Direct detection of proteins in complex environments could be used for the evaluation of interactions between gels and biopharmaceutical molecules for purposes of drug delivery. Using a commercially available gel scanner we compared the relationship between intrinsic fluorescence of activated proteins and protein mass per pixel as a function of time in the presence of 10 mg/ mL hyaluronic acid, i.e. a negatively charged, hydrophilic gel. Protein mass was measured based on excitation of fluorescence of the proteins being tested. The protein samples, prepared in PBS or citrate buffer with or without the addition of 1.5 x 10<sup>6</sup> Da hyaluronic acid, were added to 8 well slides to depths of between 2 and 6 mm, followed by activation of the protein’s fluorescence and measurement of fluorescent emissions at 384 nm. This was correlated to concentrations of protein standards. We found that protein response, on a mass basis, is independent of matrix depth (up to 6 mm), hence making it possible to determine the amount of protein based on pixel area (rather than concentration). The effects due to the hyaluronic acid, a gel-like matrix, were constant and independent of the protein added. An increase in fluorescence response was proportional to activation time, but subsequently subject to a small extent of signal decay over a 4-hour time period. The concepts were demonstrated for concentrations of 25 to 150 mg/mL of bovine IgG and BSA. Their molecular weights (150 and 66 kD) and pI’s (7.2 and 4.8, respectively) are within the range of many therapeutic proteins. This <i>in vitro</i> method provides insights into protein behavior within hydrophilic gels and enables determination of concentration maps for protein distributed within an HA matrix.

Quantification and calibration of proteins in hydrophilic gels

ACS Fall 2021

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