A long-acting factor VIII (FVIII) as a replacement therapy for hemophilia A would significantly improve treatment options for patients with hemophilia A. To develop a FVIII with an extended circulating half-life, but without a reduction in activity, we have engineered 23 FVIII variants with introduced surface-exposed cysteines to which a polyethylene glycol (PEG) polymer was specifically conjugated. Screening of variant expression level, PEGylation yield, and functional assay identified several conjugates retaining full in vitro coagulation activity and von Willebrand factor (VWF) binding. PEGylated FVIII variants exhibited improved pharmacokinetics in hemophilic mice and rabbits. In addition, pharmacokinetic studies in VWF knockout mice indicated that larger molecular weight PEG may substitute for VWF in protecting PEGylated FVIII from clearance in vivo. In bleeding models of hemophilic mice, PEGylated FVIII not only exhibited prolonged efficacy that is consistent with the improved pharmacokinetics but also showed efficacy in stopping acute bleeds comparable with that of unmodified rFVIII. In summary site-specifically PEGylated FVIII has the potential to be a long-acting prophylactic treatment while being fully efficacious for on-demand treatment for patients with hemophilia A. (Blood. 2010;116(2):270-279)

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.

Charge ladders of bovine carbonic anhydrase II, hen egg-white lysozyme, and bovine pancreatic trypsin inhibitor, prepared by partial acetylation of primary amino groups on the surface of the protein, have been analyzed by capillary electrophoresis (CE) and on-line electrospray ionization mass spectrometry (ESIMS) using solution conditions that maintain the native structure of the protein. CE was used to separate the proteins that constitute the charge ladder into individual "rungs"sprotein derivatives that have the same number of acetylated amino groups and approximately the same net charge in solution. ESI was used to produce ions in the gas phase of the proteins that constitute each rung of the charge ladder; the mass spectra of these ions were obtained and analyzed. The distributions in charge states observed in the gas phase for the groups of proteins comprising each rung of the charge ladders were narrow, consistent with the retention of a compact structure of the proteins in the gas phase, and substantially independent of the number of acetylated amino groups. The ions observed in the gas phase had surface charge densities in a relatively narrow range of ∼0.9-1.5 units of charge per 10 3 Å 2 of surface area (as estimated from crystallographic structures). These results demonstrate that the distribution of charge states for proteins produced in the gas phase by ESI do not necessarily reflect the net charge of the protein in solution or the number of amino groups on the protein.

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 combination of capillary electrophoresis (CE) with electrospray ionization (ESI) time-of-flight mass spectrometry (TOF-MS) has recently been demonstrated. When CE is combined with TOF-MS using an electrospray interface, the method provides fast, high-resolution separations with rapid mass analysis and the potential for very high sensitivity. In analyzing the data, one first reconstructs an electropherogram from the data set and then identifies peaks in the mass spectra. The mass spectra corresponding to the peaks in the electropherogram are then inspected to enable solute identification. However, background noise often prevents detection of peaks in the reconstructed electropherogram, thus interfering with the analysis. In this work we demonstrate alternative electropherogram reconstruction techniques that enhance its signal-to-noise ratio, allowing more immediate identification of the number of components in a mixture and their corresponding retention times. The techniques described here should also be adaptable for on-line analysis of CE-ESI-TOF data and the combination with other separation techniques coupled to mass spectrometry (e.g., LC/MS), as well as any multichannel detection scheme.The combination of capillary electrophoresis (CE) with electrospray ionization mass spectrometry (ESI-MS) has proven to be a very powerful technique for solving a wide diversity of chemical, biochemical, and biomedical problems.1"5 The main advantages of capillary electrophoresis6 (i.e., speed, resolution, and the ability to handle small sample volumes) can be realized only if the mass spectrometric technique employed retains these properties while providing adequate mass resolution, detection

A novel capillary electrophoresis electrospray ionization mass spectrometry (CE/ESI-MS) interface has been developed based upon an alternative means of establishing electrical contact at the CE capillary terminus. In this design, the terminus of the CE separation capillary and a short ESI emitter capillary are connected and sheathed by polysulphone micro-dialysis tubing. The electrical connection, to close the CE circuit and simultaneously establish the electrospray voltage, is provided outside the membrane via an electrode in a small liquid reservoir. In contrast to the sheath flow and liquid junction interfaces, the flow rate of liquid to the ESI source consists of the CE eluent and is not increased. The use of a 'micro-spray' type emitter produces a stable electrospray at the very low flow rates typical of CE, providing a highly sensitive, efficient interface. Post-separation acidification of analytes via the dialysis junction is also demonstrated.The separation technique of capillary electrophoresis (CE), in which analytes are separated in a small diameter ( c 100 pm) fused-silica capillary due to differences in their electrophoretic mobilities, is increasingly being employed, especially in biomedical and biochemical applications, where minute sample volumes and high-speed analyses are desirable. Mass spectrometric detection in conjunction with CE separations was recognized early as having significant potential, and the development of elecuospray ionization mass spectrometry (ESI-MS)' provided an attractive basis for CE/MS interfacing. The first CE/MS interface was demonstrated by Olivares et al. in 1987; who showed that the electrospray ionization process could provide an effective interface based upon an electrical contact established at or near the end of the CE capillary (or point of electrospray emission).' The electric contact serves to establish both the CE electric field as well as the ESI voltage.Several effective means for establishing this electrical contact have beenIn the initial report,2 a metal coating on the tip of the CE capillary made contact with a metal sheath capillary to which the CE terminus/ESI voltage was applied. Although high sensitivity and efficiency were achieved, problems included a dependence on the buffer system used, ESI instability under some operating conditions and the need to regularly replace the metal coating on the capillary tip. These issues led directly to the development, also in this laboratory, of the 'coaxial sheath-flow' approach: In this interface, a sheath liquid, generally with a small electrolyte content, is infused to the ESI source through a sheath capillary which surrounds the end of the separation capillary and terminates near the end of the separation capillary. This sheath liquid, generally flowing at a rate of a few microliters (pL) per minute, is then added to the CE effluent as it elutes from the terminus of the CE capillary, thus providing the necessary electrical contact. At the liquid flow rates used in this approach some organic content to the ...

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.

BAY 81-8973 (Kovaltry â , 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.