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Metal chelate affinity chromatography on copper-charged Chelating Sepharose has been used to purify a factor IX concentrate from 4,000- to 5,000-kg pools of human plasma, with an overall yield of 194 IU/kg. Unwanted proteins and solvent-detergent reagents added to inactivate lipid-enveloped viruses were removed during the chromatographic step. The freeze-dried product was > 80% pure factor IX with a mean specific activity of > 160 IU/mg protein. The concentrate showed no evidence of clotting factor activation by in vitro tests for potential thrombogenicity or by direct assay for activated factor IX. The concentrate did not exhibit proteolytic activity against a range of synthetic peptide chromogenic substrates. Full functional factor IX activity was retained and there was no evidence of protein degradation. Metal chelate affinity chromatography therefore appears to present less physicochemical challenge to the protein than other factor IX purification methods, while allowing the preparation of a clinical factor IX concentrate at a large scale.

Tropoelastin was isolated from the aortas of chicks rendered lathyritic by treatment with beta-aminopropionitrile. The soluble elastin was judged homogeneous by sodium dodecyl sulfate polyacrylamide gel electrophoresis and possessed an estimated molecular weight of 70000. Automated sequential analysis revealed that the N-terminal region of the chick tropoelastin is very homologous to tropoelastin isolated from copper-deficient piglets. N-terminal analysis of a trypsin digest of chick tropoelastin showed that tyrosine frequently is found adjacent to lysine residues. This positioning of tyrosine residues may be significant in terms of a possible regulatory role in elastin cross-link formation.