Drug – bio-affecting and body treating compositions – Enzyme or coenzyme containing – Hydrolases
Reexamination Certificate
2000-09-21
2002-08-20
Weber, Jon P. (Department: 1651)
Drug, bio-affecting and body treating compositions
Enzyme or coenzyme containing
Hydrolases
C435S219000
Reexamination Certificate
active
06436397
ABSTRACT:
FIELD OF THE INVENTION
The present invention is broadly directed to a method for reducing autodegradation of activated protein C during processing and purification.
BACKGROUND OF THE INVENTION
Protein C is a serine protease and naturally occurring anticoagulant that plays a role in the regulation of homeostasis by inactivating Factors V
a
and VIII
a
in the coagulation cascade. Human protein C is made in vivo primarily in the liver as a single polypeptide of 461 amino acids. This precursor molecule undergoes multiple post-translational modifications including 1) cleavage of a 42 amino acid signal sequence; 2) proteolytic removal from the one chain zymogen of the lysine residue at position 156 and the arginine residue at position 157 to make the 2-chain form of the molecule, (i.e., a light chain of 155 amino acid residues attached through a disulfide bridge to the serine protease-containing heavy chain of 262 amino acid residues); 3) vitamin K-dependent carboxylation of nine glutamic acid residues clustered in the first 42 amino acids of the light chain, resulting in 9 gamma-carboxyglutamic acid (GLA) residues; and 4) carbohydrate attachment at four sites (one in the light chain and three in the heavy chain). The heavy chain contains the well established serine protease triad of Asp 257, His 211 and Ser 360. Finally, the circulating 2-chain zymogen is activated in vivo by thrombin at a phospholipid surface in the presence of calcium ion. Activation results from removal of a dodecapeptide at the N-terminus of the heavy chain, producing activated protein C (aPC) possessing enzymatic activity. In concert with other proteins, activated protein C functions as perhaps the most important down-regulator of blood coagulation resulting in thrombosis.
Unfortunately, aPC can autodegrade, leading to decreased functionality as an anticoagulant. An art recognized degradation pathway for activated protein C is a proteolytic clip at the lysine residue at position 308 of the heavy chain yielding a 111 amino acid fragment. This degradation product is recognized in the art as the EAK fragment.
Previous attempts to reduce autodegradation have focused on minimizing the formation of the EAK fragment. Most notably, Prouty et al., EP 0 662 513, Jul. 12, 1995, teach minimizing autodegradation of aPC by controlling the pH to about 6.3 to 7.0; incubating the aPC in 3 M urea; or exposing aPC to extreme salt conditions, which are defined to be above 0.4 M or below 0.05 M.
Applicants have discovered a second important degradation pathway—autodegradation of the N-terminus of the light chain resulting in a clip on either side of the histidine residue at position 10. This degradation pathway yields two inactive products. The N-terminal clip of the first nine residues of the light chain yields des(1-9)activated protein C (SEQ ID NO:1), and the N-terminal clip of the first ten residues of the light chain yields des(1-10)activated protein C (SEQ ID NO:2). This degradation pathway, which has not been previously reported, results in loss of anticoagulant activity due to the removal of the critical GLA residues at positions 6 and 7. Therefore minimizing the level of the des(1-9)- and des(1-10)activated protein C autodegradation products is important in achieving a potent, high purity activated protein C pharmaceutical preparation. These variants were previously unknown degradation products and are exceedingly difficult, if not impossible, to remove by conventional purification techniques. The conditions to minimize their formation were previously unknown.
Identification of this important autodegradation pathway for activated protein C by Applicants has enabled the discovery of processing and formulation conditions to enhance the purity and potency of the activated protein C. Applicants have demonstrated that at a low pH (e.g. less than 6.3) the autodegradation pathway favoring the des(1-9)aPC or des(1-10)aPC predominates over the 308-309 autodegradation pathway, however use of elevated sodium chloride concentrations (greater than 150 mM) at a pH less than 6.3 substantially reduces the extent of the des(1-9)aPC and/or des(1-10)aPC autodegradation reaction.
Accordingly, the present invention provides for processing activated protein C at an ionic strength of greater than 150 mM and at a pH of about 5.5 to less than 6.3. Under these conditions the formation of des(1-9)aPC and des(1-10)aPC is significantly reduced. The present invention therefore provides an improved method for processing an aqueous solution of activated protein C without undesirable degradation.
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Baker Jeffrey Clayton
Carlson Andrew David
Huang Lihua
Sheliga Theodore Arsay
Barrett Brian P.
Caltrider Steven P.
Eli Lilly and Company
Hostettler Danica
Weber Jon P.
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