Despite major advances in modern drug discovery and development, the number of new drug approvals has not kept pace with the increased cost of their development. Increasingly, innovative uses of biomarkers are employed in an attempt to speed new drugs to market. Still, widespread adoption of biomarkers is impeded by limited experience interpreting biomarker data and an unclear regulatory climate. Key differences preclude the direct application of existing validation paradigms for drug analysis to biomarker research. Following the AAPS 2003 Biomarker Workshop (J. W. Lee, R. S. Weiner, J. M. Sailstad, et al. Method validation and measurement of biomarkers in nonclinical and clinical samples in drug development. A conference report. Pharm Res 22:499-511, 2005), these and other critical issues were addressed. A practical, iterative, "fit-for-purpose" approach to biomarker method development and validation is proposed, keeping in mind the intended use of the data and the attendant regulatory requirements associated with that use. Sample analysis within this context of fit-for-purpose method development and validation are well suited for successful biomarker implementation, allowing increased use of biomarkers in drug development.

Antibody therapy is coming of age, with 15 monoclonal antibodies approved for therapeutic use in the United States and many others currently undergoing clinical trials (1). The advent of antibody engineering over the past two decades has contributed to the recent clinical success of therapeutic antibodies. The development of chimeric (2) and humanized (3) antibodies not only reduced the potent immunogenicity of rodent antibodies in humans but also improved the serum halflives and efficacy of such therapeutics compared with rodent antibodies. Phage display (4) and other display technologies have led to the ability to increase the affinity of antibodies for their target antigens. More recently, antibody engineering has been used to modify the effector functions of antibodies by altering their binding to C1q (5) and various Fc␥ receptors (6).The neonatal Fc receptor (FcRn) 1 is a heterodimer that comprises a transmembrane ␣ chain with structural homology to the extracellular domains of the ␣ chain of major histocompatibility complex class I molecules, and a soluble light chain consisting of ␤2-microglubulin (␤2m) (7). FcRn mediates both transcytosis of maternal IgG to the fetus or neonate and IgG homeostasis in adults (8). Evidence for the latter role initially came from studies indicating an unusually short serum halflife for IgG antibodies in ␤2m-deficient mice (9 -11). This observation led to the generation of mutant mouse hinge-Fc fragments with enhanced binding to FcRn and increased serum persistence in mice (12). Recently, several studies have identified human IgG 1 mutants with enhanced FcRn binding (6, 13), although no improvement in the serum half-lives of these mutants was observed in mice (13) or reported in primates.The binding of IgG to FcRn is sharply pH-dependent; IgG binds to FcRn under mildly acidic conditions and is released under slightly basic conditions (14). It has been hypothesized that pinocytosed IgG antibodies are captured by FcRn in acidified endosomes, rescued from degradation in lysosomes, recycled back to the cell surface, and returned to the circulation (8). Mutagenesis studies have identified both the mouse (15, 16) and human (17) Fc residues believed to be important in mediating pH-dependent binding. The results of the mutagenesis studies are consistent with the interpretation of a crystallographic study of the Fc⅐FcRn interaction (18). In the current study, molecular modeling was used to identify residues in the human IgG Fc near the FcRn binding site that, when mutated, might alter binding to FcRn without affecting the pH dependence of this interaction. Following exhaustive mutagenesis at these positions, several IgG 2 mutants were identified with improved binding to FcRn at pH 6.0 that retained the property of pH-dependent release. A pharmacokinetics study in rhesus monkeys showed that two mutant IgG 2 antibodies with increased FcRn binding affinity had considerably longer serum half-lives than the wild-type antibody. EXPERIMENTAL PROCEDURESMolecular Modeling-Molecular models o...

The serum half-life of IgG Abs is regulated by the neonatal Fc receptor (FcRn). By binding to FcRn in endosomes, IgG Abs are salvaged from lysosomal degradation and recycled to the circulation. Several studies have demonstrated a correlation between the binding affinity of IgG Abs to FcRn and their serum half-lives in mice, including engineered Ab fragments with longer serum half-lives. Our recent study extended this correlation to human IgG2 Ab variants in primates. In the current study, several human IgG1 mutants with increased binding affinity to human FcRn at pH 6.0 were generated that retained pH-dependent release. A pharmacokinetics study in rhesus monkeys of one of the IgG1 variants indicated that its serum half-life was ∼2.5-fold longer than the wild-type Ab. Ag binding was unaffected by the Fc mutations, while several effector functions appeared to be minimally altered. These properties suggest that engineered Abs with longer serum half-lives may prove to be effective therapeutics in humans.

The development and validation of ligand binding assays used in the support of pharmacokinetic studies has been the focus of various workshops and publications in recent years, all in an effort to establish a guidance document for standardization of these bioanalytical methods. This summary report of the workshop from 2003 focuses on the issues discussed in presentations and notes points of discussion and areas of consensus among the participants.

The effect of the immune modulator, Cyclosporin A (CsA) on vaccinia virus replication has been examined in cell cultures. In the present study we report that CsA is anti-viral towards vaccinia virus. Viral yield was inhibited by more than 97% after 24 h postinfection in the presence of 16 microM to 40 microM CsA. An analysis of the infectious cycle in greater detail revealed that CsA did not effect the total level of [35S] methionine incorporation into vaccinia infected cells. However, both early and late viral gene expression were inhibited by CsA. Late viral protein synthesis appeared to be more sensitive to the drug. At least one late viral polypeptide of approximately Mr 38,000 was virtually undetected up to 8 h postinfection in the presence of 40 microM CsA. Host protein synthesis which is normally inhibited by the virus was not turned off until very late in infection. Viral DNA replication was also inhibited by the addition of CsA at levels comparable to those observed for late protein synthesis.

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The remaining list of author names, affiliations and Regulatory Agencies Disclaimer can be found at the end of the articleThe 2018 12 th Workshop on Recent Issues in Bioanalysis took place in Philadelphia, PA, USA on April 9-13, 2018 with an attendance of over 900 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5-day full immersion in bioanalysis, biomarkers and immunogenicity. As usual, it was specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small-and large-molecule bioanalysis involving LCMS, hybrid LBA/LCMS and LBA/cell-based assays approaches. This 2018 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2018 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations for large molecule bioanalysis, biomarkers and immunogenicity using LBA and cell-based assays. Part 1 (LCMS for small molecules, peptides, oligonucleotides and small molecule biomarkers) and Part 2 (hybrid LBA/LCMS for biotherapeutics and regulatory agencies' inputs) are published in volume 10 of Bioanalysis, issues 22 and 23 (2018), respectively.

Two distinct collagenolytic enzyme fractions have been separated from a crude Clostridium histolyticum collagenase preparation by gradient elution from a DEAE-Sephadex A50 column. The first of these fractions, though very active against native collagen and synthetic collagenase substrates, shows no activity whatsoever against the unspecific denatured collagen substrate azocoll and negligible activity only against gelatin. Both collagenase fractions are free of activity against casein, hemoglobin, elastin, leucylglycylglycine, or similar peptides, as well as benzoylarginine amide or benzoylarginine naphthylamide with and without potential activators. Ninhydrin tests for the number of peptide bonds cleaved show that both fractions have approximately the same activity against undenatured collagen whether insoluble, salt soluble, or acid soluble. Both fractions break down substrates such as Cbz-Gly-Pro-Gly-Gly-Pro-Ala; but based on the same activity against collagen, fraction I is far more active against the synthetic substrate. Against gelatin on the same basis fraction I shows only 0.01 the activity of fraction 11. Further comparison of the two fractions indicates no significant differences in p H optimum (approximately 7), electrophoretic mobility, or amino acid composition. The enzymes therefore do not appear to be isozymes but rather multiple forms of collagenases with different specificities.Collagenases by definition are enzymes capable of digesting native undenatured collagen under physiological conditions of p H and temperature (Mandl, 1961). True collagenases are very rare and have been uncontroversially confirmed only in culture filtrates of certain clostridia, in particular several strains of Clostridium histolyticum. Since collagenases are associated with many other proteolytic enzymes of lesser specificity but similar physical and chemical characteristics, their purification has at best been incomplete. Separation from nonspecific caseinolytic activity was achieved for the first time by De Bellis et al. (1954). As newer methods of purification became available and were applied, refinements in analytical techniques led to the realization that the number of constituents present in the crude collagenase preparations were far greater than originally assumed. A variety of substrates was found susceptible to what must now be considered distinct enzymatic activities. About a year ago we were able to develop two new methods of purification which allowed the preparation, reproducibly and in good yield, of relatively pure C1. histolyticum collagenase. The enzyme so obtained, although completely free from previously described activities against casein, hemoglobin, benzoylargininamide, peptides, and the like, showed an elution pattern which indicated the possibility that more than one collagenolytic enzyme might be present.We have now modified one of the two methods, ion-exchange chromatography on a DEAESephadex A50 column, substituting a different gradient-elution system, and thus obtained at least two wellsepar...