Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Blood proteins or globulins – e.g. – proteoglycans – platelet...
Reexamination Certificate
2000-12-29
2004-01-27
Low, Christopher S. F. (Department: 1653)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Blood proteins or globulins, e.g., proteoglycans, platelet...
C530S380000, C530S413000, C530S414000, C530S415000, C514S002600, C514S012200
Reexamination Certificate
active
06683159
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to improved methods for the purification of procoagulant proteins, particularly recombinant production of Factor VIII and related proteins.
BACKGROUND OF THE INVENTION
Hemophilia is an inherited disease which has been known for centuries, but it is only within the last few decades that it has been possible to differentiate among the various forms; hemophilia A and hemophilia B. Hemophilia A is caused by strongly decreased level or absence of biologically active coagulation factor VIII, which is a protein normally present in plasma.
Until recently, therapeutic factor VIII concentrates have been prepared by fractionation of plasma. However, in recent years, DNA sequences encoding the human coagulation cofactor, Factor VIII:C (FVIII), became known in the art [see e.g., Toole et al, 1984, Nature 312:312-317; Wood et al, 1984,Nature 312:330-337; Vehar et al, 1984, Nature 312:337-342], as well as methods for expressing them to produce recombinant FVIII [see e.g. Toole, U.S. Pat. No. 4,757,006; WO 87/04187, WO 88/08035 and WO 88/03558]. Active variants and analogs of FVIII protein, and DNA sequences encoding them, have also been reported [see e.g. Toole, U.S. Pat. No. 4,868,112; EP 0 786 474; WO 86/06101 and WO 87/07144]. Generally, such variants and analogs are modified such that part or all of the B domain are missing and/or specific amino acid positions are modified, for example, such that normally protease-labile sites are resistant to proteolysis, e.g. by thrombin or activated Protein C. Other analogs include modification at one or more lysine and/or tyrosine residues.
It has been previously surprisingly found that the B domain is dispensable for the procoagulant activity of FVIII, and that active procoagulant protein can be expressed and secreted by expression of a FVIII-encoding DNA in which the nucleotide region encoding part or all of the B domain is lacking. Not only is active protein of these variants produced and secreted, it accumulates in the media at higher levels than when expressed by the full-length DNA. The reduced level of active procoagulant FVIII protein in the media has been attributed, at least in part to several factors [see e.g. WO 87/04187, WO 88/08035 and WO 88/03558]. In Toole et al.,
Exploration of Structure-Function Relationships in Human Factor VIII by Site-directed Mutagenesis
, Cold Spring Harbor Symposium on Quantitative Biology, 51:543 (1986), it was reported that recombinant FVIII could be purified by a combination of monoclonal antibody affinity chromatography and ion-exchange chromatography. U.S. Pat. No. 5,470,954 describes a similar process in which FVIII is passed directly from immunoaffinity purification to the ion exchange column. In that document it is stated that changing the ionic strength of the eluted polypeptide solution increases the chance that monoclonal antibodies will remain bound to the FVIII polypeptide and co-purify.
SUMMARY OF THE INVENTION
In the present invention, it has been found that diluting the eluate from the monoclonal antibody column provides certain advantages in yield and/or reduced monoclonal antibody contamination of the FVIII protein being purified therefrom. Accordingly, the present invention provides improved methods for the purification of procoagulant proteins, including both FVIII and variants thereof, which may be produced by recombinant techniques in higher yield and/or in more homogeneous form.
The present invention provides improved methods of purification of FVIII protein from cell cultures, preferably from recombinant cell cultures. The methods provide for obtaining FVIII protein of a higher purity than methods currently in use. In one embodiment, the methods of the present invention comprise diluting the eluate from the immunoaffinity column with a solution of higher ionic strength than the eluate solution. In another embodiment, the methods of the present invention comprise diluting the eluate from an immunoaffinity column with a solution containing lower amounts of ethylene glycol than contained in the eluate solution.
DETAILED DESCRIPTION OF THE INVENTION
Accordingly, the present invention provides improved methods for purification of a Factor VIII polypeptide comprising:
a) adding a mixture containing Factor VIII polypeptide to be purified to an immunoaffinity matrix which binds by hydrophobic attraction to the FVIII polypeptide;
b) eluting the Factor VIII polypeptide from the immunoaffinity matrix with a desorbing solution which causes desorption of the Factor VIII polypeptide, which is released in an elution solution;
c) diluting the elution solution with a solution comprising higher ionic strength than that of the elution solution, resulting in a diluted Factor VIII solution;
d) passing the diluted Factor VIII solution through an ion exchange column capable of binding to the Factor VIII polypeptide, thereby binding the Factor VIII polypeptide while allowing contaminants to pass through the ion exchange column; and
e) eluting the purified Factor VIII polypeptide from the ion exchange column.
The desorbing solution of step (b) may contain no salt, or very low salt. The dilution of step (c) is preferably performed using a solution comprising from about 5 to about 20 mM NaCl, most preferably about 5 to about 15 mM NaCl. The eluting solution is preferably diluted with salt-containing solution from about 3-fold to about 5-fold, most preferably about 3-fold.
In another embodiment, the present invention comprises improved methods for purification of a Factor VIII polypeptide comprising:
a) adding a mixture containing Factor VIII polypeptide to be purified to an immunoaffinity matrix which binds by hydrophobic attraction to the FVIII polypeptide;
b) eluting the Factor VIII polypeptide from the immunoaffinity matrix with a desorbing solution which causes desorption of the Factor VIII polypeptide, which is released in an elution solution, wherein the desorbing solution comprises a non-polar agent;
c) diluting the elution solution with a solution comprising lower concentration of the non-polar agent than that of the desorbing solution, resulting in a diluted Factor VIII solution;
d) passing the diluted Factor VIII solution through an ion exchange column capable of binding to the Factor VIII polypeptide, thereby binding the Factor VIII polypeptide while allowing contaminants to pass through the ion exchange column; and
e) eluting the purified Factor VIII polypeptide from the ion exchange column.
Preferably, the desorbing solution of step (b) contains ethylene glycol, more preferably about 50% (v/v) ethylene glycol, and the dilution of step (c) is performed using a solution comprising less than about 50% (v/v) ethylene glycol, such that the final concentration of ethylene glycol is from about 17% to about 33% (v/v). In a preferred embodiment, the desorbing solution of step (b) contains 50% (v/v) ethylene glycol, and the dilution of step (c) is performed using a solution comprising no ethylene glycol, such that the final concentration of ethylene glycol is from about 17 to about 33% (v/v), most preferably about 33% (v/v) ethylene glycol. Preferably, the elution solution is diluted from about 1.5-fold to about 3-fold, most preferably about 1.5 fold, or 2:3.
The Factor VIII polypeptide of the present invention is generally produced recombinantly, but may also be purified from plasma. The recombinant Factor VIII polypeptide may be natural full length Factor VIII polypeptide, or a variant, such as a B-domain deleted variant of Factor VIII, including the VIII:SQ variant.
The immunoaffinity columns useful in the present invention may be any industrially acceptable column and resin, to which is adsorbed one or more monoclonal or polyclonal antibodies which are capable of binding to a Factor VIII polypeptide and in which the Factor VIII polypeptide may later be released using standard methods and reagents. Suitable monoclonal antibodies, for example, are disclosed in Fass et al.,
Blood,
59:594-600 (1982).
Useful as the
Dalborg Johanna
Kelley Brian D.
Petersson Anna
Vunnum Suresh
Genetics Institute LLC
Kam Chin-Min
Low Christopher S. F.
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