Date on Master's Thesis/Doctoral Dissertation

8-2005

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Chemical Engineering

Committee Chair

Kang, Kyung A.

Committee Co-Chair (if applicable)

Starr, Thomas L.

Committee Member

Watters, James C.

Committee Member

Maurer, Muriel C.

Committee Member

Zamborini, Frank P.

Committee Member

Bruley, Duane F.

Author's Keywords

Protein C; Cohn Fraction IV-1; Miniantibody

Subject

Blood proteins

Abstract

Protein C (PC) is an important anticoagulant, antithrombotic, and antiinflammatory in blood plasma. PC deficiency can lead to severe venous thrombotic events, including lung embolism, stroke, and heart attack. In the body, PC is activated only when it is needed, and, therefore, PC does not cause bleeding problems that currently available anticoagulants may do. Purification of PC from plasma is currently done by immunoaffinity chromatography using monoclonal antibodies (Mabs), which is very expensive. Single chain variable regions (mini-Mab) with a PC binding capability, which can be produced in recombinant E. coli , were developed for PC purification. Compared to Mabs, mini-Mabs are easier to control the contamination during the production, with lower production cost, and easier to scale-up the process. In this Ph.D. research, the effect of media conditions on the PC mini-Mab production, the mini-Mab purification yield from the production media, and the PC purification performance of the mini-Mab from Cohn Fraction IV-1 (an inexpensive PC source) were studied. The optimum conditions for mini-Mab production medium were determined to be 0.1 % glucose, 0.1 mM IPTG, initial pH 5.5~6.0, 23~30 °C of media temperature during the production, and 18 hours of mini-Mab production time. The final mini-Mab production level became 450 mg/L. Mini-Mab in the production medium broth was purified using protein A affinity chromatography. A purification yield of 34 % was obtained using the elution pH of 3.0. For PC purification using the PC mini-Mab, among four commercially available affinity chromatography matrices, NHS Sepharose showed the best performance. The PC mini-Mab immobilized NHS Sepharose, when Cohn Fraction IV-1 is used as the source material, showed a PC purification yield of 16 %. High human serum albumin (HAS) content in the source material was found to reduce PC purification yield significantly. Therefore, anion exchange chromatography (DEAE) was used for the pretreatment of the Cohn Fraction IV-1 before the affinity chromatography. DEAE chromatography removed over 99 % of the human serum albumin from Cohn Fraction IV-l. The PC purification yield from the eluate of DEAE chromatography of Cohn Fraction IV-1 was approximately 25 %, increasing by approximately 10 %. PC production cost using the mini-Mab immobilized NHS-Sepharose matrix was estimated to be approximately 100 times cheaper than that using regular Mab.

Download

Share

COinS