Examination of capillary zone electrophoresis, capillary isoelectric focusing and sodium dodecyl sulfate capillary electrophoresis for the analysis of recombinant tissue plasminogen activator.
The microscale techniques of CZE, cIEF and SDS capillary electrophoresis have been evaluated for the analysis of a complex glycoprotein, recombinant tissue plasminogen activator (rtPA). A series of omega-amino acid buffers (pH approximately 5) was found suitable for the CZE separation of rtPA on coated capillaries. rtPA could be resolved into a series of major and minor peaks in an epsilon-aminocaproic acid buffer containing 0.01% (v/v) Tween 80. For cIEF, a two step method with pressure mobilization was utilized. Using a commercial instrument, either a polymer solution with a 50 microns I.D. capillary or narrow bore capillaries without a polymer solution (25 microns I.D.) were employed. rtPA was resolved into at least eight species within a pI range of 6.4-9.2 using Ampholine 3.5-10. Migration time precision for the major peaks ranged from 0.2% for CZE to < or = 2-3% R.S.D. for cIEF. Total recovery of rtPA from the capillary was also demonstrated for both methods. Analysis of rtPA, rtPA Type I, rtPA Type II and the desialylated forms resulted in the expected elution profiles. Finally, the potential of SDS capillary electrophoresis using a coated capillary for an rtPA Type I/Type II purity assay was shown.
Thorne
J M
JM
Northeastern University, Barnett Institute, Boston, MA, USA.
Goetzinger
W K
WK
Chen
A B
AB
Moorhouse
K G
KG
Karger
B L
BL
D000085:Acetates / Q000737:chemistry; D000178:Acrylamides / Q000737:chemistry; D000327:Adsorption; D000595:Amino Acid Sequence; D000665:Ampholyte Mixtures / Q000737:chemistry; D000818:Animals; D004586:Electrophoresis / Q000379:methods*; D019075:Electrophoresis, Capillary / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004797:Enzyme-Linked Immunosorbent Assay; D006041:Goats / Q000276:immunology; D007106:Immune Sera / Q000276:immunology; D007525:Isoelectric Focusing / Q000379:methods; D007526:Isoelectric Point; D008969:Molecular Sequence Data; D009439:Neuraminidase / Q000378:metabolism; D010958:Plasminogen / Q000378:metabolism; D011142:Polyvinyl Alcohol / Q000737:chemistry; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D015203:Reproducibility of Results; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D013501:Surface-Active Agents / Q000737:chemistry; D010959:Tissue Plasminogen Activator / Q000032:analysis* / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism
D000085:Acetates; D000178:Acrylamides; D000665:Ampholyte Mixtures; D007106:Immune Sera; D011994:Recombinant Proteins; D013501:Surface-Active Agents; D012967:Sodium Dodecyl Sulfate; D010958:Plasminogen; D011142:Polyvinyl Alcohol; D009439:Neuraminidase; D010959:Tissue Plasminogen Activator