A method for routine analysis of recombinant immunoglobulins (rIgGs) by capillary isoelectric focusing (cIEF).
A capillary isoelectric focusing (cIEF) method was developed for routine analysis of recombinant immunoglobulins (rlgGs). The cIEF method used a dimethyl siloxane-coated capillary and a separation matrix of 2% ampholytes in 0.4% methylcellulose (MC). The rIgGs, and internal pI marker protein standards, were mixed with carrier ampholyte in MC, focused using high voltage, and then the protein bands were mobilized past a UV detector by simultaneous application of low pressure and voltage. Qualitatively and quantitatively equivalent rIgG focusing profiles were obtained via cIEF and gel-based IEF, with individual isoform peak area percentages and calculated peak pI values being comparable for the same samples. Linear relationships were obtained for peak area response versus sample concentration, and for the pI gradient developed between the internal pI marker standards. The relative standard deviation (RSD) in rIgG peak areas was less than 2% intra-day and less than 8% inter-day (72 h). The RSD for the mobilization times of rIgG peaks was less than 1% intra-day and less than 3% inter-day (72 h). There was no observed decrease in the performance of the capillary over 150 analyses. cIEF offers several important advantages over gel IEF, e.g. direct, quantitative detection of proteins by intrinsic UV absorbance at 280 nm, rapid analyses ( < or = 30 min), capability of automation, and one-step, electronic data analysis and archival. These data demonstrate the superiority of the cIEF method for routine analysis of rIgGs.
Tang
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S
Department of Bioanalytical Sciences, SmithKline Beecham Pharmaceuticals, King of Prussia, PA 19406, USA.
Nesta
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DP
Maneri
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LR
Anumula
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KR
D002021:Buffers; D002138:Calibration; D005069:Evaluation Studies as Topic; D006863:Hydrogen-Ion Concentration; D007136:Immunoglobulins / Q000032:analysis*; D007525:Isoelectric Focusing / Q000295:instrumentation / Q000379:methods; D020033:Protein Isoforms / Q000032:analysis*; D011994:Recombinant Proteins / Q000032:analysis*; D015203:Reproducibility of Results; D012833:Siloxanes / Q000737:chemistry; D013997:Time Factors
D002021:Buffers; D007136:Immunoglobulins; D020033:Protein Isoforms; D011994:Recombinant Proteins; D012833:Siloxanes