"Elucidating Structure-Function Rules for an Adsorbed Protein Layer"

Susan Daly (Advisors: Robert Tilton and Todd Przybycien)

Polyethylene glycol (PEG) modification of pharmaceutical proteins is becoming more commonplace due to improved therapeutic effect upon delivery of the conjugates. As these conjugates will encounter interfaces in manufacture, purification and end-use, and adsorption to these interfaces may impact achievable production yields and in vivo efficacies, it is important to understand how PEG modification affects protein adsorption behavior. To this end, we are currently studying the adsorption of wild type and PEG modified ribonuclease A to polylactide co glycolide (PLG) biodegradable copolymers. RNase A functions as a catalyst for RNA cleavage, rendering mRNA’s encoded information indecipherable. Upon systemic delivery, RNase A is inactive, however, when coupled to PEG, the monomeric RNase A exhibits antitumoral effects in mice [1]. We envision that incorporation of the PEG modified RNase into biodegradable polymeric microspheres could be a means for controlled, slow delivery of protein to maximize the length of time that drug concentration remains within the therapeutic window. PLG is commonly used to form microspheres that slowly biodegrade in water and release protein drug over a period of weeks to months, however incomplete release is common. Our results discussed below indicate that PEG conjugation to protein will alter the adsorbed layer structure in a manner that will likely improve protein release from the microspheres.

Using optical reflectometry, we observe that PEG modification decreases the amount of RNase A adsorbed to PLG and that the initial adsorption kinetics are controlled by PEG chains. The adsorption of both wild type and PEG modified RNase A to PLG is irreversible. Hence, RNase A release from microspheres should be more complete upon PEGylation, as the ratio of bound to entrapped RNase decreases. As PLG ages in water and begins to degrade, its surface structure changes, so RNase A adsorptive behavior changes. Streaming current measurements show increasing magnitude of the negative surface charge of PLG upon aging, likely due to the generation of carboxyl end groups upon PLG degradation. RNase A adsorption increases as the PLG surfaces age in water, likely due to increased electrostatic attraction and increased available surface area. On the contrary, the extent of PEG-RNase A adsorption decreases significantly with aging, despite increased electrostatic attraction and increased surface area of the adsorbing substrate. We propose that PLG aging increases the number of binding sites for PEG causing the chains to adopt a more spread configuration. PEG chains subsequently occupy more surface area and preclude additional adsorption via protein – PLG interaction. The cartoons illustrate the proposed layer structures for PEGylated RNase A adsorbed to fresh and aged PLG.

(1)Martin, M.; Matousek, J.; Vogel, J.; Salvik, T.; Langer, K.; Cinatl, J.; Kreuter, J. Schwabe, D. Cinatl, J. Anti-Cancer Drugs 2000, 11, 5, 369.