Optimization of an sds-page method for determination of the exact molecular weight of monoclonal antibodies
,1 Zeinab ahsani
,2 Pegah naeemi
,3 Hossein behrouz
1. Biopharmaceutical Research Center, AryoGen Pharmed Inc., Alborz University of Medical Sciences, Karaj, Iran
2. Biopharmaceutical Research Center, AryoGen Pharmed Inc., Alborz University of Medical Sciences, Karaj, Iran
3. Biopharmaceutical Research Center, AryoGen Pharmed Inc., Alborz University of Medical Sciences, Karaj, Iran
4. Biopharmaceutical Research Center, AryoGen Pharmed Inc., Alborz University of Medical Sciences, Karaj, Iran
Gel electrophoresis of proteins with a polyacrylamide matrix, commonly called polyacrylamide gel electrophoresis (page) is undoubtedly one of the most widely used techniques to characterize complex protein mixtures. it is a convenient, fast and inexpensive method because requires only the order of micrograms quantities of protein. the proteins have a net electrical charge if they are in a medium having a ph different from their isoelectric point and therefore have the ability to move when subjected to an electric field. in these cases, the proteins are denatured by adding a detergent such as sodium dodecyl sulfate (sds) to separate them exclusively according to molecular weight. when using traditional tris-glycine method, all monoclonal antibodies show molecular weight higher than 200 kd with several bands close to each other which is not a correct estimation of molecular weight and purity of protein for ipqc tests. our main goal in this study is to evaluate the purity and exact molecular weight of monoclonal antibodies by developing and optimizing a new sds-page method.
As tris-acetate sds page is used for larger proteins, we used this method to evaluate if monoclonal antibody bands are detected better in this method. we evaluated samples position in the gel whilst discontinuous buffer systems use a gel separated into two sections: a large- pore stacking gel (ph = 6.8) on top of small-pore resolving gel (ph = 7.0), in this method we used lds sample buffer (4x), tris-acetate sds running buffer (20x) and resolving buffer 3 m tris–acetate, ph 7 ± 0.1. when we did the test according to first step, the position of bands were above compare to last band of molecular weight of marker. to solve this problem, the tricine system uses a low ph of the gel buffer and replaces the trailing glycine ion with a fast moving tricine ion in the running buffer. in this study we used the tricine gels that are based on the tricine system. in this buffer system, tricine substitutes glycine in the running buffer the smaller proteins that migrate with the stacked ds micelles in the tris-glycine system are now well separated from dodecylsulfate (ds) ions in the tricine system resulting in sharper bands and higher resolution subsequently we changed condition of gradient gel (5-15%). in each run the position of band and band intensity were determined accurately by image lab software of bio-rad densitometer.
The analysis of fractional factorial design by half-normal plot reveals that ph of running buffer is the most important factor affecting band position response and change the voltage is most important factor affecting band intensity. hence, ph of running buffer and voltage should precisely adjusted in sds-page of monoclonal antibodies.
By using tris-acetate gel and using tricine in running buffer, monoclonal antibody bands exactly placed at 147-150 kd with a single band that this method gives a real and suitable estimation of molecular weight and purity.
Sds-page, tris-acetate, monoclonal antibody