Therapeutic proteins with increased half-life and methods of preparing same

A method of preparing a therapeutic protein conjugate comprising the step of

• a) contacting a therapeutic protein, or biologically-active fragment thereof, with a thiol reductant and a water-soluble polymer, or functional derivative thereof, under conditions that produce a reduced cysteine sulfhydryl group on the therapeutic protein, and allow conjugation of the water-soluble polymer to the reduced cysteine sulfhydryl group;
• the therapeutic protein having an amino acid sequence with no more than one accessible cysteine sulfhydryl group;
• wherein the therapeutic protein is A1PI (alpha-I proteinase inhibitor);
• the water soluble polymer is derivatized to contain maleimide (MAL);
• the therapeutic protein, water-soluble polymer and thiol reductant are incubated together in a single vessel, wherein the reduction of an oxidized sulfhydryl group and the conjugation reaction is carried out simultaneously;
• and
• at least 70% of the therapeutic protein comprises a single water-soluble polymer.

A method of preparing an A1PI conjugate comprising the steps of:

• a) contacting an A1PI (alpha-1 proteinase inhibitor), or biologically-active fragment thereof, with TCEP under conditions that allow the reduction of a sulfhydryl group on the A1PI, and
• b) contacting a linear PEG derivatized to contain a MAL group with the A1PI under conditions that allow conjugation of the linear PEG to the reduced sulfhydryl group;
• c) the A1PI comprising only one cysteine residue which comprises an accessible sulfhydryl group that is completely or partially oxidized, the only one cysteine residue is not involved in a di-sulfide bond with another cysteine residue in the A1 PI's amino acid sequence;
• d) the TCEP concentration is between 3 and 4-fold molar excess relative to the A1PI concentration;
• e) the reduction of the sulfhydryl group and the conjugation reaction is carried out simultaneously;
• f) wherein at least 70% of the A1PI comprises a single linear PEG;
• g) the A1PI conjugate having an increased half-life relative to native A1PI;
• and
• h) the A1PI conjugate retaining at least 60% biological activity relative to native A1PI.