Neurons from embryonic (E18) rat hippocampus were chosen to identify and characterize neurite growth-stimulating proteins accumulating in serum-free conditioned media (CM) obtained from primary or secondary cultures of cerebral astrocytes (less than 5% nonglial cells) using a quantitative cell culture bioassay. CM were fractionated by FPLC on an anion exchange column (Mono Q) and by gel filtration (Superose 6). Column fractions were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, immunoblotting and enzyme-linked immunosorbent assay (ELISA) using antibodies to laminin (LN) and fibronectin (FN). The neurite-promoting activity (NPA) was tested by incubating aliquots of the eluted fractions with poly-L-lysine precoated glass coverslips prior to addition of neurons suspended in chemically defined medium. We provide evidence that the NPA in astroglial CM could be assigned mainly to a negatively charged, highly sulfated LN complex consisting predominantly of the B-chains of LN and presumably a sulfated proteoglycan that was sensitive for chondroitinase and to a lower degree to heparinase degradation. In addition, a smaller proportion of the NPA was associated with uncomplexed LN and free FN. FN reached approximately 10 times the concentration of LN in astroglial CM. As revealed by immunofluorescence microscopy, both LN and FN are simultaneously expressed by cultured astrocytes; however, only the production of FN, measured by ELISA, increased during the time astrocytes were in culture, whereas the release of LN remained unchanged. We conclude that, besides the most active LN complex, FN bound to a polycationic matrix is able to induce neurite growth in hippocampal neurons in vitro.

Ultraviolet-C (UVC) irradiation is a pathogen inactivation method used for disinfection of pharmaceutical products derived from human blood. Previous studies have shown that UVC can potentially damage proteins through photolysis or can generate reactive species resulting in protein thiol oxidation. In this study, two fluorescence-based quantitative proteomic approaches were used to assess the effects of a novel UVC-disinfection strategy on human plasma fractions. We show minimal changes in protein content, but gross alterations in protein thiol reactivity, indicative of oxidative damage. We identify a number of the damaged proteins by mass spectrometry, including serum amyloid P component, and further demonstrate UVC-induced photolysis of its disulphide bond.

SummaryBaxter has developed a recombinant therapy for treating von Willebrand‘s disease. Recombinant VWF is co-expressed with the rFVIII in CHO cells used to produce the rFVIII product Advate. This rVWF is used as a drug component for a rVWF-rFVIII complex drug product. CHO cells produce partially processed and partially un-processed rVWF still containing the pro-peptide. In order to make a consistent preparation containing mature and processed rVWF only, rVWF is exposed to recombinant furin to remove the pro-peptide. Recombinant

Covalent modification of therapeutic proteins by polyethylene glycol derivatives is an established method for improving pharmacokinetic properties of therapeutic proteins. Highly purified rVWF expressed in CHO cells, was chemically modified via PEGylation of lysine residues at mild alkaline pH with PEG succinimidyl succinate (linear 5 kDa PEG). Increasing the amount of PEG used in the coupling procedure, the molecular size of VWF increased, as demonstrated in SDS-PAGE and by agarose gel electrophoreses indicating an increase in size of the bands resembling the VWF multimers. PEGylation of rVWF reduced platelet-aggregating and collagen-binding functions by 60% (VWF:RCo activity) and 40% (VWF:CB activity). While FVIII-binding capacity, measured by a FVIII binding ELISA, was reduced and reduction correlated with the amount of PEG bound to VWF, there was almost no effect on FVIII binding affinity which remained in the same order of magnitude as measured with non-PEGylated VWF. Similar results were obtained when rVWF was PEGylated via carbohydrate moieties after oxidation and subsequent derivatization with monomethoxy-PEG hydrazide. PEGylated rVWF was applied to VWF-deficient mice at a dose of 40 VWF:Ag U/kg and plasma levels were monitored for up to 24 hours. As a control, non-modified rVWF was applied to the animals. PEGylated VWF had substantially prolonged survival in the circulation compared with non-modified rVWF with an increase of the AUC by a factor of >10. VWD mice substituted with human VWF show a secondary rise in FVIII bringing them into the FVIII levels measured in C57Bl/6 control mice. This secondary rise was sustained after treatment with PEGylated rVWF where FVIII levels above the starting level were measurable even 48 hours after injection while in the control group base line FVIII levels were reached already after 24 hours. rVWF is the largest protein ever PEGylated and PEGylation results in prolonged survival in the circulation while maintaining FVIII stabilizing functions of the VWF molecule in vivo.

The high specific activity of the A1PI preparation achieved with this process should allow a reduction of the A1PI total protein load necessary to achieve clinically relevant effects.