Mapping and partial characterization of proteases expressed by a CHO production cell line.

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.

Structural and functional characterization of B-domain deleted recombinant factor VIII.

A new high-purity recombinant factor VIII preparation has been developed for the treatment of hemophilia A. Structurally, this factor VIII preparation, B-domain deleted recombinant factor VIII (BDDrFVIII), differs from other recombinant and plasma-derived factor VIII preparations in that most of the B-domain has been deleted. To ensure that BDDrFVIII contains the requisite structural and functional features, it has been subjected to detailed biochemical and biophysical characterization in comparison to the plasma-derived form of factor VIII. Laboratory studies have shown that the primary, secondary, and tertiary structures of BDDrFVIII and the posttranslational modifications are similar to those of the [80 + 90]-kd form of plasma-derived factor VIII. In addition, BDDrFVIII has full biologic activity compared with full-length factor VIII preparations.

Measurement of factor VIII activity of B-domain deleted recombinant factor VIII.

The factor VIII activity of B-domain deleted recombinant factor VIII (BDDrFVIII) measured by activated partial thromboplastin time (APTT)-based one-stage assays is approximately 50% of the activity obtained by the chromogenic assay. Similar results have been reported for the two licensed full-length recombinant factor VIII products. In view of these findings, comprehensive studies have been undertaken to find the cause of the assay differences. Only the phospholipid reagent, used as a platelet substitute in the one-stage assay, proved to be crucial for explaining the assay difference. When platelet-rich plasma was used as the source of phospholipid in the one-stage assay, the factor VIII activity assay results correlated well with those measured by the chromogenic assay. Similar results were obtained when the platelets were replaced by liposomes prepared using platelet factor 3 (PF3) as a model that has a low content (5% to 10%) of phosphatidylserine (PS). In contrast, the use of liposomes with 20% to 30% PS, as in the crude lipid extracts used in ordinary APTT reagents, resulted in underestimation of the factor VIII activity. Antigen measurements using an enzyme-linked immunosorbent assay (ELISA) method demonstrated a good correlation between the antigen and chromogenic activity, but not always between antigen and one-stage activity results. Based on these findings and the clinical data, it can be concluded that the chromogenic assay most accurately measures the functional activity of BDDrFVIII. However, modifications of the one-stage assay, such as the use of a product-specific standard or development of a PF3-like phospholipid reagent, could address the observed assay discrepancies.

B-domain deleted recombinant factor VIII formulation and stability.

B-domain deleted recombinant factor VIII (BDDrFVIII) is a deletion form of human coagulation factor VIII. A lyophilized formulation of highly purified BDDrFVIII has been developed that does not require the use of blood-derived products such as human serum albumin (HSA). By avoiding the use of blood-derived products, the BDDrFVIII formulation minimizes the risk of transmitting blood-borne pathogens that may be present in plasma-derived factor VIII or in other recombinant factor VIII products that contain HSA in their formulation. Upon reconstitution with saline (4 mL), the composition of the reconstituted product (62.5 to 250 IU/mL BDDrFVIII) is 18 mg/mL sodium chloride, 3.0 mg/mL sucrose, 1.5 mg/mL L-histidine, 0.25 mg/mL calcium chloride dihydrate, and 0.1 mg/mL polysorbate 80. The optimal combination of these excipients in the lyophilized BDDrFVIII formulation provides long-term stability, as measured by a variety of analytical methods. The formulation preserves factor VIII activity of lyophilized BDDrFVIII during storage for at least 24 months at 8 degrees C, and for up to 6 months at room temperature (25 degrees C). The reconstituted product retains its factor VIII potency for at least 100 hours at 25 degrees C, which would allow it to be continuously administered via an infusion pump, assuming the product is handled under aseptic conditions.

Structural and functional characteristics of the B-domain-deleted recombinant factor VIII protein, r-VIII SQ.

Recombinant 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 x 10(6) M(-1)s(-1). Two dissociation rate constants were found, 1 X 10(-2)s(-1) and 4 x 10(-4)s(-1). The results extend the present knowledge that the factor VIII B-domain is dispensable for the factor VIII cofactor function in hemostasis.

p49, a putative HLA class I-specific inhibitory NK receptor belonging to the immunoglobulin superfamily.

NK cells display several killer inhibitory receptors (KIR) specific for different alleles of MHC class I molecules. A family of KIR are represented by type I transmembrane proteins belonging to the immunoglobulin superfamily (Ig-SF). Besides cDNA encoding for these KIR, additional cDNA have been identified which encode for Ig-SF receptors with still undefined specificity. Here we analyze one of these cDNA, termed cl.15.212, which encodes a type I transmembrane protein characterized by two extracellular Ig-like domains and a 115-amino acid cytoplasmic tail containing a single immuno-receptor tyrosine-based inhibitory motif (ITIM) which is typical of KIR. cl.15.212 cDNA displays approximately 50 % sequence homology with other Ig-SF members. Different from the other KIR, cl.15.212 mRNA is expressed by all NK cells and by a fraction of KIR+ T cell clones. cl.15.212 cDNA codes for a membrane-bound receptor displaying an apparent molecular mass of 49 kDa, thus termed p49. To determine the specificity of the cl.15.212-encoded receptor, we generated soluble fusion proteins consisting of the ectodomain of p49 and the Fc portion of human IgG1. Soluble molecules bound efficiently to 221 cells transfected with HLA-G1, -A3, -B46 alleles and weakly to -B7 allele. On the other hand, they did not bind to 221 cells either untransfected or transfected with HLA-A2, -B51, -Cw3 or -Cw4. The binding specificity of soluble p49-Fc was confirmed by competition experiments using an anti-HLA class I-specific monoclonal antibody. Finally, different cDNA encoding for molecules homologous to cl.15.212 cDNA have been isolated, two of which lack the sequence encoding the transmembrane portion, thus suggesting they may encode soluble molecules.

Influence of phospholipids on the assessment of factor VIII activity.

In view of reported discrepancies between different factor VIII assays, the influence of phospholipids on the performance of one-stage clotting (OS) and chromogenic substrate (CS) assays was evaluated. The B domain deleted recombinant factor VIII, rVIII SQ, two full-length recombinant products and a plasma derived factor VIII concentrate were each diluted into severe haemophilia A plasma and assayed against a plasma standard. The one-stage activity was 50, 80, 75 and 106%, respectively, of the chromogenic result. Variations in the phospholipid concentration did not affect the chromogenic assay, except at very low levels where the apparent activity increased. In contrast, dilution of the phospholipid reagent had a substantial influence on the activity measured by OS assays, especially in the case of rVIII SQ. At low levels of phospholipid, the one-stage activity of rVIII SQ exceeded the chromogenic result. When mixtures of phosphatidylserine (PS) and phosphatidyl-choline (PC) were used as a source of phospholipid, the OS results for rVIII SQ agreed well with the CS activity as long as the content of PS was below 10%, i.e., closer to the physiological level. At higher levels of PS, as in most commercial APTT reagents, the OS activity decreased. When the APTT reagent was replaced by platelets in the OS assay, the results compared well with those obtained by the CS assay for both t-VIII SQ and full-length factor VIII products.

Development of freeze-dried albumin-free formulation of recombinant factor VIII SQ.

PURPOSE: To develop a stable freeze-dried formulation of recombinant factor VIII-SQ (r-VIII SQ) without the addition of albumin. METHODS: Different formulations were evaluated for their protective effect during sterile filtration, freeze-thawing, freeze-drying, reconstitution and long term storage. Factor VIII activity (VIII:C), visual inspection, clarity, solubility, moisture content and soluble aggregates and/ or fragments were assayed. RESULTS: A combination of non-crystallising excipients (L-histidine and sucrose), a non-ionic surfactant (polysorbate 80) and a crystalline bulking agent (sodium chloride) was found to preserve the factor VIII activity during formulation, freeze-drying and storage. Calcium chloride was included to prevent dissociation of the heavy and light chains of r-VIII SQ. Sodium chloride was chosen as the primary bulking agent since the concentration of sodium chloride necessary for dissolution of r-VIII SQ in the buffer will inhibit the crystallization of many potential cake formers. It was found that L-histidine, besides functioning as a buffer, also protected r-VIII SQ during freeze-drying and storage. A pH close to 7 was found to be optimal. Some potential macromolecular stabilisers, PEG 4000, Haes-steril and Haemaccel, were evaluated but they did not improve the recovery of VIII:C. The freeze-dried formulation was stable for at least two years at 7 degrees C and for at least one year at 25 degrees C. The reconstituted solution was stable for at least 100 hours at 25 degrees C. CONCLUSIONS: The albumin-free formulation resulted in consistently high recovery of VIII:C, very low aggregate formation and good storage stability. The stability of the reconstituted solution makes the formulation suitable for continuous administration via infusion pump. The formulation strategy described here may also be useful for other proteins which require a high ionic strength.

Recombinant factor VIII SQ--inactivation kinetics in aqueous solution and the influence of disaccharides and sugar alcohols.

PURPOSE: To investigate the influence of various nonreducing disaccharides and sugar alcohols on the inactivation kinetics of recombinant factor VIII SQ (r-VIII SQ) in aqueous solution not containing albumin as a stabiliser. METHODS: The stability of r-VIII SQ was followed using measurement of activity (VIII:C) and HPLC gel filtration at different temperatures. The thermal stability was investigated using differential scanning calorimetry (DSC). RESULTS: The decline in VIII:C followed pseudo-first order kinetics. However, the Arrhenius plot was not linear for formulations without carbohydrate, demonstrating a distinct, reproducible curvature. The reaction rate at 5 degrees C was faster than expected from the Arrhenius kinetics. The energy of activation (Ea) for formulations without added carbohydrates, derived from the linear part of the Arrhenius plot, varied between 77 and 86 kJ/mole in the temperature range 20-37 degrees C. The addition of 600 mg/ml sucrose increased the Ea to 104 kJ/mole. DSC measurements showed that Tm' was 64.2 +/- 0.2 degrees C for r-VIII SQ without stabiliser. This value increased linearly with increasing concentrations of carbohydrate. This stabilising effect is most probably explained by the theory of preferential hydration. CONCLUSIONS: The inactivation kinetics of r-VIII SQ in aqueous solution without addition of carbohydrates followed pseudo-first order kinetics but the Arrhenius plot was nonlinear. Sucrose and sorbitol both had highly stabilising effects on r-VIII SQ at concentrations above 300 mg/ml. The preparation containing 600 mg/ml sucrose was stable for at least 12 months at 5 degrees C and 6 months at 25 degrees C.

Determination of total protein in highly purified factor IX concentrates.

Protein determination by the methods of Kjeldahl, Biuret, Bradford and UV absorbance at 280 nm have been studied in regard to accuracy, precision and simplicity. A reference preparation of a highly purified factor IX concentrate, Nanotiv, reconstituted to 1/5 of ordinary volume was used in the study in order to make a comparison between the different procedures. The Kjeldahl method resulted in a protein concentration of 3.7 mg/ml, whereas the Biuret, Bradford (BSA) and UV absorbance at 280 nm resulted in protein concentrations of 3.6, 2.5 and 2.8 mg/ml, respectively. The corresponding values for specific activity were 136, 140, 200 and 179 IU/mg, respectively. These results demonstrate a great variation in the response obtained by different methods for determination of total protein.

Human factor VIII: a calcium-linked protein complex.

The possible role of Ca2+ as an essential constituent part of the human factor VIII complex has been investigated by stability studies, metal determinations, and gel filtration experiments. In citrated plasma, the factor VIII coagulant activity (VIII:C) deteriorated during storage in a biphasic manner. Collection of blood in heparin, instead of chelating anticoagulants, or neutralization of citrate by addition of CaCl2 to heparinized citrate phosphate dextrose (CPD) plasma rendered VIII:C noticeably stable. At physiologic levels of ionized calcium, VIII:C was almost completely stable during incubation of plasma for 6 hr at 37 degrees C. The influence of other divalent ions was also studied. Highly purified factor VIII complex was subjected to atomic absorption spectrophotometric analysis and found to contain about 1.0 mole calcium per 220,000 daltons. This intrinsic calcium could be readily removed by EDTA. When heparin plasma and CPD plasma were chromatographed on Sepharose CL-6B at 37 degrees C, all the factor-VIII-related activities eluted together as large protein complexes. In contrast, factor VIII coagulant antigen (VIII:CAg) and factor-VIII-related antigen (VIIIR:Ag) were completely dissociated upon exposure to EDTA. From these observations it is concluded that human factor VIII circulates in normal plasma as a calcium-linked protein complex.

The effect of intranasal DDAVP on coagulation and fibrinolytic activity in normal persons.

DDAVP (1-desamino-8-D-arginine vasopressin) was administered intranasally to 20 normal persons and blood was collected at various intervals up to 72 h. Plasma concentrations of VIII:C, VIIIR:Ag and fibrinolytic activity were increased by DDAVP with peak levels observed 1 h after the administration. Factors V, IX, XI, XII, XIII, P-APT time, platelets, platelet factor 3, P&P, AT III, alpha 2AP, fibrinogen and FDP were unchanged by the treatment. No side effects or changes in plasma Na or osmolality were observed. The present data demonstrate the selectivity of the effect of DDAVP on the coagulation system and points towards the possibility of using DDAVP in blood donors to increase plasma contents of factor VIII.

Factor VIII concentrate prepared from blood donors stimulated by intranasal administration of a vasopressin analogue.

With an interval of eight weeks between collections, blood was drawn twice from 120 blood donors. At one of the donations, 0.25 ml of synthetic vasopressin, (DDAVP, 1300 micrograms/ml), was administered intranasally 60 minutes prior to collection of the blood. No drug was given at the other donation. The yield of factor VIII clotting activity (VIII:C) and factor VIII antigen (VIIIR:Ag) was compared in blood drawn from the treated and untreated donors. To prevent degradation of VIII:C by fibrinolysis, tranexamic acid was added to the plasma from treated donors immediately after separation from the red blood cells. Plasma from treated donors and the derived Cohn fraction I-O contained approximately twice as much VIII:C as plasma and fraction I-O from untreated control donors. The concentration of VIIIR: Ag was also increased in fraction I-O made from plasma from treated donors, however to a lesser degree. The in vivo properties of factor VIII concentrates made from each group of donors were studied. Half-life in plasma and recovery of VIII:C were identical. Thus, intranasal synthetic vasopressin may be used to increase the yield of VIII:C in production of factor VIII concentrates.

Factor VIII concentrate prepared from DDAVP stimulated blood donor plasma.

Human fraction I-0 (AHF-Kabi) was prepared from plasma from blood donors who had received an i.v. injection of DDAVP (0.2 microgram per kg b.w.) and tranexamic acid (0.01 g per kg b.w.) 15 min before collection of the blood. The factor VIII preparation from such plasma contained twice as much VIII:C,VIIIR:Ag, and VIIIR:RFC as normal fraction I-0. Normal fraction I-0 and DDAVP fraction I-0 were given to 2 patients with severe haemophilia A. The in vivo response of the DDAVP fraction I-0 corresponded to the in vitro values. No differences in survival time were seen. Hence, it is possible to produce factor VIII concentrates with at least double the yield by increasing the factor VIII level in blood donors by i.v. injection of DDAVP.