Method of producing recombinant vitamin k dependent proteins

a technology of recombinant technology and dependent proteins, which is applied in the direction of instruments, peptide/protein ingredients, extracellular fluid disorders, etc., can solve the problems of limited -carboxylation system of cho cells, limited recombinant production of other vitamin k dependent proteins, and limited success in achieving high levels of functional vitamin k-dependent proteins

Inactive Publication Date: 2012-11-01
CNJ HLDG
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

Achieving high levels of functional vitamin K-dependent proteins by recombinant technology has been limited by the structural complexity of these proteins and the inability to create genetically engineered cell systems that overcome the inherent deficiencies in the enzymatic activities required for efficient and complete post-translational modification to occur.
Other vitamin K dependent proteins have been produced recombinantly with limited success.
They hypothesized that the γ-carboxylation system of CHO cells is limited and that only a certain level of protein can be efficiently processed.
Among the problems encountered in recombinant systems is that in order to produce biologically active Factor IX and Factor VII / VIIa, substantial gamma-carboxylation of glutamic acid residues in the amino terminal region of the protein referred to as the gla-domain, is needed.
The amount of vitamin K in the human diet is limited.
Furthermore, treatment may include acts that may worsen the patient's overall feeling of well-being or appearance.

Method used

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  • Method of producing recombinant vitamin k dependent proteins
  • Method of producing recombinant vitamin k dependent proteins

Examples

Experimental program
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Effect test

example 1

Determination of Factor IX Antigen Level

[0092]A gene for Factor IX was synthesized, operably linked to the CHEF-1 promoter, and transfected into CHO cells. The primary transfectants were grown up in a E-well microtiter plate and were assayed in triplicate to give an average level of antigen expression for the cells in the well. As presented in Table 1, the cells were designated primary transfectant line T-335. It should be kept in mind that this is an average production for the primary transfectants. Some of the cells produce no Factor IX, some produce a moderate level and some cells produce high levels of the protein: The primary transfectants were followed by measuring the amount of Factor IX antigen produced. Biological activity was not measured at this early stage of expression. As a control, a Factor IX gene construct in which the wild type Factor IX Propeptide sequence was replaced with the Propeptide sequence from another Vitamin K dependent protein, Protein C, was transfecte...

example 2

[0096]For comparison, the wild type Factor IX gene of Example 1 was cloned into a construct using the CMV promoter and transfected into HEK293 cells. As in Example 1 above, the primary transfectants were grown up in a 6-well microliter plate. The titer for these samples was less than 0.2 mg / L (data not shown). Further experiments were conducted using the CHEF-1 promoter.

example 3

[0097]In order to identify and characterize clones with high levels of Factor IX expression, primary transfectants were cloned by limit dilution into 96-well plates. As a consequence of the low surface area for cells to grow the level of expression was less that that reported in Example 1 where cells were grown on 6-well microliter plates. One hundred and fifty two single cell clones were identified from the T-335 primary transfectant experiment. The Factor IX antigen expression level for each clone was measured by ELISA analysis. The distribution of Factor IX expression levels from the clones are shown in FIG. 1.

[0098]The T-335 clones were separated into clones that expressed Factor IX at a level of greater than 0.4 mg / L and those that produced Factor IX at lower levels. Clones expressing Factor IX at levels higher than 0.4 mg / L were considered high expresser clones. Clones with expression levels lower than 0.4 mg / L were considered low expressers. The range of expression for Factor...

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Abstract

Methods for producing cell lines with high levels of biologically active recombinant vitamin K dependent proteins are described. The transfected cell lines do not include heterologous genes for processing enzymes and are not subject to selection pressure such as methotrexate resistance. Cell lines producing Factor VII / VIIa and Factor IX are described. These cell lines can be used for isolation of Factor VII / VIIa and / or Factor IX for treatment of Hemophilia.

Description

RELATED APPLICATIONS[0001]This application claims priority to provisional application No. 61 / 256,802, filed Oct. 30, 2009, which is incorporated herein by reference.BACKGROUND OF THE INVENTION[0002]1. Field of the Invention[0003]The invention related to recombinant vitamin K dependent proteins and methods of preparing the protein in a mammalian cell without the use of heterologous post-translational modification enzymes.[0004]2. Description of the Related Art[0005]Bleeding disorders can result from a deficiency in the functional levels of one or more of the blood proteins, collectively known as blood coagulation factors, that are required for normal hemostasis, i.e. blood coagulation. The severity of a given bleeding disorder is dependent on the blood level of functional coagulation factors. Mild bleeding disorders are generally observed when the functional level of a given coagulation factor reaches about 5% of normal, but if the functional level falls below 1%, severe bleeding is ...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N9/50A61P7/04C12P21/00C12N5/10G01N33/573A61K38/48C12N9/74
CPCA61K38/36C12N9/50C07K14/745A61P7/04C12N9/64C12P21/02
Inventor DROHAN, WILLIAM N.DROHAN, MARIAN J.GRIFFITH, MICHAEL J.
Owner CNJ HLDG
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