Use of high-performance capillary electrophoresis to monitor charge heterogeneity in recombinant-DNA derived proteins.
The separation of charge variants of recombinant DNA-derived (rDNA) proteins by high-performance capillary electrophoresis (HPCE) has been explored with the following examples: human growth hormone (rhGH), a soluble form of a T4 receptor protein (rCD4) and tissue plasminogen activator (rt-PA). The separation of rhGH and deamidated variants were examined over the range of low pH (less than 2.5) to high pH (8.0) on a coated silica capillary. No resolution was observed at pH 2.5 while at pH values of 6.5 or greater the deamidated species were separated. At pH 3.5 the variant was partially resolved but no detector signal was observed at pH 4.5 and 5.0. HPCE was also used to monitor a glycoprotein (rt-PA) with charge heterogeneity presumably due to variable sialic acid content. At a pH value of 4.5, the charge heterogeneity was only observed as peak broadening. For rCD4 multiple peaks were observed at pH 5.5 but no signal was observed at pH 6.5 or above (the pI of rCD4 is 8). These results suggest that HPCE will prove to be a valuable technique for the analysis of charged variants present in rDNA products either as a consequence of natural microheterogeneity or due to degradative processes such as deamidation.
Wu
S L
SL
Genetech, Inc., Medicinal and Analytical Chemistry, South San Francisco, CA 94080.
Teshima
G
G
Cacia
J
J
Hancock
W S
WS
D015704:CD4 Antigens / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D004274:DNA, Recombinant / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D004586:Electrophoresis / Q000379:methods*; D013006:Growth Hormone / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D010959:Tissue Plasminogen Activator / Q000032:analysis / Q000235:genetics / Q000378:metabolism
D015704:CD4 Antigens; D004274:DNA, Recombinant; D013006:Growth Hormone; D010959:Tissue Plasminogen Activator