ABSTRACT
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.
Subject(s)
Chelating Agents , Chromatography, Affinity/methods , Copper , Factor IX/isolation & purification , Chromatography, High Pressure Liquid , Drug Stability , Electrophoresis, Polyacrylamide Gel , Factor IX/chemistry , Factor IX/metabolism , Factor IXa/analysis , Freeze Drying , Humans , Molecular WeightABSTRACT
A therapeutic concentrate of factor XIII containing both A and B sub-units has been prepared from 300 kg pools of human plasma. The process starts from a cold-ethanol fraction from cryoprecipitate supernatant and therefore does not interfere with the recovery of other clinically valuable plasma proteins. Factor XIII is purified approximately 600-fold from plasma by precipitation with sodium citrate and by the removal of fibrinogen by brief heating. The product has been pasteurised in sorbitol solution to inactivate blood-borne viruses, ultrafiltered to remove sorbitol, adsorbed with bentonite and freeze-dried in a formulation meeting requirements for intravenous injection.
Subject(s)
Factor XIII/isolation & purification , Drug Contamination , Factor XIII/adverse effects , Factor XIII/therapeutic use , Factor XIII Deficiency/drug therapy , Freeze Drying , Hepatitis, Viral, Human/prevention & control , Humans , Sorbitol , SterilizationSubject(s)
Heparin/blood , Kaolin , Absorption , Blood Coagulation Tests/methods , Diagnostic Errors , Humans , ThromboplastinSubject(s)
Abnormalities, Multiple/diagnosis , Dwarfism/complications , Eyelid Diseases/complications , Muscles/pathology , Muscular Dystrophies/complications , Abnormalities, Multiple/enzymology , Abnormalities, Multiple/pathology , Acetylcholinesterase/metabolism , Child , Chromosome Aberrations , Chromosome Disorders , Diagnosis, Differential , Dwarfism/enzymology , Dwarfism/pathology , Electromyography , Histocytochemistry , Humans , Male , Microscopy, Electron , Muscles/enzymology , Muscles/physiopathology , Muscular Dystrophies/enzymology , Muscular Dystrophies/pathology , Muscular Dystrophies/physiopathology , SyndromeABSTRACT
An Elliott 903 computer with 8K central core store and magnetic tape backing store has been operated for approximately 20 months in a clinical chemistry laboratory. Details of the equipment designed for linking AutoAnalyzers on-line to the computer are described, and data presented concerning the time required by the computer for different processes. The reliability of the various components in daily operation is discussed. Limitations in the system's capabilities have been defined, and ways of overcoming these are delineated. At present, routine operations include the preparation of worksheets for a limited range of tests (five channels), monitoring of up to 11 AutoAnalyzer channels at a time on a seven-day week basis (with process control and automatic calculation of results), and the provision of quality control data. Cumulative reports can be printed out on those analyses for which computer-prepared worksheets are provided but the system will require extension before these can be issued sufficiently rapidly for routine use.
