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Charge-reversed, polymer-coated capillary column for the analysis of a recombinant chimeric glycoprotein
Journal of Chromatography, 594 (1992) 317-324 Elsevier Science Publishers B.V.. Amsterdam
CHROM.
23 878
Charge-reversed, polymer-coated capillary column for the analysis of a recombinant chimeric glycoprotein Kiyoshi
Tsuji* and Richard
J. Little
Control Biotechnology, Pharmaceutical Product Control Division, The Upjohn Company, Kalamazoo, MI 49001 (USA) (First received September
6th, 1991; revised manuscript
received
November
12th, 1991)
ABSTRACT Fused-silica capillary columns coated with an amphiphatic polymer were used to characterize a recombinant, basic, chimeric glycoprotein. Composition, ionic strength, and pH of the electrophoretic buffers were found to affect significantly the selectivity, resolution, and rate of migration of the basic glycoprotein peaks. Out of seven buffers evaluated, sodium citrate-acetic acid buffer gave the best peak resolution while maintaining peak migration at less than 20 min. The peak resolution (R,) was greater than 1 .O above a pH of 5.0; however, the R, was near zero below a pH of 4.5. Peak migration time increased exponentially with increase in pH of the running buffer. The R, increases linearly with increase in the concentration of the running buffer from 20 to 60 mM, the number of theoretical plates peaked at about 50 mM buffer concentrations. The condition selected for electrophoretic analysis of the glycoprotein uses a 50 mM concentration of sodium citrate-acetic acid buffer at pH of about 5.2. The standard curve for the analysis of the glycoproteins is linear over the range from 50 to 200 pg/ml glycoprotein with the number of theoretical plates over 60 000 per meter. The relative standard deviation of the assay method is approximately 4.6% and that of the peak migration time is about 3%. The polymer-coated capillary column electrophoretic analysis method has been demonstrated to be capable of monitoring degradation of the chimeric, basic FG glycoprotein.
INTRODUCTION
Analysis of recombinant proteins, especially of basic glycoproteins, by fused-silica capillary column electrophoresis presents a unique challenge due to non-specific adsorption of the proteins on the capillary wall. Unless the active binding sites on the surface of the capillary wall are masked, the proteins strongly interact with the wall resulting in severely skewed peaks or no migration of the protein. Conventionally, electrophoretic separation of proteins by the capillary column uses buffers of very low ( < 2.5) or very high pH (> 10) to minimize the protein-to-wall interaction. However, this strategy limits the selection of an electrophoretic buffer and severely compromises peak resolution and selectivity. Recent advancements in capillary wall modification technologies to decrease the protein-wall interactions have resulted in the commercialization of
columns and column coating reagents. These technologies include coating of the capillary wall by amphipathic polymers [l], inclusion of zwitterions in the running buffer [2], and partially deactivating columns by derivatization with hydrocarbons and neutral hydrophilic compounds [3]. Samples of a recombinant glycoprotein used in this experiment were manufactured by The Upjohn Company (Kalamazoo, MI, USA). The glycoprotein, FG, is a chimeric protein composed of the fusion protein (F) and the receptor protein (G) of respiratory syncytial virus (RSV) [4]. The terms F and G are the designation of peptide segments in the RSV glycoprotein. The portion of the genes coding for the anchor region of the F and G gene were truncated and the remaining portions of the genes fused. The FG glycoprotein contains amino acids 1 to 489 of F and 97 to 279 of G and the amino acid sequence translated from the DNA sequence is presented in Table I. The F portion of FG consists of
K. TSUJI.
324
000000
Y
700000
:
REFERENCES J. E. Wiktorowiez and _I. C. Colburn. ~?%~rrophoreris. II (1990) 769-773. M. Merion, B. Bell-Alden, E. Grover, U. Neue and J. Petersen, presented at the 3rd Inrernu~iorzul .SJwr~x~~iun~on
1 2
600000
HiXII-PcrfbmlNncc
.
:
400000
~~~~0 100
200
Time
400
(minutes)
Fig. 6. Peak area of a chimeric FG glycoprotein analyzed at 60-min intervals indicating evaporation of the sample solution.
improve selectivity was attained.
of the column.
No improvement
Cnpillar>,
El~ctrophorrsis.
San Dicyo, CA.
3-6. IYYI, Poster 69. A. M. Dougherty, C. L. Wooley. D. L. Williams. D. F. Swaile. R. 0. Cole and M. J. Sepaniak. J. Liq. Chromurogr., 14( 1991) 907~-92 1. M. W. Wathen, R. J. Brideau. D. R. Thomsen and B. R. Murphy. J. Gen Viral., 70 (1989) 2625 2635. D.,K. Wagner, P. Muelenaer, F. W. Henderson, M. H. Snyder, C. B. Reimer, E. E. Walsh, L. J. Anderson, D. L. Nelson and B. R. Murphy. J. C/in. .Microhiol., 27 (1989) 5899592. K. Tsuji, J. Chromutogr., 550 (1991) X23-830. A. D. Tran, S. Park, P. J. Lisi. Q. T. Huynh. R. R. Ryall and P. A. Lane, J. Clrronzcrtogr., 542 (1991) 454-471. K. Tsuji, L. Baczynskyj and G. E. Bronson, Awl. Clw~.. submitted for publication Fcbruar~
0
R. J. LITTLE
CHROM.
23 878
Charge-reversed, polymer-coated capillary column for the analysis of a recombinant chimeric glycoprotein Kiyoshi
Tsuji* and Richard
J. Little
Control Biotechnology, Pharmaceutical Product Control Division, The Upjohn Company, Kalamazoo, MI 49001 (USA) (First received September
6th, 1991; revised manuscript
received
November
12th, 1991)
ABSTRACT Fused-silica capillary columns coated with an amphiphatic polymer were used to characterize a recombinant, basic, chimeric glycoprotein. Composition, ionic strength, and pH of the electrophoretic buffers were found to affect significantly the selectivity, resolution, and rate of migration of the basic glycoprotein peaks. Out of seven buffers evaluated, sodium citrate-acetic acid buffer gave the best peak resolution while maintaining peak migration at less than 20 min. The peak resolution (R,) was greater than 1 .O above a pH of 5.0; however, the R, was near zero below a pH of 4.5. Peak migration time increased exponentially with increase in pH of the running buffer. The R, increases linearly with increase in the concentration of the running buffer from 20 to 60 mM, the number of theoretical plates peaked at about 50 mM buffer concentrations. The condition selected for electrophoretic analysis of the glycoprotein uses a 50 mM concentration of sodium citrate-acetic acid buffer at pH of about 5.2. The standard curve for the analysis of the glycoproteins is linear over the range from 50 to 200 pg/ml glycoprotein with the number of theoretical plates over 60 000 per meter. The relative standard deviation of the assay method is approximately 4.6% and that of the peak migration time is about 3%. The polymer-coated capillary column electrophoretic analysis method has been demonstrated to be capable of monitoring degradation of the chimeric, basic FG glycoprotein.
INTRODUCTION
Analysis of recombinant proteins, especially of basic glycoproteins, by fused-silica capillary column electrophoresis presents a unique challenge due to non-specific adsorption of the proteins on the capillary wall. Unless the active binding sites on the surface of the capillary wall are masked, the proteins strongly interact with the wall resulting in severely skewed peaks or no migration of the protein. Conventionally, electrophoretic separation of proteins by the capillary column uses buffers of very low ( < 2.5) or very high pH (> 10) to minimize the protein-to-wall interaction. However, this strategy limits the selection of an electrophoretic buffer and severely compromises peak resolution and selectivity. Recent advancements in capillary wall modification technologies to decrease the protein-wall interactions have resulted in the commercialization of
columns and column coating reagents. These technologies include coating of the capillary wall by amphipathic polymers [l], inclusion of zwitterions in the running buffer [2], and partially deactivating columns by derivatization with hydrocarbons and neutral hydrophilic compounds [3]. Samples of a recombinant glycoprotein used in this experiment were manufactured by The Upjohn Company (Kalamazoo, MI, USA). The glycoprotein, FG, is a chimeric protein composed of the fusion protein (F) and the receptor protein (G) of respiratory syncytial virus (RSV) [4]. The terms F and G are the designation of peptide segments in the RSV glycoprotein. The portion of the genes coding for the anchor region of the F and G gene were truncated and the remaining portions of the genes fused. The FG glycoprotein contains amino acids 1 to 489 of F and 97 to 279 of G and the amino acid sequence translated from the DNA sequence is presented in Table I. The F portion of FG consists of
K. TSUJI.
324
000000
Y
700000
:
REFERENCES J. E. Wiktorowiez and _I. C. Colburn. ~?%~rrophoreris. II (1990) 769-773. M. Merion, B. Bell-Alden, E. Grover, U. Neue and J. Petersen, presented at the 3rd Inrernu~iorzul .SJwr~x~~iun~on
1 2
600000
HiXII-PcrfbmlNncc
.
:
400000
~~~~0 100
200
Time
400
(minutes)
Fig. 6. Peak area of a chimeric FG glycoprotein analyzed at 60-min intervals indicating evaporation of the sample solution.
improve selectivity was attained.
of the column.
No improvement
Cnpillar>,
El~ctrophorrsis.
San Dicyo, CA.
3-6. IYYI, Poster 69. A. M. Dougherty, C. L. Wooley. D. L. Williams. D. F. Swaile. R. 0. Cole and M. J. Sepaniak. J. Liq. Chromurogr., 14( 1991) 907~-92 1. M. W. Wathen, R. J. Brideau. D. R. Thomsen and B. R. Murphy. J. Gen Viral., 70 (1989) 2625 2635. D.,K. Wagner, P. Muelenaer, F. W. Henderson, M. H. Snyder, C. B. Reimer, E. E. Walsh, L. J. Anderson, D. L. Nelson and B. R. Murphy. J. C/in. .Microhiol., 27 (1989) 5899592. K. Tsuji, J. Chromutogr., 550 (1991) X23-830. A. D. Tran, S. Park, P. J. Lisi. Q. T. Huynh. R. R. Ryall and P. A. Lane, J. Clrronzcrtogr., 542 (1991) 454-471. K. Tsuji, L. Baczynskyj and G. E. Bronson, Awl. Clw~.. submitted for publication Fcbruar~
0
R. J. LITTLE