Functional characteristics of N8, a new recombinant FVIII.

The aim of this study was to evaluate the in vitro function of the new recombinant factor VIII (FVIII) compound, N8. The specific activity of N8 as measured in a FVIII:C one-stage clot assay was 9300±400 IU mg(-1) based on the analysis of seven individual batches. The ratio between the FVIII:C activity measured in clot and chromogenic assays was 1.00 (95% confidence interval 0.97-1.03). N8 bound to von Willebrand factor with Kd values of 0.2 nm when measured by ELISA and by surface plasmon resonance. FVIIIa cofactor activity was determined from the kinetic parameters of factor IXa-catalysed factor X (FX) activation. The rate of activation of N8 by thrombin as well as Km and kcat for FX activation was in the same range as those observed for Advate®. The rate of activated protein C (APC)-catalysed inactivation was similar for activated N8 and Advate®. N8 improved thrombin generation in a dose-dependent manner and induced similar rates of thrombin generation as Advate® and the plasma-derived FVIII product Haemate®. Using thromboelastography (TEG®), N8 was shown to improve the clot formation and clot stability in whole blood from haemophilia A patients. Comparable potency and efficacy of N8 and Advate® was found based on TEG® parameters. Finally, similar binding profiles to immobilized lipoprotein receptor-related protein (LRP) of N8 and Advate® were observed. The study demonstrated that N8 is fully functional in a variety of assays measuring FVIII activity. No functional differences were found between N8 and comparator compounds.

Purification and characterization of a new recombinant factor VIII (N8).

SUMMARY: A new recombinant factor VIII (FVIII), N8, has been produced in Chinese hamster ovary (CHO) cells. The molecule consists of a heavy chain of 88 kDa including a 21 amino acid residue truncated B-domain and a light chain of 79 kDa. The two chains are held together by non-covalent interactions. The four-step purification includes capture, affinity purification using a monoclonal recombinant antibody, anion exchange chromatography and gel filtration. The specific clotting activity of N8 was 8800-9800 IU mg(-1). Sequence and mass spectrometry analysis revealed two variants of the light chain, corresponding to two alternative N-terminal sequences also known from plasma FVIII. Two variants of the heavy chain are present in the purified product, namely with and without the B-domain linker attached. This linker is removed upon thrombin activation of N8 rendering an activated FVIII (FVIIIa) molecule similar to plasma FVIIIa. All six known tyrosine sulphations of FVIII were confirmed in N8. Two N-linked glycosylations are present in the A3 and C1 domain of the light chain and two in the A1 domain of the heavy chain. The majority of the N-linked glycans are sialylated bi-antennary structures. An O-glycosylation site is present in the B-domain linker region. This site was glycosylated with a doubly sialylated GalNAc-Gal structure in approximately 65% of the product. In conclusion, the present data show that N8 is a pure and well-characterized FVIII product with biochemical properties that equal other FVIII products.

Purification and characterisation of tyrosine decarboxylase and aromatic-L-amino-acid decarboxylase.

Microbial tyrosine decarboxylase (EC 4.1.1.25) and mammalian aromatic-L-amino-acid decarboxylase (EC 4.1.1.28) catalyse the formation of tyramine from L-tyrosine. These enzymes were characterised after isolation to purity by methods including fast polymer liquid chromatography (FPLC). Tyrosine decarboxylase was isolated from Streptococcus faecalis by FPLC anion exchange chromatography (11-times purification; 72% recovery; 23.2 U/mg protein). FPLC on Phenyl-Superose resulted in purification to 115 U/mg protein. Aromatic-L-amino-acid decarboxylase was isolated from pig kidney by ammonium sulfate fractionation, DEAE chromatography, and FPLC anion exchange chromatography (21-times purification; 22% recovery; 0.71 U/mg protein). By FPLC chromatofocusing, tyrosine decarboxylase eluted at pH 4.3 and aromatic-L-amino-acid decarboxylase at pH 5.0. Isoelectric focusing of tyrosine decarboxylase gave two bands (pI 4.4 and 4.5). With pyridoxal 5'-phosphate removed by ultrafiltration, only one band (pI 4.4) appeared, and SDS polyacrylamide electrophoresis confirmed the purity. FPLC gel filtration resulted in molecular weights 143,000 and 86,000, respectively, for tyrosine decarboxylase and aromatic-L-amino-acid decarboxylase. In SDS electrophoresis, tyrosine decarboxylase had the monomer molecular weight 75,000, showing a dimer structure for the enzyme.

Analysis of the site-specific asparagine-linked glycosylation of recombinant human coagulation factor VIIa by glycosidase digestions, liquid chromatography, and mass spectrometry.

The two asparagine-linked glycosylation sites of recombinant coagulation factor VIIa have been characterized by glycosidase digestions, size-exclusion chromatography (SEC), and mass spectrometry (MS). Nine structures were characterized as core fucosylated bi-and triantennary structures with 0-3 sialic-acid residues which were alpha 2-3 linked to galactose exclusively. Three of the structures had one or two galactose residues substituted by N-acetylgalactosamine. Significant differences were found between the oligosaccharide profiles for the two glycosylation sites in rFVIIa. At Asn322, the degree of sialylation was lower and higher amounts of structures containing N-acetylgalactosamine were found compared to Asn145.

Analysis of the glycoforms of human recombinant factor VIIa by capillary electrophoresis and high-performance liquid chromatography.

The carbohydrate-dependent microheterogeneity of recombinant coagulation factor VIIa (rFVIIa) has been characterized by capillary electrophoresis (CE) of the native protein and by high-performance liquid chromatography (HPLC) of tryptic peptides and of oligosaccharides released by hydrazinolysis. The development of the CE analysis is reported here. We have found that application of 1,4-diaminobutane (putrescine) as additive to the CE separation buffer is essential for the separation of the various glycoforms. Under optimum conditions rFVIIa migrates as a cluster of six peaks or more. By CE of neuraminidase-treated rFVIIa a faster-moving double peak is observed. This indicates that the separation obtained is primarily based upon the different content of N-acetyl-neuraminic acid of the oligosaccharide structures in rFVIIa. By reversed-phase HPLC of tryptic digested neuraminidase treated rFVIIa the glycopeptides containing the heavy chain N-glycosylated site elute as two peaks compared to the four peaks corresponding to glycopeptides with 0 to 3 N-acetyl-neuraminic acids seen for untreated rFVIIa. In high-pH anion-exchange HPLC of the oligosaccharides released from native rFVIIa by hydrazinolysis the major peaks elute as oligosaccharides with 1 or 2 N-acetyl-neuraminic acids. Oligosaccharides released from neuraminidase treated rFVIIa elute earlier compared to oligosaccharides from native rFVIIa, but separated into several peaks, indicating heterogeneity for the oligosaccharide structures without N-acetyl-neuraminic acid.

Formation of biogenic amines in herring and mackerel.

The formation of biogenic amines (histamine, cadaverine, putrescine and spermidine) was followed during vacuum packed storage at 2 degrees C or 10 degrees C in the scombroid fish mackerel and in the non-scombroid fish herring. Also the changes in the content of free amino acids and in the organoleptic and microbiological qualities were followed. At 10 degrees C the amine contents were 2-20 times higher at the time of rejection as compared with samples stored at 2 degrees C. In herring and mackerel similar amounts of histamine were accumulated, whilst cadaverine was formed at much higher levels in mackerel compared with herring. The high contents of cadaverine in mackerel can possibly explain why mackerel and not herring are often implicated in incidents of scombrotoxic poisoning.

Characterization of glycopeptides from recombinant coagulation factor VIIa by high-performance liquid chromatography and capillary zone electrophoresis using ultraviolet and pulsed electrochemical detection.

Four glycopeptide (GP) fractions containing glycosylated Asn 322 were isolated from a tryptic digest of recombinant coagulation factor VII by reversed-phase-HPLC (RP-HPLC). Characterization of the GPs by enzymatic desialylation and RP-HPLC as well as by enzymatic deglycosylation, RP-HPLC, and high-pH anion-exchange chromatography indicated that the four GPs consisted of the same decapeptide but with 0, 1, 2, or 4 residues of sialic acid. In comparison to HPLC, capillary zone electrophoresis (CZE) using uv and pulsed electrochemical detection (PED) afforded improved separation of GPs from each other and from contaminants. CZE-uv and CZE-PED of the desialylated GPs and deglycosylated GPs corroborated the results obtained with the chromatographic methods.

Characterisation of a trisulphide derivative of biosynthetic human growth hormone produced in Escherichia coli.

A novel protein derivative has been found during process development of biosynthetic human growth hormone; it has been characterised as human growth hormone with a Cys182-Cys189 trisulphide bridge. We have not been able to find a previous report in the literature about this kind of derivative. The characterisation was obtained partly on the full-length derivative and partly on a tryptic fragment of the derivative. The full-length derivative was characterised by reduction with 1,4-dithiothreitol followed by electrospray mass spectrometry, treatment with cysteine and measurement of hydrogen sulphide liberation upon cysteine treatment. The tryptic fragment from peptide mapping was characterised by amino acid analysis, amino acid sequencing and mass spectrometry. All data indicated an extra sulphur atom in the Cys182-Cys189 cystine bridge.

Synthesis of stereoisomers and isoforms of a tryptic heptapeptide fragment of human growth hormone and analysis by reverse-phase HPLC and capillary electrophoresis.

The amino acid sequence Asn-Gly has at pH 7 a tendency to induce deamidation of asparagine to aspartic acid via the formation of a cyclic imide. This imide opens up to yield Asp-Gly or the isoaspartic acid (isoAsp) form, isoAsp-Gly. Both isomers may be found in their L-form or D-form. Like Asn-Gly, the sequence Asp-Gly has a tendency for isomerization and racemization via the formation of a cyclic imide intermediate. When human growth hormone is digested with trypsin, one of the fragments is a heptapeptide (amino acid residues 128-134) containing the amino acid sequence Asp-Gly (amino acid residues 130 and 131). This heptapeptide, as well as stereoisomers and isoforms where L-Asp was replaced by D-Asp, L-isoAsp, D-isoAsp or the L-cyclic imide, respectively, has been synthesized and used as a standard to achieve separation of the five forms by capillary electrophoresis and by reverse-phase HPLC. Capillary electrophoresis analysis was performed in uncoated capillaries by the use of aspartic acid/cyclodextrin buffers at low pH. The elution order of the aspartic-acid-containing heptapeptides was D-Asp, L-Asp, L-isoAsp, D-isoAsp and L-cyclic imide. Reverse-phase HPLC analysis was performed on a C18 column by the use of a shallow acetonitrile gradient in trifluoroacetic acid/water. The elution order was D-isoasp, L-isoASp, L-Asp, D-Asp and L-cyclic imide. Human growth hormone samples were degraded by incubation at high temperature and analyzed for their potential content of isomerization and racemization products. Only L-forms of aspartic acid and isoaspartic acid of the heptapeptide fragment were found.

Recombinant activated factor VII (rFVIIa): characterization, manufacturing, and clinical development.

Recombinant activated coagulation factor VII (rFVIIa) (NovoSeven) was developed for treatment of bleeding in hemophilia patients with inhibitors (antibodies) against factors VIII or IX. rFVIIa initiates the coagulation cascade by binding to tissue factor at the site of injury and causes the formation of sufficient amounts of thrombin to trigger coagulation. Patients with a variety of other coagulation deficiencies than hemophilia characterized by an impaired thrombin generation and life-threatening bleeding have been reported as successfully treated with rFVIIa. Data are now entered into clinical registries established to further monitor this experimental treatment with NovoSeven. rFVIIa is produced free of any added human protein. The amino acid sequence of rFVIIa is identical to plasma-derived FVIIa (pdFVIIa). Posttranslational modifications (i.e., gamma-carboxylations, N- and O-glycosylations) are qualitatively identical in pdFVIIa and rFVIIa although some quantitative differences exist. The activities of rFVIIa and pdFVIIa are indistinguishable. Manufacturing of rFVIIa involves expression in baby hamster kidney (BHK) cells followed by purification, including three ion-exchange and one immunoaffinity chromatography steps. The last anion-exchange chromatography step ensures completion of the autoactivation of recombinant factor VII (rFVII) to rFVIIa. This review describes the mechanism of action, characterization, manufacturing, and preclinical and current clinical evidence for the efficacy and safety of rFVIIa.