Recombinant molecules similar to the smallest active plasma-derived factor VIII molecule, a complex of an 80-kDa and a 90-kDa polypeptide chain lacking the B domain, have been produced using various factor VIII cDNA constructs in order to obtain primary translation products which were efficiently processed into the 80+90-kDa complex. Three types of single-chain cDNAs encoding B-domain-deleted derivatives factor VIII were designed, taking account of sites at Arg740 and Glu1649, assumed to be important for processing factor VIII.In the type 1 constructs, either Arg747, Arg752, or Arg776 in the N-terminal region of factor VIII B domain was fused to the N-terminus (Glu1649) of the 80-kDa subunit. In the type 2 construct r-VIII SQ, Ser743 was fused to Gln1638, creating a link of 14 amino acids between the C-terminus (Arg740) of the 90-kDa chain and N-terminus of the 80-kDa chain, whereas in type 2 r-VIII RH, Arg747 was fused to His1646. In the type 3 constructs, the B-domain was completely removed or replaced with 1-4 Arg residues.After expression in Chinese hamster ovary cells, the type 1 derivatives and the type 3 derivatives with 0-2 Arg residues inserted were found to be only partially processed and contained a large amount of the 170-kDa primary translation product. In contrast, most of the type 2 derivatives r-VIII SQ and r-VIII RH and the type 3 derivatives r-VIII R4 and r-VIII R5 containing three or four extra Arg residues preceding the N-terminus of the 80-kDa chain were processed into the desired 80+90-kDa chain complexes. The feature common to the most efficiently processed factor VIII deletion derivatives was that they contained the recognition motif for proteolytic cleavage by the membrane-bound subtilisin-like protease furin, which is expressed in most types of cells; that is, basic amino acid residues at positions -1 and -4 relative to the cleavage site at Glu1649. Biochemical studies of r-VIII SQ and r-VIII R5, two of the most effectively processed factor VIII derivatives, showed that both proteins had a normal factor VIII cofactor function, and had N-and C-termini of the 80-kDa and 90-kDa chains corresponding to those found in plasma-derived factor VIII.

The antibacterial and cytotoxic activity of the human cathelicidin peptide LL-37 is inhibited by plasma. Because LL-37 does not undergo rapid degradation in human plasma, we postulated that this inhibition results from binding of LL-37 to unidentified proteins. An LL-37 binding plasma protein has now been isolated by affinity chromatography. SDS-polyacrylamide gel electrophoresis of proteins that bound to an LL-37 column revealed one band with a molecular mass of about 26 kDa, and amino acid sequence analysis identified the protein as apolipoprotein A-I (apoA-I). Biomolecular interaction analysis using surface plasmon resonance showed that LL-37 and isolated apoA-I bind with an apparent K d in the low micromolar range. 50 M of apoA-I inhibits the antibacterial activity of 50 M LL-37 by about 50% of the inhibition exhibited by plasma. In addition, anti-apoA-I IgG completely blocks the plasma inhibition of LL-37 antibacterial activity up to a peptide concentration of 25 M and blocks most of the plasma inhibition at higher LL-37 concentrations. These results indicate that apoA-I is the main LL-37 binding protein in human plasma and may work as a scavenger of LL-37, thus suggesting a novel mechanism involved in the regulation of a cathelicidin peptide.

The proteolytic activities expressed by a Chinese hamster ovary (CHO) cell line in the cultivation supernatant during the production of recombinant factor VIII were mapped with a broad spectrum protease assay and a series of different types of protease inhibitors. The destabilizing effect on the product emanated from a metalloproteinase, which could be effectively blocked by chelating agents to lead to product stabilization. Amino acid sequences of the isolated metalloproteinase were found to have sequence homology with matrix metalloproteinases (MMPs) MMP3, MMP10, and MMP12. Several species with metalloproteinase activity were characterized and found to be related to each other. The results indicate that an MMP pro-enzyme of >/=200 kDa was released from the CHO cells during the production phase. The enzyme expressed collagenase/gelatinase activity when activated. Due to autoproteolysis, a number of smaller, less specific MMPs were formed with the smallest form, a 19.4 kDa protein, being the most active. These results may be of particular relevance for other production processes using CHO cells for the expression of recombinant proteins.

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SummaryRecombinant factor VIII SQ (r-VIII SQ), ReFacto®, is a recombinant factor VIII product similar to the smallest active factor VIII protein found in plasma-derived factor VIII (p-VIII) concentrates. The protein comprises two polypeptide chains of 80 and 90 kDa and lacks the major part of the heavily glycosylated B-domain i.e. amino acids Gln744 to Ser1637. r-VIII SQ retains six potential glycosylation sites for N-linked oligosaccharides at asparagine residues 41, 239, 582, 1685, 1810 and 2118.We describe a thorough comparison of the characteristics of r-VIII SQ with those of p-VIII. The primary and secondary structures of r-VIII SQ were in good agreement with that of B-domain-deleted p-VIII (p-VIII-LMW) as shown by SDS-PAGE, Western blotting with antifactor VIII antibodies, tryptic mapping, amino acid sequence analysis and circular dichroism spectroscopy. A few divergences also existed. Thus r-VIII SQ was shown to contain a small amount of the single chain primary translation product of 170 kDa and also the product specific sequence of 14 amino acids, the SQ-link, in the C-terminal end of the 90 kDa chain. It was shown that r-VIII SQ had a high specific activity of about 14,000 IU VIII:C/mg as determined by use of a chromogenic substrate assay. The r-VIII SQ protein was comparable to p-VIII forms with a retained B-domain, in terms of potency measured by a chromogenic substrate or a two-stage clotting assay, in interactions with thrombin, and with activated protein C (APC) in combination with Protein S. The ability of r-VIII SQ to participate as a cofactor in factor Xa generation in a mixture of factors IXa and X, phospholipid and calcium was in conformity with that of p-VIII. Furthermore r-VIII SQ had a good binding capacity for phospholipid vesicles and von Willebrand factor (vWF) as shown in gel filtration studies. The same kinetics in binding to von Willebrand factor was found for r-VIII SQ and p-VIII as determined by real-time biospecific interaction analysis (BIA) with use of the BIAcore® instrument. The apparent association rate constant was 4 × 106 M−1s−1. Two dissociation rate constants were found, 1 × 10−2s−1 and 4 × 10−4s−1. The results extend the present knowledge that the factor VIII B-domain is dispensable for the factor VIII cofactor function in hemostasis.