Protein microarrays are considered an enabling technology, which will significantly expand the scope of current protein expression and protein interaction analysis. Current technologies, such as two-dimensional gel electrophoresis (2-DE) in combination with mass spectrometry, allowing the identification of biologically relevant proteins, have a high resolving power, but also considerable limitations. As was demonstrated by Gygi et al. (Proc. Nat. Acad. Sci. USA 2000,97, 9390-9395), most spots in 2-DE, observed from whole cell extracts, are from high abundance proteins, whereas low abundance proteins, such as signaling molecules or kinases, are only poorly represented. Protein microarrays are expected to significantly expedite the discovery of new markers and targets of pharmaceutical interest, and to have the potential for high-throughput applications. Key factors to reach this goal are: high read-out sensitivity for quantification also of low abundance proteins, functional analysis of proteins, short assay analysis times, ease of handling and the ability to integrate a variety of different targets and new assays. Zeptosens has developed a revolutionary new bioanalytical system based on the proprietary planar waveguide technology which allows us to perform multiplexed, quantitative biomolecular interaction analysis with highest sensitivity in a microarray format upon utilizing the specific advantages of the evanescent field fluorescence detection. The analytical system, comprising an ultrasensitive fluorescence reader and microarray chips with integrated microfluidics, enables the user to generate a multitude of high fidelity data in applications such as protein expression profiling or investigating protein-protein interactions. In this paper, the important factors for developing high performance protein microarray systems, especially for targeting low abundant messengers of relevant biological information, will be discussed and the performance of the system will be demonstrated in experimental examples.

The 2016 10th Workshop on Recent Issues in Bioanalysis (10th WRIB) took place in Orlando, Florida with participation of close to 700 professionals from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations, and regulatory agencies worldwide. WRIB was once again a weeklong event - A Full Immersion Week of Bioanalysis for PK, Biomarkers and Immunogenicity. As usual, it is specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small and large molecules involving LCMS, hybrid LBA/LCMS, and LBA approaches, with the focus on PK, biomarkers and immunogenicity. This 2016 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. This White Paper is published in 3 parts due to length. This part (Part 3) discusses the recommendations for large molecule bioanalysis using LBA, biomarkers and immunogenicity. Parts 1 (small molecule bioanalysis using LCMS) and Part 2 (Hybrid LBA/LCMS and regulatory inputs from major global health authorities) have been published in the Bioanalysis journal, issues 22 and 23, respectively.

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Insulin-like growth factor I (IGF-I) has important anabolic and homeostatic functions in tissues like skeletal muscle, and a decline in circulating levels is linked with catabolic conditions. Whereas IGF-I therapies for musculoskeletal disorders have been postulated, dosing issues and disruptions of the homeostasis have so far precluded clinical application. We have developed a novel IGF-I variant by site-specific addition of polyethylene glycol (PEG) to lysine 68 (PEG-IGF-I). In vitro, this modification decreased the affinity for the IGF-I and insulin receptors, presumably through decreased association rates, and slowed down the association to IGF-I-binding proteins, selectively limiting fast but maintaining sustained anabolic activity. Desirable in vivo effects of PEG-IGF-I included increased half-life and recruitment of IGF-binding proteins, thereby reducing risk of hypoglycemia. PEG-IGF-I was equipotent to IGF-I in ameliorating contraction-induced muscle injury in vivo without affecting muscle metabolism as IGF-I did. The data provide an important step in understanding the differences of IGF-I and insulin receptor contribution to the in vivo activity of IGF-I. In addition, PEG-IGF-I presents an innovative concept for IGF-I therapy in diseases with indicated muscle dysfunction.

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