A comparative analysis of heterogeneity in commercially available recombinant factor VIII products.

INTRODUCTION: Advances in analytical technologies enable investigation of possible correlations between molecular structure, aggregation and subvisible particle content. Regulatory agencies place increasing attention on potential risks associated with protein aggregates in the micron range in biological therapeutics. AIM: Assess the heterogeneity, high-molecular-weight protein (HMWP) species, subvisible particle content and posttranslational modifications in six commercially available recombinant FVIII (rFVIII) products. METHODS: Three B-domain-deleted (BDD) or B-domain truncated rFVIII products (turoctocog alfa, simoctocog alfa and moroctocog alfa) and three full-length rFVIII products (octocog alfa FS and two octocog alfa) were analysed. HMWP content, amount of micron range subvisible particles, tyrosine-1680 sulphation and N-glycan analysis were investigated. RESULTS: The B-domain-modified products had more protein size homogeneity vs the full-length products. Size exclusion-high-performance liquid chromatography data indicated no association between B-domain structure and aggregate content or size of the products tested. The rFVIII products showed large variation in subvisible particle concentration, with turoctocog alfa and simoctocog alfa having the lowest numbers (1000-1600 and 1800-2400 particles/100 IU, respectively). Turoctocog alfa and simoctocog alfa displayed the most complete tyrosine sulphation (>99.5%). CONCLUSION: Overall, there was no association between molecular structure (full-length B-domain, BDD or truncated) and subvisible particle or HMWP content. Dissimilarities may be related to production and product handling differences. In this study, turoctocog alfa, such as simoctocog alfa, had one of the lowest levels of subvisible particles and HMWP content, and high protein size homogeneity.

Processing, disulfide pattern, and biological activity of a sugar beet defensin, AX2, expressed in Pichia pastoris.

AX2 is a 46-amino-acid cysteine-rich peptide isolated from sugar beet leaves infected with the fungus Cercospora beticola (Sacc.). AX2 strongly inhibits the growth of C. beticola and other filamentous fungi, but has little or no effect against bacteria. AX2 is produced in very low amounts in sugar beet leaves, and to study the protein in greater detail with respect to biological function and protein structural analysis, the methylotrophic yeast Pichia pastoris was used for large-scale production. The amino acid sequence, processing of the signal peptide, disulfide bridges, and biological activity of the recombinant protein were determined and compared with that of the authentic AX2. In P. pastoris, the protein was expressed with an additional N-terminal arginine. The disulfide bonding was found to be identical to that of the authentic AX2. However, when tested in in vitro bioassay, the biological activity of the recombinant protein was slightly lower than that measured for the authentic protein. Furthermore, the recombinant protein was significantly more sensitive to Ca(2+) than the authentic protein. This is most probably due to the extra arginine, since no other differences between the two proteins have been found.

Substrate specificities of three members of the human UDP-N-acetyl-alpha-D-galactosamine:Polypeptide N-acetylgalactosaminyltransferase family, GalNAc-T1, -T2, and -T3.

Mucin-type O-glycosylation is initiated by UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferases (GalNAc-transferases). The role each GalNAc-transferase plays in O-glycosylation is unclear. In this report we characterized the specificity and kinetic properties of three purified recombinant GalNAc-transferases. GalNAc-T1, -T2, and -T3 were expressed as soluble proteins in insect cells and purified to near homogeneity. The enzymes have distinct but partly overlapping specificities with short peptide acceptor substrates. Peptides specifically utilized by GalNAc-T2 or -T3, or preferentially by GalNAc-T1 were identified. GalNAc-T1 and -T3 showed strict donor substrate specificities for UDP-GalNAc, whereas GalNAc-T2 also utilized UDP-Gal with one peptide acceptor substrate. Glycosylation of peptides based on MUC1 tandem repeat showed that three of five potential sites in the tandem repeat were glycosylated by all three enzymes when one or five repeat peptides were analyzed. However, analysis of enzyme kinetics by capillary electrophoresis and mass spectrometry demonstrated that the three enzymes react at different rates with individual sites in the MUC1 repeat. The results demonstrate that individual GalNAc-transferases have distinct activities and the initiation of O-glycosylation in a cell is regulated by a repertoire of GalNAc-transferases.

Determination of isoforms, N-linked glycan structure and disulfide bond linkages of the major cat allergen Fel d1 by a mass spectrometric approach.

The domestic cat (Felis domesticus) is an important source of indoor allergens, the major allergen being Fel d1 (formerly cat allergen 1). Fel d1 is responsible for cat allergy and has also been established to cause cat-induced asthma. The allergen is a 38 kDa dimer composed of two 19 kDa subunits. Each 19 kDa subunit comprises two disulfide linked polypeptide chains, a light alpha-chain and a heavy beta-chain containing an N-linked oligosaccharide. In this study a variety of endoproteinase digestions of the native allergen in combination with HPLC and matrix-assisted laser desorption mass spectrometry was used to determine the position of the disulfide bridges and to demonstrate that the peptide chains are linked in an anti parallel way. Enzymatic digestion of the reduced and alkylated peptides located the N-glycan to Asn33. Moreover, Fel d1 is found to be partially truncated and to exist in several isoforms. Sequential degradation of the glycosylated peptide with specific glycosidases monitored by mass spectrometry, shows that the glycan is a heterogeneous triantennary complex type structure. The heterogeneity is caused by terminal sialic acid and a fucose residue attached to a beta-galactose residue.

UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase. Identification and separation of two distinct transferase activities.

Using a defined acceptor substrate peptide as an affinity chromatography ligand we have developed a purification scheme for a unique human polypeptide, UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase (GalNAc-transferase) (White, T., Bennett, E.P., Takio, K., Sørensen, T., Bonding, N., and Clausen, H. (1995) J. Biol. Chem. 270, 24156-24165). Here we report detailed studies of the acceptor substrate specificity of GalNAc-transferase purified by this scheme as well as the Gal-NAc-transferase activity, which, upon repeated affinity chromatography, evaded purification by this affinity ligand. Using a panel of acceptor peptides, a qualitative difference in specificity between these separated transferase activities in four rat organs and two human organs also revealed qualitative differences in specificity. The results support the existence of multiple Gal-NAc-transferase activities and suggest that these are differentially expressed in different organs. As the number of GalNAc-transferases existing is unknown, as is the specificity of the until now cloned and expressed GalNAc-transferases (T1 and T2), it is as yet impossible to relate the results obtained to specific enzyme proteins. The identification of acceptor peptides that can be used to discriminate GalNAc-transferase activities is an important step toward understanding the molecular basis of GalNAc O-linked glycosylation in cells and organs and in pathological conditions.