BAY 81-8973, a full-length recombinant factor VIII: manufacturing processes and product characteristics.

BAY 81-8973 (Kovaltry® , Bayer, Berkeley, CA, USA) is an unmodified, full-length recombinant human factor VIII (FVIII) approved for prophylaxis and on-demand treatment of bleeding episodes in patients with haemophilia A. The BAY 81-8973 manufacturing process is based on the process used for sucrose-formulated recombinant FVIII (rFVIII-FS), with changes and enhancements made to improve production efficiency, further augment pathogen safety, and eliminate animal- and human-derived raw materials from the production processes. The baby hamster kidney cell line used for BAY 81-8973 was developed by introducing the gene for human heat shock protein 70 into the rFVIII-FS cell line, a change that improved cell line robustness and productivity. Pathogen safety was enhanced by including a 20-nm filtration step, which can remove viruses, transmissible spongiform encephalopathy agents and potential protein aggregates. No human- or animal-derived proteins are added to the cell culture process, purification or final formulation. The BAY 81-8973 manufacturing process results in a product of enhanced purity with a consistently high degree of sialylation of N-linked glycans on the molecular surface. The innovative manufacturing techniques used for BAY 81-8973 yield an effective rFVIII product with a favourable safety profile for treatment of haemophilia A.

Characterization of tyrosine sulfate residues in antihemophilic recombinant factor VIII by liquid chromatography electrospray ionization tandem mass spectrometry and amino acid analysis.

Recombinant Factor VIII (rFVIII) is involved in the cascade of biochemical reactions leading to blood coagulation and is used for the treatment of haemophilia A. Plasma-derived FVIII (pdFVIII) has been reported to be post-translationally modified by sulfation of tyrosine residues at positions 346, 1664, 1680, 718, 719 and 723. This report describes the quantitation of tyrosine sulfate residues in BHK-derived, human rFVIII by amino acid composition analysis and the identification of their positions in the polypeptide sequence using a combination of liquid chromatography and electrospray ionization mass spectrometry in the analysis of proteolytic digests of the protein.

Characterization of the microdialysis junction interface for capillary electrophoresis/microelectrospray ionization mass spectrometry.

A capillary electrophoresis/electrospray ionization mass spectrometry (CE/ESI-MS) interface, based on an electric circuit across a microdialysis membrane surrounding a short capillary segment closely connected to the separation capillary terminus, is demonstrated to be sensitive, efficient, and rugged. A microspray type ionization emitter produces a stable electrospray at the low flow rates provided by CE and thus avoids both the need for a makeup liquid flow provided by liquid junction or sheath flow interfaces and the subsequent dilution and reduction in sensitivity. Reproducibility studies and comparisons with CE/UV and the CE/sheath flow interface with ESI-MS are presented. Additionally, postrun acidification via the microdialysis junction interface is demonstrated and shown to be capable of denaturing the holomyoglobin protein noncovalent complex while maintaining separation efficiency.

The interface of capillary electrophoresis with high performance Fourier transform ion cyclotron resonance mass spectrometry for biomolecule characterization.

The interfacing of capillary electrophoresis (CE) with Fourier transform ion cyclotron resonance-mass spectrometry (FTICR-MS) and the factors which dictate obtainable performance (i.e., sensitivity, mass resolution, scan rate and duty cycle) are described. We demonstrate the current status of the technique with examples of capillary zone electrophoresis (CZE) and capillary isotachophoresis (CITP) with FTICR analyses of proteins and oligonucleotides, and describe current limitations on sensitivity and scan speed. The first on-line interfacing of capillary isoelectric focusing (CIEF) with FTICR is also demonstrated and shown to be effective for separating minor components of protein mixtures for on-line mass spectral analysis. Finally, the potential for greatly improved performance based upon recent advances in FTICR instrumentation and methods is briefly described.

Analysis of single cells with capillary electrophoresis electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry.

Recent results with on-line capillary electrophoresis (CE) electrospray ionization (ESI) Fourier transform ion cyclotron resonance (FTICR) mass spectrometry suggest that CE/ESI-FTICR can provide a powerful technique for micro-sample analyses owing to the inherent sensitivity of the technique and the enhanced information content derived from high-performance mass measurements. Using micro-sampling methods and ion accumulation techniques based on quadrupolar excitation, we demonstrate that adequate sensitivity exists to characterize the hemoglobin from a single human erythrocyte (approximately 450 amol). In these studies mass spectra with average mass resolution in excess of 45 000 (FWHM) were obtained for both the alpha- and beta chain of hemoglobin following in-column lysing of a single erythrocyte.

Proton-transfer reactions of mass-selected multiply charged ions.

A triple-quadrupole spectrometer has been used to study proton-transfer reactions of multiply charged ions generated by electrospray ionization. Doubly and triply charged ions generated from the peptides Arg-Lys-Glu-Val-Tyr and Met-Lys-bradykinin, respectively, were found to undergo proton-transfer reactions with ammonia molecules contained in the RF-only quadrupole collision-gas cell of the spectrometer. With horse-heart myoglobin in the source, ions having charges of 20+, 19+, 16+ and 14+ were selected in turn by the first quadrupole and their proton-transfer reactions with ammonia investigated. For each ion, numerous product ions were detected having charges (n-1)+, (n-2)+, (n-3)+ ... where n was the charge on the reacting parent ion. The possibility of using the experimental technique to measure approximately the proton affinities of multiply charged ions is discussed. Also, a procedure is outlined for identifying the charge states of product ions resulting from collision-induced dissociation of multiply charged ions.