Glycosimilarity assessment of biotherapeutics 1: Quantitative comparison of the N-glycosylation of the innovator and a biosimilar version of etanercept.

The carbohydrate moieties on the polypeptide chains in most glycoprotein based biotherapeutics and their biosimilars play essential roles in such major mechanisms of actions as antibody-dependent cell-mediated cytotoxicity, complement-dependent cytotoxicity, anti-inflammatory functions and serum clearance. In addition, alteration in glycosylation may influence the safety and efficacy of the product. Glycosylation, therefore, is considered as one of the important critical quality attributes of glycoprotein biotherapeutics, and consequently for their biosimilar counterparts. Thus, the carbohydrate moieties of such biopharmaceuticals (both innovator and biosimilar products) should be closely scrutinized during all stages of the manufacturing process. In this paper we introduce a rapid, capillary gel electrophoresis based process to quantitatively assess the glycosylation aspect of biosimilarity (referred to as glycosimilarity) between the innovator and a biosimilar version of etanercept (Enbrel® and Benepali®, respectively), based on their N-linked carbohydrate profiles. Differences in sialylated, core fucosylated, galactosylated and high mannose glycans were all quantified. Since the mechanism of action of etanercept is TNFα binding, only mannosylation was deemed as critical quality attribute for glycosimilarity assessment due to its influence on serum half-life.

Quantitative assessment of mAb Fc glycosylation of CQA importance by capillary electrophoresis.

The attached carbohydrates at the highly conserved asparagine-linked glycosylation site in the CH 2 domain of the fragment crystallizable (Fc) region of monoclonal antibody therapeutics can play an essential role in their mechanism of action, including ADCC, CDC, anti-inflammatory functions, and serum half-life. Thus, this particular glycosylation represents one of the important critical quality attributes (CQA) of therapeutic monoclonal antibodies, which should be closely monitored and controlled during all stages of biopharmaceutical manufacturing. To study Fc glycosylation related quantitative critical quality attributes, the N-glycan pool of adalimumab (Humira® ) was spiked with increasing amounts of mannose-5 oligosaccharide, a glycan with high CQA importance. The method enabled precise quantitative CQA assessment with high detection sensitivity.

Assessment of the Airway Smooth Muscle Relaxant Effect of Drotaverine.

BACKGROUND: Drotaverine, a type 4 cyclic nucleotide phosphodiesterase (PDE4) inhibitor, blocks the degradation of 3',5'-cyclic adenosine monophosphate. However, published receptor binding data showed that drotaverin also binds to the L-type voltage-operated calcium channel (L-VOCC). Based on these molecular mechanisms of action, a direct and indirect (by blocking the constrictor response) relaxant effect on airway smooth muscle can be predicted, which has not yet been assessed. SUMMARY: Accordingly, drotaverine and reference agents were tested both on the histamine-, methacholine-, or KCl-induced contraction response and on precontracted guinea pig tracheal preparations. It was found that drotaverine not only relaxed the precontracted tracheal preparations but also decreased mediator-induced contraction. These effects of drotaverine were concentration dependent, with a significantly higher potency on the KCl-induced response, than on either the histamine or methacholine induced one. A similar result was noted for nifedipine. The PDE inhibitor, theophylline, also relaxed the precontracted preparations but was ineffective on the mediator-induced contraction in a physiologically relevant concentration range. Moreover, theophylline did not show selectivity and was the least potent relaxant among the 3 tested molecules. Key Message: These results show that drotaverine is a more potent airway smooth muscle relaxant molecule than theophylline. This enhanced potency on relaxation and inhibition of the constrictor response, at least partly, may be explained by the combined L-VOCC blocking and PDE inhibitory potential of drotaverine.

High-throughput analysis of therapeutic and diagnostic monoclonal antibodies by multicapillary SDS gel electrophoresis in conjunction with covalent fluorescent labeling.

Capillary gel electrophoresis (CGE) in the presence of sodium dodecyl sulfate (SDS) is a well-established and widely used protein analysis technique in the biotechnology industry, and increasingly becoming the method of choice that meets the requirements of the standards of International Conference of Harmonization (ICH). Automated single channel capillary electrophoresis systems are usually equipped with UV absorbance and/or laser-induced fluorescent (LIF) detection options offering general applicability and high detection sensitivity, respectively; however, with limited throughput. This shortcoming is addressed by the use of multicapillary gel electrophoresis (mCGE) systems with LED-induced fluorescent detection (LED-IF), also featuring automation and excellent detection sensitivity, thus widely applicable to rapid and large-scale analysis of biotherapeutics, especially monoclonal antibodies (mAb). The methodology we report in this paper is readily applicable for rapid purity assessment and subunit characterization of IgG molecules including detection of non-glycosylated heavy chains (NGHC) and separation of possible subunit variations such as truncated light chains (Pre-LC) or alternative splice variants. Covalent fluorophore derivatization and the mCGE analysis of the labeled IgG samples with multi-capillary gel electrophoresis are thoroughly described. Reducing and non-reducing conditions were both applied with and without peptide N-glycosidase F mediated deglycosylation.

Candidate gene copy number analysis by PCR and multicapillary electrophoresis.

Genetic polymorphisms are often considered as risk factors of complex diseases serving as valuable and easily detectable biomarkers, also stable during the whole lifespan. A novel type of genetic polymorphism has been identified just recently, referred to as gene copy number variation (CNV) or copy number polymorphism. CNV of glycogen synthase kinase 3 beta and its adjacent gene, Nr1i2 (pregnane X receptor isoform), has been reported to associate with bipolar depression. In our study we introduced multicapillary electrophoresis for gene copy number analysis as an affordable alternative to real-time PCR quantification with TaqMan gene probes. Our results show the reliability of the developed method based on conventional PCR followed by separation of products by multicapillary electrophoresis with quantitative evaluation. This method can be readily implemented for the analysis of candidate gene CNVs in high throughput clinical laboratories and also in personalized medicine care of depression-related risk factors.