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Molecular Weight Determination

Creative Biolabs has established a platform to analyze the molecular weight of therapeutic drug candidates, strictly following the ICH Topic Q6b guideline. Molecular weight (or size) should be identified utilizing size exclusion chromatography, SDS-polyacrylamide gel electrophoresis (under reducing and/or non-reducing conditions), mass spectrometry and/or other appropriate methodologies.

Molecular weight is a crucial physiochemical parameter for drug pharmacokinetic. There is a linear relationship between MW (246–19,000) and absorption by lymphatic system. Molecules with higher MW than 16,000 were primarily absorbed by the lymphatic system, while molecules with a MW less than 1,000 are absorbed mainly through the blood. Therapeutic proteins demonstrate slow absorption rate when compared to small peptides, depending on MW. For example, following subcutaneous administration, time required for maximum concentration in blood is a few days for full length mAb while compared to few hours for small proteins. According to their molecular size and weight, peptides are absorbed by different organs and the most affected one is kidney. Biotherapeutics with molecular weights of less than 20,000 have large renal uptake, which are close to the glomerular filtration rate, while those with MWs greater than 70,000 show little urinary excretion. Depending on the MW, proteins are subjected to degradation in the liver, and the degradants with low molecular weight were subsequently excreted into bile.

Given its importance in determining the molecular weight of a therapeutic protein sample, Creative Biolabs utilizes line Liquid Chromatography with Electrospray-Mass Spectrometric detection (LC/ES-MS, Q-TOF) for protein assessment. In the case of monoclonal antibody based products, samples are processed with reduction, de-N-glycosylation, followed by mass spectrometry. In addition, Creative Biolabs also utilizes  SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) method to determine molecular weight, with only small amount of highly purified protein sample. We separate the sample on the same gel with a set of molecular weight standards. After running the gel, we determine the relative migration distance (Rf) of the protein standards and the given protein samples. Based on the values obtained for the bands of the standard on the gel, the logarithm of the molecular weight of an SDS-denatured protein vs. its relative migration distance (Rf) is plotted into a graph. The molecular weight of the protein sample band is determined from interpolating the value from this graph. All the proteins are characterized in house in 2-3 weeks.


All listed services and products are For Research Use Only. Do Not use in any diagnostic or therapeutic applications.

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