Bio-Rad Gel Filtration Standard, 20 µL injection. Peak 1: 6.091, Peak 2: Thyroglobulin – 6.498, Peak 3: 7.541, Peak 4: Gamma globulin – 8.742, Peak 5: Ovalbumin – 10.304, Peak 6 – Myoglobin – 11.353, Peak 7: Vitamin B12 – 13.013
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The Bio-Rad gel filtration standard is a lyophilized mixture of molecular weight markers with a range of 1,350 to 670,000 daltons. It is used as a calibration and system suitability standard for size exclusion chromatography (SEC). The mixture contains thyroglobulin, γ-globulin, ovalbumin, myoglobin, and vitamin B12. Vitamin B12 and myoglobin are visible when applied to the column and this ensures that the column is properly packed and the sample is eluting evenly. The standard is supplied as a set of 6 vials, and each vial is reconstituted with 0.5 mL of deionized water prior to use. Each vial contains approximately 18 mg of protein.
The primary function of our lab is the analysis of biologic drug substances and drug product samples in support of clinical trials. We perform SEC analysis on these samples using HPLC to determine their purity and to confirm that the results are within specifications. We are in a regulated environment (GMP) using validated methods, so it is critical that we document that our analytical instrumentation is working properly. Each analysis requires a calibration and/or system suitability check at the beginning of the run. The Bio-Rad gel filtration standard has proven to be a reliable, reproducible way to achieve this. By checking the resolution and theoretical plates, we can determine at a glance whether or not we can proceed with the experiment. The Bio-Rad gel filtration standard is regarded as the industry standard (no pun intended!) and has always given us reliable and reproducible results.
Size exclusion chromatography (SEC)
A drug substance or drug product sample along with a reference is prepared as per our method to a concentration of 10 mg/mL using formulation buffer. The Bio-Rad gel filtration standard is prepared as described above.
The HPLC is set up with a phosphate/salt mobile phase (isocratic run) using a Tosoh G3000 column. Flow rate is 1 mL/min and detection is @ 280 nm. 20µL of the gel filtration standard is injected at the beginning of the run, followed by injections of the reference and samples.
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Resolution and theoretical plates are calculated from the gel filtration standard injection. If these results pass specifications it tells you right away that the HPLC/column system is working properly and that the reference and sample results obtained are valid. Then we calculate the purity of the reference and samples as % monomer, % high molecular weight (HMW) species and % low molecular weight (LMW) species.
The Bio-Rad gel filtration standard has proven to be an extremely useful tool for our SEC analysis. We use it for all of our SEC methods and it gives reliable, reproducible results. The MW range encompasses anything from large peptides/small proteins to larger proteins and can be used in a wide variety of applications. I would definitely recommend it to anyone doing similar analyses to our own.
Product Name: Gel Filtration StandardSupplier Page