DETERMINATION OF THE DOMAINS OF FACTOR VIII ESSENTIAL FOR PROCOAGULANT ACTIVITY

1987 ◽  
Author(s):  
M Ph Verbeet ◽  
R F Evers ◽  
A Leyte ◽  
H L Lamain ◽  
A J J Van Ooyen ◽  
...  
Keyword(s):  
Factor Viii ◽  
Recombinant Proteins ◽  
Cleavage Site ◽  
Specific Activity ◽  
Expression System ◽  
Full Length ◽  
Procoagulant Activity ◽  
Expression Vectors ◽  
Recombinant Fviii

Factor VIII (FVIII) consists of an obvious domain structure that can be represented as A1-A2-B-A3-C1-C2 (Vehar et al., 1984, Nature 312, 337). In order to determine the domains involved in the procoagulant activity of FVIII, we constructed mutant FVIII cDNAs containing deletions in the coding sequence of the full-length molecule. In one of the mutants a large part of the B domain is deleted. In another one we made a deletion in the B domain that extends beyond the thrombine cleavage site. We used pSV-2 derived expression vectors and COS-1 cells in a transient expression system for the full-length and mutant recombinant proteins. Conditioned media (CM) were harvested.In accordance with the described mutants of recombinant FVIII (Toole et al., 1986, PNAS 83, 5939), we demonstrated an increase in activity in the CM for these mutants as compared to the full-length activity. We also found that the specific activity of the mutants is similar to that of plasma FVIII. So, shorter chains lead to an increased amount of procoagulant protein.

SUSTAINED EXPRESSION OF FULL LENGTH AND VARIANT RECOMBINANT FACTOR VIII IN GENETICALLY ENGINEERED CELLS

1987 ◽  
Author(s):  
Nava Sarver ◽  
George A Ricca
Keyword(s):  
Factor Viii ◽  
Recombinant Dna ◽  
Expression System ◽  
Genetically Engineered ◽  
Full Length ◽  
Cdna Insert ◽  
Mammalian Expression ◽  
Coagulant Activity ◽  
Recombinant Fviii

A major effort is presently underway to provide factor VIII (FVIII) in a form free of viral pathogens via a recombinant DNA approach. We have constructed two chimeric FVIII cDNA vectors based on the bovine papillomavirus mammalian expression system. The first vector (FVIII) contained a full length FVIII cDNA; the second vector (AFVIII) contained a cDNA insert with an extensive deletion, corresponding to amino acid residues 747 to 1560 in the region encoding the "B" domain. This internal region is removed during activation of the parental FVIII molecule and is believed not to be required for coagulant activity. We have found that recombinant FVIII produced by stable cell lines harboring either the full length or the variant FVIII was capable of restoring coagulant activity to FVIII deficient plasma in. vitro. This expressed activity was neutralized by anti-FVIII antibodies. Similar to observations with FVIII derived from human plasma, the two recombinant FVIII forms were (i) inactivated by the chelating agent EDTA, (ii) demonstrated a biphasic response of an initial activation followed by a decay in activity when treated with thrombin, and (iii) presented the expected peptide banding pattern by western blot analyses. A higher percentage of ΔFVIII transformants were isolated expressing coagulant activity compared to transformants harboring the complete FVIII cDNA. Among the positive transformants isolated, those harboring ΔFVIII produced higher levels of coagulant activity than their full length counterparts. Comparable steady state levels of FVIII specific transcripts were detected in FVIII and ΔFVIII transformants indicating that the difference in expression levels is due to a post transcriptional event(s). Our study demonstrates the efficacy of a full length and an abridged recombinant FVIII produced by stably transformed cells in correcting coagulation deficiency in. vitro. It further suggests the potential usefulness of other molecular variants for efficient expression in genetically engineered cells.

Development of a Novel Recombinant Factor VIII Product through Transgene Engineering and Lentiviral-Driven Expression

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3072-3072
Author(s):  
Keith W Kerstann ◽  
Pete Lollar ◽  
H. Trent Spencer ◽  
Gabriela Denning ◽  
Lajos Baranyi ◽  
...  
Keyword(s):  
Factor Viii ◽  
Hemophilia A ◽  
Specific Activity ◽  
Phase 1 ◽  
Expression System ◽  
Scale Up ◽  
High Expression ◽  
Enzyme Linked Immunosorbent Assay ◽  
Slow Decay ◽  
Recombinant Fviii

Abstract The increased cost of goods associated with low-level expression of factor VIII (fVIII) is considered a significant factor in the pricing of recombinant fVIII products. Worldwide treatment of hemophilia A is limited due to the cost of fVIII products, which easily can exceed $100,000 per year. Using state-of-the-art commercial production facilities and technology, recombinant human (h)-fVIII expresses at 100 – 1000-fold lower levels than other plasma proteins. We have developed a novel recombinant fVIII product that overcomes the primary limitation to the manufacture of currently available hemophilia A pharmaceuticals. There are two components to this product: a humanized highexpression hybrid-human/porcine (HP) fVIII transgene, designated ET-801i, and the LENTIMAX™ lentiviral production system. The ET-801i transgene contains elements of porcine fVIII (p-fVIII) sequence in the A1 and A3 domains that facilitate more efficient secretion from production cell lines and is 90% identical in amino acid sequence to h-fVIII. Additionally, lentiviral introduction of the transgene allows achievement of an optimal number of transgene copies and the lentiviral vectors typically integrate into sites of active chromatin, thus facilitating high-levels of transgene transcription. In the current study, we demonstrated that high-expression porcine sequence elements enable 20 – 100- fold superior expression over any previously described fVIII transgene variant. Using a stable baby hamster kidney cell-derived expression system, ET-801i was expressed at a level indistinguishable from p-fVIII, which both were significantly greater than h-fVIII (<0.001, Mann-Whitney U test). After scale up, ET-801i was purified using the same two-step ion exchange procedure that has been used previously to purify p-fVIII and other high-expression HP-fVIII constructs. This purification scheme does not involve an immunoaffinity step, which greatly simplifies the process and downstream quality control. The purity of ET-801i was assessed to be greater than 95% by SDS-PAGE and ET-801i displayed a specific activity of 3,400 units/nmol. Treatment of ET-801i with thrombin and endoglycosidase PNGase F resulted in a decrease in Mr for the A1 and A3-C1-C2 (light chain) domain fragments. No change in the Mr of the A2 domain was observed. These data suggest correct glycosylation of ET-801i. Using an enzyme-linked immunosorbent assay, ET-801i was demonstrated to bind von Willebrand factor indistinguishably from p-fVIII. Therefore, ET-801i should display similar pharmacokinetics to recombinant p-fVIII, which has been studied in phase 1 and 2 clinical trials. Furthermore, thrombinactivated ET-801i displays the slow decay property of activated p-fVIII (T1/2 = 6 min), which is approximately 3-fold slower than h-fVIII. It is likely that slow decay increases the in vivo functionality of activated fVIII and may allow lower dosing in hemophilia A patients leading to further cost reduction and an improved safety profile by reducing immunogenicity. These results indicate that the combined technological advancements of high-expression fVIII elements and lentiviral-driven gene transfer and expression can be utilized to provide a potentially safer recombinant fVIII at a lower cost than current h-fVIII products and thus better support patients with hemophilia A.

scholarly journals A human expression system based on HEK293 for the stable production of recombinant erythropoietin

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Christine Lin Chin ◽  
Justin Bryan Goh ◽  
Harini Srinivasan ◽  
Kaiwen Ivy Liu ◽  
Ali Gowher ◽  
...  
Keyword(s):  
Recombinant Proteins ◽  
High Titer ◽  
Expression System ◽  
Mass Spectrometry Analysis ◽  
Hek293 Cells ◽  
Expression Vectors ◽  
Methionine Sulfoximine ◽  
Mammalian Host ◽  
Recombinant Erythropoietin ◽  
Cellular Expression

AbstractMammalian host cell lines are the preferred expression systems for the manufacture of complex therapeutics and recombinant proteins. However, the most utilized mammalian host systems, namely Chinese hamster ovary (CHO), Sp2/0 and NS0 mouse myeloma cells, can produce glycoproteins with non-human glycans that may potentially illicit immunogenic responses. Hence, we developed a fully human expression system based on HEK293 cells for the stable and high titer production of recombinant proteins by first knocking out GLUL (encoding glutamine synthetase) using CRISPR-Cas9 system. Expression vectors using human GLUL as selection marker were then generated, with recombinant human erythropoietin (EPO) as our model protein. Selection was performed using methionine sulfoximine (MSX) to select for high EPO expression cells. EPO production of up to 92700 U/mL of EPO as analyzed by ELISA or 696 mg/L by densitometry was demonstrated in a 2 L stirred-tank fed batch bioreactor. Mass spectrometry analysis revealed that N-glycosylation of the produced EPO was similar to endogenous human proteins and non-human glycan epitopes were not detected. Collectively, our results highlight the use of a human cellular expression system for the high titer and xenogeneic-free production of EPO and possibly other complex recombinant proteins.

Quantitative Evaluation of Factor VIII in Factor VIII Products.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4012-4012
Author(s):  
Saulius Butenas ◽  
Behnaz Parhami-Seren ◽  
Matthew T. Gissel ◽  
Edward D. Gomperts ◽  
Kenneth G. Mann
Keyword(s):  
Factor Viii ◽  
Protein Content ◽  
Quantitative Evaluation ◽  
Hemophilia A ◽  
Specific Activity ◽  
Full Length ◽  
Recombinant Factor Viii ◽  
Factor Viii Activity

Abstract Several factor VIII products, recombinant and natural, have been used for hemophilia A treatment worldwide. Typically, two activity-based assays (factor Xase and aPTT) are used for the assessment of factor VIII concentration in these products. Frequently, the results are dependent upon the assay and its modifications in different laboratories. In this study, we evaluated five pharmacologic factor VIII products (three lots of each) in three activity-based assays and in two immunoassays for the concentration and activity of factor VIII protein. Two factor VIII products were plasma-derived (Immunate and Hemofil M) and three were recombinant; two of these contained full-length factor VIII (Recombinate and Kogenate) and one was B-domainless (ReFacto). Albumin-free full-length recombinant factor VIII was used as a standard in all assays. In the factor Xase assay, all recombinant factor VIII products and Immunate at 1U/ml (indicated by manufacturer) showed activity similar to that of 0.7nM (1U/ml) standard, whereas activity of Hemofil M was 64–68% of the standard. In the aPTT assay both full-length recombinant products and Hemofil M displayed activity similar to the standard, whereas Immunate had increased (142% of standard) and ReFacto decreased (83% of standard) activity. In synthetic plasma, all three recombinant products had standard-like activity, whereas Hemofil M and Immunate were slightly more active than standard. The ELISA immunoassay revealed that the factor VIII protein content in Recombinate, Kogenate and Hemofil M corresponded to the units assigned by manufacturers (1.4–1.6x1012U/mol vs1.4x1012U/mol calculated for standard), whereas the specific activity of Immunate was 50% of that expected (0.7x1012U/mol). In contrast, the specific activity of ReFacto was almost 3-fold that of full-length factor VIII (4.0x1012U/mol). The data of this study indicate that: 1) factor VIII activity estimated in different assays gives dissimilar results; 2) the specific activity of factor VIII in various factor VIII products is different and, as a consequence, administration of an equal factor VIII activity in U/ml means the administration of different amounts of factor VIII protein.

Epitope Mapping of a Monoclonal Antibody against VWF Which Diminishes the Proteolytic Cleavage of VWF by ADAMTS13 Under Flow.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2133-2133
Author(s):  
Jingyu Zhang ◽  
Zhenni Ma ◽  
Ningzheng Dong ◽  
Jian Su ◽  
Anyou Wang ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Proteolytic Activity ◽  
Recombinant Proteins ◽  
Expression System ◽  
Guanidine Hydrochloride ◽  
Proteolytic Cleavage ◽  
Terminal Region ◽  
Full Length ◽  
Western Blots ◽  
A2 Domain

Abstract Abstract 2133 Poster Board II-110 Introduction: In our former study, we have found that SZ-34, a monoclonal antibody to Von Willebrand factor (VWF), can inhibit the proteolysis of VWF by ADAMTS13 under shear stress. But the precise epitope of this antibody (SZ-34) on VWF is not clear for it is generated by immunizing mouse with native full-length VWF purified from pooled human normal plasmas. Thus, the objective of this study is to map the epitope of SZ-34 and to explore the effect of VWF structrue on the proteolytic activity by ADAMTS13. Materials and Methods: Firstly we constructed and expressed a series of recombinant proteins of different domains or polypeptide fragments of human VWF in prokaryotic cell expression system, including A1A2A3, D′D3, A1, A2, A3, A1A2, A2A3 and five sub-fragments of A2 domain. Then native VWF and these recombinant proteins or polypeptide fragments were subjected to polyacrylamide gel electrophoresis (PAGE) and analyzed by Western blots with SZ-34. Results: Different recombinant proteins of VWF were successfully expressed and purified. Results of Western blot showed that SZ-34 could bind specifically some recombinant proteins, such as full-length VWF, A1A2A3, A2 and GST-D1459D1596 in which the last was a fusion protein of a sub-fragment of A2 domain with GST. But SZ-34 couldn't bind to others, including A1, A3, D′D3, GST-D1459E1554, GST-E1554D1596, GST-D1596R1668 (VWF73) and GST- E1554R1668. In addition, the reacting activity of SZ-34 with native VWF was significantly stronger than with unfolded VWF, such as heat-treated or 1.5M guanidine hydrochloride-treated VWF. Conclusions: The epitope of SZ-34 is located within N-terminal region fore-VWF73 inside VWF-A2 domain. Besides, SZ-34 maybe is a conformation-specific monoclonal antibody. Combining with our former findings that SZ-34 inhibits the proteolytic cleavage of VWF by ADAMTS13, we can conclude that N-terminal region fore-VWF73 inside VWF-A2 domain also regulates the proteolytic activity of VWF by ADAMTS13, although VWF73 is considered as the minimal substrate for ADAMTS13. Disclosures: No relevant conflicts of interest to declare.

Confocal microscopy analysis of native, full length and B-domain deleted coagulation factor VIII trafficking in mammalian cells

2004 ◽  
Vol 92 (07) ◽  
pp. 23-35 ◽  
Author(s):  
Sven Becker ◽  
Jeremy Simpson ◽  
Rainer Pepperkok ◽  
Stefan Heinz ◽  
Christian Herder ◽  
...  
Keyword(s):  
Factor Viii ◽  
Golgi Complex ◽  
Mammalian Cells ◽  
Laser Scanning ◽  
Coagulation Factor ◽  
Full Length ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Microscopy Analysis ◽  
Recombinant Fviii

SummaryIn mammalian cells, factor VIII (FVIII) secretion depends upon its interaction with chaperones of the endoplasmic reticulum (ER) and requires a unique ATP-dependent step to dissociate aggregates formed within the ER. To further elucidate mechanisms which might account for the inefficient secretion of recombinant FVIII (rFVIII), we have analyzed the pathways of recombinant full length (rFVIII-FL) and B-domain deleted (rFVIIIΔB) FVIII and compared these to the secretion route of native FVIII in primary hepatocytes. Using confocal laser scanning microscopy in combination with a pulse chase of a known secretion marker, we describe the trafficking route of FVIII, which upon release from the ER – where it colocalizes with calnexin – is transported to the Golgi complex in vesiculartubular transport complexes (VTCs) which could be further identified as being COP I coated. However, a large portion of rFVIII is retained in the ER and additionally in structures which could not be assigned to the ER, Golgi complex or intermediate compartment. Moderate BiP transcription levels indicate that this observed retention of FVIII does not reflect cellular stress due to an overexpression of FVIII-protein in transduced cells. Moreover, a pulse of newly synthesized rFVIII protein is released within 4 hrs, indicating that once rFVIII is released from the ER there is no further limitation to its secretion. Our data provide new details about the secretory route of FVIII, which may ultimately help to identify factors currently limiting the efficient and physiological expression of FVIII in gene therapy and manufacture.

scholarly journals Towards Designer Cellulosomes in Clostridia: Mannanase Enrichment of the Cellulosomes Produced by Clostridium cellulolyticum

2004 ◽  
Vol 186 (19) ◽  
pp. 6544-6552 ◽  
Author(s):  
Stéphanie Perret ◽  
Anne Bélaich ◽  
Henri-Pierre Fierobe ◽  
Jean-Pierre Bélaich ◽  
Chantal Tardif
Keyword(s):  
Guar Gum ◽  
Specific Activity ◽  
Full Length ◽  
Expression Vectors ◽  
Leader Peptide ◽  
Crystalline Cellulose ◽  
Glycosyl Hydrolases ◽  
Truncated Form ◽  
E Coli ◽  
Clostridium Cellulolyticum

ABSTRACT The man5K gene of Clostridium cellulolyticum was cloned and overexpressed in Escherichia coli. This gene encodes a 424-amino-acid preprotein composed of an N-terminal leader peptide, followed by a dockerin module and a C-terminal catalytic module belonging to family 5 of the glycosyl hydrolases. Mature Man5K displays 62% identity with ManA from Clostridium cellulovorans. Two forms of the protein were purified from E. coli; one form corresponds to the full-length enzyme (45 kDa), and a truncated form (39 kDa) lacks the N-terminal dockerin module. Both forms exhibit the same typical family 5 mannanase substrate preference; they are very active with the galactomannan locust bean gum, and the more galacto-substituted guar gum molecules are degraded less. The truncated form, however, displays fourfold-higher activity with galactomannans than the full-length enzyme. Man5K was successfully overproduced in C. cellulolyticum by using expression vectors. The trans-produced protein was found to be incorporated into the cellulosomes and became one of the major enzymatic components. Modified cellulosomes displayed 20-fold-higher specific activities than control fractions on galactomannan substrates, whereas the specific activity on crystalline cellulose was reduced by 20%. This work clearly showed that the composition of the cellulosomes is obviously regulated by the relative amounts of the enzymes produced and that this composition can be engineered in clostridia by structural gene cloning.

scholarly journals An Alkane-Responsive Expression System for the Production of Fine Chemicals

1999 ◽  
Vol 65 (6) ◽  
pp. 2324-2332 ◽  
Author(s):  
Sven Panke ◽  
Andreas Meyer ◽  
Caroline M. Huber ◽  
Bernard Witholt ◽  
Marcel G. Wubbolts
Keyword(s):  
Specific Activity ◽  
Styrene Oxide ◽  
Expression System ◽  
Regulatory System ◽  
Dry Weight ◽  
Pseudomonas Oleovorans ◽  
Structural Genes ◽  
Biotechnological Processes ◽  
Terminal Amino

ABSTRACT Membrane-located monooxygenase systems, such as thePseudomonas putida mt-2-derived xylene oxygenase, are attractive for challenging transformations of apolar compounds, including enantiospecific epoxidations, but are difficult to synthesize at levels that are useful for application to biotechnological processes. In order to construct efficient biocatalysis strains, we utilized the alkane-responsive regulatory system of the OCT plasmid-located alk genes of Pseudomonas oleovorans GPo1, a very attractive system for recombinant biotransformation processes. Determination of the nucleotide sequence of alkS, whose activated gene product positively regulates the transcription of the structural genes alkBFGHJKL, on a 3.7-kb SalI-HpaI OCT plasmid fragment was completed, and the N-terminal amino acid sequence of an AlkS-LacZ fusion protein was found to be consistent with the predicted DNA sequence. The alkS gene and the alkBp promoter were assembled into a convenient alkane-responsive genetic expression cassette which allowed expression of the xylene oxygenase genes in a recombinant Escherichia coli strain at a specific activity of 91 U per g (dry weight) of cells when styrene was the substrate. This biocatalyst was used to produce (S)-styrene oxide in two-liquid-phase cultures. Volumetric productivities of more than 2 g of styrene oxide per h per liter of aqueous phase were obtained; these values represented a fivefold improvement compared with previous results.

Absence of Inhibitors in Previously Untreated Patients with Severe Haemophilia A after Exposure to a Single Intermediate Purity Factor VIII Product

1997 ◽  
Vol 78 (03) ◽  
pp. 1027-1029 ◽  
Author(s):  
T T Yee ◽  
M D Williams ◽  
F G H Hill ◽  
C A Lee ◽  
K J Pasi
Keyword(s):  
Factor Viii ◽  
High Purity ◽  
Specific Activity ◽  
Haemophilia A ◽  
Severe Haemophilia ◽  
True Incidence ◽  
Conventional Fractionation ◽  
Heat Treated ◽  
Recombinant Fviii ◽  
Von Willebrand

SummaryUse of high purity and recombinant factor VIII (FVIII) concentrates has been thought to be associated with an increased incidence of FVIII inhibitors in patients with severe haemophilia A. Comparison with comparable historical control groups has suggested that the true incidence of inhibitors in patients with severe haemophilia A was ~20-25%, similar to the incidence seen with new high purity and recombinant FVIII products.We have conducted a study of inhibitor development in a cohort of 37 boys with severe haemophilia A (VIII: C <2 u/dl) exposed only to a single FVIII concentrate (BPL 8Y) with no previous blood or blood product exposure. This factor VIII concentrate is an intermediate purity product with a specific activity of ~2 IU/mg protein and contains well preserved von Willebrand factor multimers. It is manufactured by conventional fractionation technologies and terminally dry heat treated at 80° C for 72 h.

THE EFFECT OF CALCIUM ON THE STABILITY OF PURIFIED FACTOR VIII

1987 ◽  
Author(s):  
P R Qanz ◽  
E S Tackaberry ◽  
D S Palmer ◽  
B Malchy ◽  
G Rock
Keyword(s):  
Factor Viii ◽  
Specific Activity ◽  
Procoagulant Activity ◽  
Factor X ◽  
Single Chain ◽  
Rate Of Decay ◽  
The Stability ◽  
The One ◽  
Polyethylene Glycol Precipitation ◽  
And Function

The involvement of calcium and phospholipid in the activation of Factor X to Xa by Factor IXa and Factor VIII has been well documented. Although we and others have shown that maintenance of physiological concentrations of calcium has a positive effect on the stability of Factor VIII in plasma, calcium’s role in the structure and function of Factor VIII remains to be fully elucidated. To this end, we examined the effect of calcium on the stability of highly purified Factor VIII. Homogeneous Factor VIII (specific activity approximately 5,200 U/mg) was prepared from heparinized blood using a six-step purification procedure including cryoprecipitation, polyethylene glycol precipitation, Affi-Gel Blue, Aminohexyl, polyelectrolyte E5 and immunoaffinity chromatography. This yielded a single chain high molecular weight species of approximately 260,000. The protein was tested for stability using the one stage assay over 6h of incubation at 4°C in buffers containing 0 mM, 5 mM, and 10 mM CaCl2. Addition of 5 mM and 10 mM CaCl2 to desalted, purified Factor VIII resulted in an immediate 12% (for 5 mM CaCl2) and 23% (for 10 mM CaCl2), enhancement of procoagulant activity compared to samples containing no added calcium. The calculated half-life (T1/2) of activity of Factor VIII in buffers containing no added calcium was 3.8h, whereas the Tl/2 for preparations incubated in the presence of 5 mM and 10 mM CaCl2 were increased to 5h and 5.5h respectively. Although the addition of calcium improved the recovery of activity over the first 0.5h of incubation, at later times the rate of decay in the calcium containing preparations was similar to Factor VIII preparations without added calcium. Our results suggest that removal of calcium from the microenvironment of purified Factor VIII by desalting, results in an immediate loss of procoagulant activity, which can be partially restored within the first 0.5h following readdition of calcium. The decay in Factor VIII activity observed at later times in the 0 mM, 5 mM and 10 mM CaCl2 containing buffers likely reflects calcium-independent denaturation of the protein.

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