A galactopoiesis accordant yield of functional recombinant human factor IX from homozygous transgenic pigs requires a large amount of vitamin K supplementation

AbstractIntravenous administration of recombinant human factor IX (rhFIX) acutely corrects the coagulopathy in hemophilia B dogs. To date, 20 of 20 dogs developed inhibitory antibodies to the xenoprotein, making it impossible to determine if new human FIX products, formulations, or methods of chronic administration can reduce bleeding frequency. Our goal was to determine whether hemophilia B dogs rendered tolerant to rhFIX would have reduced bleeding episodes while on sustained prophylactic rhFIX administered subcutaneously. Reproducible methods were developed for inducing tolerance to rhFIX in this strain of hemophilia B dogs, resulting in a significant reduction in the development of inhibitors relative to historical controls (5 of 12 versus 20 or 20, P < .001). The 7 of 12 tolerized hemophilia B dogs exhibited shortened whole blood clotting times (WBCTs), sustained detectable FIX antigen, undetectable Bethesda inhibitors, transient or no detectable antihuman FIX antibody titers by enzyme-linked immunosorbent assay (ELISA), and normal clearance of infused rhFIX. Tolerized hemophilia B dogs had 69% reduction in bleeding frequency in year 1 compared with nontolerized hemophilia B dogs (P = .0007). If proven safe in human clinical trials, subcutaneous rhFIX may provide an alternate approach to prophylactic therapy in selected patients with hemophilia B. (Blood. 2003;102:4393-4398)

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Investigations into, and development of, a lyophilized and formulated recombinant human factor IX produced from CHO cells

Biotechnology Letters ◽

10.1007/s10529-017-2353-y ◽

2017 ◽

Vol 39 (8) ◽

pp. 1109-1120 ◽

Cited By ~ 1

Author(s):

Aline G. Almeida ◽

Rodrigo C. V. Pinto ◽

C. Mark Smales ◽

Leda R. Castilho

Keyword(s):

Cho Cells ◽

Human Factor ◽

Factor Ix ◽

Human Factor Ix ◽

Recombinant Human Factor Ix

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RECOGNITION SITE DIRECTING GAMMA-CARBOXYLATION RESIDES ON THE PROPEPTIDES OF FACTOR IX AND PROTRROMBIN

10.1055/s-0038-1643992 ◽

1987 ◽

Author(s):

B C Furis ◽

M J Jorgensem ◽

M J Rabiet ◽

A B Contor ◽

C L Brown ◽

...

Keyword(s):

Vitamin K ◽

Human Factor ◽

Chinese Hamster ◽

Factor Ix ◽

Absolute Amount ◽

Native Structure ◽

Mutant Proteins ◽

Specific Antibodies ◽

Human Factor Ix ◽

Carboxyglutamic Acid

Factor IX and prothrombin vitamin K-dependent proteins that participate in blood coagulation undergo post-translationalmodification in which glutamic acid residues in the amino terminus of the protein are converted to gamma-carboxyglutamic acid residues. This modification confers divalent metal ion binding ability upon the proteins.As a consequence of binding divalent metal ions these proteins undergoconformational changes necessary for biological function.The vitamin K-dependent proteins are synthesized with an NH2-terminal extension. The region distal to the NH2-terminus of the mature protein is a prototypic signal sequence while the proximal region is a propeptide with homology among the vitamin K-dependent proteins. The boundary between the pre and pro sequences has been established for factor IX by analysis of three naturally occurring factor IX mutants factor IX Cambridge factor IX Oxford-3 and factor IX San Dimas, in which processing is incomplete.For human factor IX the propeptide extends from residue -18 to -1. The homology among the propeptides of vitamin K-dependent proteins suggests that the propeptide may designate adjacent gamma-carboxyglutamic acids for carboxylation. To test this hypothesis alterations in sequence were introduced into the propeptide region of human factor IX cDNA by oligonucleotide directed site specific mutagenesis.Mutated genes were expressed in Chinese hamster ovary cells. Rapid and efficient isolationof the mutant proteins by immunoaffinity chromatography permitted detailed analysis of the mutants on quantities of protein easily obtainable at low expression levels. The extent of gamma-carboxylation was assessed by the ability of the mutant proteins to interact with conformation specific antibodies directed against the gamma-carboxyglutamic acid-dependent metal stabilized native structure of factor IX as well as by direct amino acid analysis. Unmodified recombinant factor IX contained, on average, 9 gamma-carboxyglutamic acid residues, as compared to 12 for plasma factor IX. About 70% of the recombinant wild type factor IX bound to the conformation specific antibodies. Deletion of the propiece or point mutations at residues -10 or -16 led to secretion of uncarboxylated factor IX unreaotive with antibodies specific for the native structure but with the NH2-terminus of mature factor IX. In order to assess the universality of these observations we have recently cloned human prothrombin cDNA and expressed the gene in the same Chinese hamster ovary cell system used for factor IX. In contrast to factor IX, at low levels ofexpressionof the prothrombin gene, the prothrombin is fully carboxylated relative to a plasma prothrombin standard.The recombinant prothrombin exhibits the same specific clotting activity as plasma derivedprothrombin and is fully native as evaluated by conformation specific antibodies. At high levels of expression the capacityof the cells to carboxylate prothrombin can be exceeded leading to secretion of under carboxylated prothrombin. However, the absolute amount of fully carboxylated prothrombin that can be produced in this system appears to be a least fivefold greater that the absolute amount of highly carboxylated factor IX that can be synthesized.The elimination of carboxylation observed upon mutation of the propiece of factor IX suggest that the propiece contains a recognition element required for carboxylation of the protein. Assignment of a functional role to the propiece of factor IX represents the first determination of function for any pro sequence. It is anticipated that extension of these studies to prothrombin will demonstrate that this recognition signal is used by all the members of this class of proteins. In order to determine if the propiece is sufficient to designate a protein for gamma-carboxylation we are currently constructing chimeric proteins incorporating the propieceof prothrombin into the cDNA of normally uncarboxylated proteins.

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Characterization of recombinant human factor IX expressed in transgenic mice and in derived trans-immortalized hepatic cell lines.

The EMBO Journal ◽

10.1002/j.1460-2075.1990.tb07529.x ◽

1990 ◽

Vol 9 (10) ◽

pp. 3295-3301 ◽

Cited By ~ 41

Author(s):

S. Jallat ◽

F. Perraud ◽

W. Dalemans ◽

A. Balland ◽

A. Dieterle ◽

...

Keyword(s):

Transgenic Mice ◽

Cell Lines ◽

Human Factor ◽

Hepatic Cell ◽

Factor Ix ◽

Human Factor Ix ◽

Recombinant Human Factor Ix ◽

Hepatic Cell Lines

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Recombinant Human Factor IX Produced from Transgenic Porcine Milk

BioMed Research International ◽

10.1155/2014/315375 ◽

2014 ◽

Vol 2014 ◽

pp. 1-8 ◽

Cited By ~ 4

Author(s):

Meng-Hwan Lee ◽

Yin-Shen Lin ◽

Ching-Fu Tu ◽

Chon-Ho Yen

Keyword(s):

Human Factor ◽

Specific Activity ◽

Active Form ◽

High Specific Activity ◽

Factor Ix ◽

Scale Production ◽

Sialic Acid Content ◽

Human Factor Ix ◽

Recombinant Human Factor Ix ◽

Porcine Milk

Production of biopharmaceuticals from transgenic animal milk is a cost-effective method for highly complex proteins that cannot be efficiently produced using conventional systems such as microorganisms or animal cells. Yields of recombinant human factor IX (rhFIX) produced from transgenic porcine milk under the control of the bovineα-lactalbumin promoter reached 0.25 mg/mL. The rhFIX protein was purified from transgenic porcine milk using a three-column purification scheme after a precipitation step to remove casein. The purified protein had high specific activity and a low ratio of the active form (FIXa). The purified rhFIX had 11.9γ-carboxyglutamic acid (Gla) residues/mol protein, which approached full occupancy of the 12 potential sites in the Gla domain. The rhFIX was shown to have a higher isoelectric point and lower sialic acid content than plasma-derived FIX (pdFIX). The rhFIX had the sameN-glycosylation sites and phosphorylation sites as pdFIX, but had a higher specific activity. These results suggest that rhFIX produced from porcine milk is physiologically active and they support the use of transgenic animals as bioreactors for industrial scale production in milk.

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Production of recombinant human factor IX by propeptide modification in Drosophila S2 cell line

Biotechnology Letters ◽

10.1007/s10529-019-02643-z ◽

2019 ◽

Vol 41 (3) ◽

pp. 347-355

Author(s):

Samira Bahrami ◽

Mahmoud Ghaffari ◽

Alireza Zomorodipour

Keyword(s):

Cell Line ◽

Human Factor ◽

Factor Ix ◽

Human Factor Ix ◽

Recombinant Human Factor Ix

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