Infrared spectroscopy for biopharmaceutical protein analysis.

A mid-infrared transmission spectroscopic method with the possibility of high sample throughput was developed and validated on the basis of GMP requirements for protein therapeutics. In comparison with the method predominantly used, i.e. the surface-sensitive attenuated total reflection (ATR), we have shown that transmission measurements in solution possess several advantages, such as quantitative analysis on the basis of Lambert-Beer's law, determination of identity and purity based on specifically developed identity criteria, characterization of protein structure and structural changes including aggregation in solution and study of ligand binding to the protein. The usefulness of this method is exemplified by the characterization of the drug substance of ONCOHIST (recombinant human histone H1.3 and bis-Met-histone H1.3) and its interaction with phosphate ions. Our conclusion is that transmission mid-infrared spectroscopy is a powerful tool for protein analysis in biotechnology and supplements the current analytical techniques for biopharmaceutical quality control of therapeutic proteins.

An alternative structure model for the polypentapeptide in elastin.

Polypentapeptides (GVGVP)n which are designed in analogy to the connective tissue protein elastin are reported to transform various kinds of energy into mechanical work by the so-called deltaT(t)-mechanism in cross-linked macroscopic polypentapeptide (PPP) films. In the literature, the responsible element of conformation in such polypeptides is described as a beta-spiral and the deltaT(t) effect is explained as a sudden change of macroconformation of single polypeptide molecules from an extended but not regular state below a transition temperature T(t) to the beta-spiral above T(t). We examined the secondary structure of the linear PPP C(GVGVP)6 in solution with DSC, CD, UV absorption, FTIR and NMR spectroscopy. The results suggest that the beta-spiral is not present in the conformational structure of the PPP molecules. The antiparallel beta-sheet is proposed to be the basic regular part of conformation because it agrees with all spectroscopic data. As a consequence, the elasticity of natural elastin must be considered from a new perspective.

Immunoglobulins and cytokines level in follicular fluid in relation to etiology of infertility and their relevance to IVF outcome.

OBJECTIVE: The aims of the present study were to (i) determine the presence and concentration of albumin fractions (alpha1, alpha2, beta, gamma), immunoglobulins (IgA, IgG, IgM) and cytokines [interleukin (IL)-6, IL-8, granulocyte-macrophage colony-stimulating factor (GM-CSF)] in periovulatory ovarian follicular fluid (FF) of in vitro fertilization (IVF) patients, (ii) examine the relationship between these parameters and the etiology of infertility as well as the IVF outcome and (iii) find out if these parameters in FF could be used as a predictive factor of IVF outcome. DESIGN: The levels of albumin fractions, immunoglobulin and cytokines were measured from women who underwent IVF therapy for various indications and the results were compared between the patient groups and IVF outcome. MATERIALS AND METHODS: Follicular fluid was obtained from 160 IVF patients. A total of 79 patients underwent controlled ovarian hyperstimulations (COH) either with follicle-stimulating hormone (FSH) or HMG. Whereas, the HMG was used for the second set of patients (n=81) - after down regulation with gondotropin-releasing hormone agonists (Gn-RHa) - the protein fractions were determined using electrophoresis separation. Immunoglobulins were measured using a commercial kits and the concentration of cytokines was determined by the highly sensitive enzyme-linked immunosorbent assay (ELISA) methods. RESULTS: The stimulation regimens used have no effect on albumin (alpha1, alpha2, beta, gamma) and immunoglobulin (IgA, IgG, IgM) concentrations, as no significant difference was observed between the two groups. Besides, no specific relationship was found between the concentration of these investigated parameter in FF and etiology of infertility or fertilization, cleavage and pregnancy rate. Besides, there were no significant differences between the groups for any cytokine investigated. Moreover, there were no correlations between the concentration of IL-6, IL-8 and GM-CSF in FF and steroid hormone concentration in the blood at the day of oocytes retrieval or IVF outcome. IN CONCLUSION: Total protein, albumin fraction, immunoglobulins and cytokines level in FF of patients undergoing COH for IVF therapy for various etiology of infertility could not be a useful parameter for predicting IVF outcome.