(115c) Diffusion of High Concentrations of Unlabeled IgG and BSA in a Hydrophilic, Hyaluronic Acid Gel

The direct detection of unlabeled proteins in hydrophilic gels could prove to be an important tool for fundamental studies of protein / hydrophilic polymer interactions related to the movement of injectable biologics within subcutaneous environments. We report development of a method for direct imaging of the spreading of protein in an isotropic, 1.5 x 10⁶ D, hyaluronic acid (HA) matrix formulated in PBS (pH 7.4) at a HA concentration of 10 mg/mL. The proteins, at initial concentrations of between 12 to 120 mg protein /mL, are injected into either 2 or 6 mm deep matrices of hyaluronic acid which resemble a gel and that have been placed onto a slide. This approach differs from that of Bown et al. (2018) and Lueng et al. (2017). They indirectly measured diffusion of proteins or insulin and Naproxen out of hyaluronic acid or agarose gels, respectively, into large volumes of buffers at pH 7.4 where protein concentration was measured as a function of time. The approach presented here measures diffusion within the matrix itself.

Imaging is based on excitation of protein fluorescence and capturing images in a BioRad gel scanner. Together with the principle of optical dilution, these properties enable protein mass at high concentrations to be directly analyzed. The protocol consists of placing a 20 to 50 µL sample of the protein, dissolved in PBS (pH 7.4) or citrate buffer (pH 5.5), into the matrix. After injection, protein florescence is activated and changes in protein area and intensity are subsequently imaged at 384 nm as a function of distance from the injection point over a 4-hour time period. The protein spreads out from an initial area of as little as 10 mm² to a final area of up to 300 mm².

We discuss a device that facilitates reproducible injection of dialyzed protein into the matrix and a protocol for tracking the injected sample when the device is placed in a commercially available gel scanner (Freeby et al., 2017). Comparison of the intensity of the images on a pixel by pixel basis against protein standards enables concentration maps to be generated from which protein movement within the matrix may be determined. We demonstrate the key concepts for bovine IgG (MW 150 kD, pI 7.2), and BSA (MW 66 kD, pI 4.8). A first estimate of effective diffusion coefficients, calculated from the rate of area increase across the circular protein pattern, is compared to literature values for these proteins. Bovine IgG and BSA represent molecular weights and charges (i.e., pI’s) encountered with biotherapeutic proteins, and give insights into protein behavior at high concentrations within a hydrophilic matrix.

References

Bown, H. K., C. Bonn, S. Yohe, D. B. Yadav, T. W. Patapoff, A. Daugherty, R. J. Mrsny, In vitro model for predicting bioavailability of subcutaneously injected monoclonal antibodies, J. Controlled Release, 273, 13-20 (2018).

Freeby, S., N. Liu, K. MacDonald, A. Paulus, A. Posch, US 9,606,111 B2, Stain free protein quantification and normalization, March 28, 2017.

Leung, D. H., Y. Kapoor, C. Alleyne, E. Walsh, A. Leithead, B. Habulihaz, G. M. Salituro, A. Bak, T. Rhodes, AAPS Pharm Sci Tech, 18(6), 2203-2213 (2017).