MS/MS) or IA-LC/MRM. These techniques offer competitive advantages with respect to sensitivity and selectivity over traditional LC/MS and are complementary to ligand binding assays (LBA) or ELISA's. However, these techniques are not entirely straightforward and there are several tips and tricks to routine sample analysis.

Elevated aggrecanase activity in a rat model of joint injury is attenuated by an aggrecanase specific inhibitor.

Objective: To evaluate aggrecanase activity after traumatic knee injury in a rat model by measuring the level of aggrecanase-generated Ala-Arg-Gly-aggrecan (ARG-aggrecan) fragments in synovial fluid, and compare with ARG-aggrecan release into joint fluid following human knee injury. To evaluate the effect of small molecule inhibitors on induced aggrecanase activity in the rat model.

Method: An enzyme-linked immunosorbent assay (ELISA) was developed to measure ARG-aggrecan levels in animal and human joint fluids.

An immunoaffinity liquid chromatography-tandem mass spectrometry assay for detection of endogenous aggrecan fragments in biological fluids: Use as a biomarker for aggrecanase activity and cartilage degradation.

The degradation of articular cartilage by aggrecanases (ADAMTS-4 and ADAMTS-5) plays a significant role in the pathology of osteoarthritis (OA). To monitor aggrecanase activity in OA, we have developed a sensitive, accurate, and versatile assay for detection of two specific cleavage sites on aggrecan. The assay uses an immunoaffinity-based liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to detect cleavage at the (374)ARGS site and the (1820)AGEG site.

Geldanamycin-induced PCNA degradation in isolated Hsp90 complex from cancer cells.

Hsp90 is a molecular chaperone involved in the folding and proteolytic turnover of many regulatory proteins associated with it. Some of the Hsp90 client proteins are known to be involved in tumorigenesis. An Hsp90-specific inhibitor, geldanamycin, is shown to bind to the ATP binding site of the chaperone to induce degradation of many client proteins, and results in antitumor activities.

Pharmacology of PF-4191834, a novel, selective non-redox 5-lipoxygenase inhibitor effective in inflammation and pain.

5-Lipoxygenase (LOX) is an important arachidonic acid-metabolizing enzyme producing leukotrienes and other proinflammatory lipid mediators with potent pathophysiological functions in asthma and other inflammatory diseases. 4-(3-(4-(1-Methyl-1H-pyrazol-5-yl)phenylthio)phenyl)-tetrahydro-2H-pyran-4-carboxamide (PF-4191834) is a novel, selective non-redox 5-lipoxygenase inhibitor effective in inflammation and pain. In vitro and in vivo assays were developed for the evaluation of a novel 5-LOX inhibitor using conditions of maximal enzyme activity.

Development and validation of an LC-MS/MS method for quantification of cyclic guanosine 3',5'-monophosphate (cGMP) in clinical applications: a comparison with a EIA method.

An LC-MS/MS method was developed and validated to quantify endogenous cyclic guanosine 3',5'-monophosphate (cGMP) in human plasma. The LC-MS/MS and competitive enzyme immunoassay (EIA) assays were compared. cGMP concentrations of 20 human plasma samples were measured by both methods.

A rat air pouch model for evaluating the efficacy and selectivity of 5-lipoxygenase inhibitors.

The 5-lipoxygenase (5-LOX) pathway has been associated with a variety of inflammatory diseases including asthma, atherosclerosis, rheumatoid arthritis, pain, cancer and liver fibrosis. Several classes of 5-LOX inhibitors have been identified, but only one drug, zileuton, a redox inhibitor of 5-LOX, has been approved for clinical use. To better evaluate the efficacy of 5-LOX inhibitors for pharmacological intervention, a rat model was modified to test the in vivo efficacy of 5-LOX inhibitors.

Discovery and development of a type II collagen neoepitope (TIINE) biomarker for matrix metalloproteinase activity: from in vitro to in vivo.

Destruction of cartilage by matrix metalloproteinases (MMPs) plays a significant role in the pathology of osteoarthritis (OA). A translatable biomarker of MMP activity would enable development of MMP inhibitors for the treatment of OA and potentially the improved diagnosis of OA. A directed approach to identifying specific MMP cleavage products as potential biomarkers has been undertaken.

Host limits to accurate human growth hormone production in multiple plant systems.

Human growth hormone (hGH) is not only a valuable recombinant therapeutic protein for hormone deficiency indications, but is also an extensively characterized molecule both from recombinant bacterial systems and as circulating in humans. We describe the characterization of hGH produced in three different plant systems: tobacco cell culture, soy seed, and maize seed. The data indicate highest production in the maize seed system, with continued productivity over multiple generations, and when bred to a new host genotype for improved productivity.

Selective site-specific fenton oxidation of methionine in model peptides: evidence for a metal-bound oxidant.

The metal-catalyzed oxidation (MCO) of proteins represents an important pathway for protein degradation. Although many mechanistic details of MCO are currently unknown, such mechanistic information would greatly benefit formulation scientists in the rational design and analysis of protein formulations. Here, we describe the Fenton oxidation (by Fe(2+)/H(2)O(2)) of several Met-, Tyr-, and His containing model peptides, including one derivative containing a conformationally restricted norbornyl Met analogue (Nor), Nor-Gly-His-Met-NH(2).

Mass spectrometry compatibility of two-dimensional gel protein stains.

As proteomic technology evolves, protein staining sensitivity is constantly being improved, enabling researchers to better visualize the proteome of their system. The current challenge is to balance the limits of detection of protein visualization with those of the mass spectrometric methods. In this report, mass spectra generated from human serum or rat liver proteins stained with either colloidal Coomassie blue, Daiichi silver, SYPRO Orange, SYPRO Red, SYPRO Ruby, or SYPRO Tangerine are compared.