Rapid Determination of Spectrophotometric Absorptivity by Analytical Ultracentrifugation


Spectrophotometric determination of protein concentrations is an important analytical tool for nearly all phases of protein drug development and manufacturing. Determinations of absorptivities (A0.1%) and molar extinction coefficients (ε) for protein solutions require at least one initial, accurate measurement of protein concentration. Traditionally, this crucial concentration has been determined by dry weight analysis, total nitrogen analysis, or quantitative amino acid analysis. Unfortunately, these techniques are labor intensive, may require relatively large sample size and can give unreliable results.

It has been suggested that the refractive increment of a protein is relatively insensitive to its amino acid composition and therefore may be used universally to determine protein concentration. An analytical ultracentrifuge equipped with interference optics has been used previously to determine protein concentration. This publication describes the rapid determination of the absorptivity for a recombinant IgG monoclonal antibody using the Optima XL-I analytical ultracentrifuge from Beckman Coulter equipped with both Raleigh interference and UV absorbance optical systems. The analytical ultracentrifuge data for determining spectrophotometric absorptivities is compared to experimental data from quantitative amino acid analysis and an enzymatic digestion method.

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