Protein Adsorption Study
Single-use systems are increasingly used in downstream processing, final formulation, and filling to process fluids of a critical nature, including protein-containing solutions. These systems gained acceptance for storage and processing at manufacturing scale of recombinant proteins or monoclonal antibodies in liquid or frozen forms. Container-protein interactions may include potential adsorption of the protein onto the container surfaces.
In this study, traditional glassware used in analytical and bioprocess applications was also tested.
The quantities of adsorbed protein were low, thus RP-HPLC was selected for its sensitive and quantitative method of amino acid analysis.
The major driving forces influencing the adsorption of proteins onto solid surfaces are hydrophobic and electrostatic interactions. These interactions are responsible for nonspecific protein binding on a variety of surfaces. Interaction factors between the surface and protein are modulated by the physical state of the surface, the composition and pH of the solution, the storage conditions (temperature, contact time) and the concentration and structural and conformational properties of the protein.
Test conditions. Protein adsorption levels are dependent on a variety of different parameters, chosen according to standards used in the field. The flexible single-use containers were made of ethylene vinyl acetate copolymer (Evam and Stedim 71) or low-density polyethylene (Stedim 40). The study was performed under worst-case conditions in respect to contact surface-to-volume ratio.
Two model proteins commonly used in biotechnology were chosen: bovine serum albumin (BSA) and bovine polyclonal Immunoglobulin G (IgG). 10 mg/mL BSA and 1 mg/mL bovine IgG solutions were prepared in a phosphate-buffered saline at pH 7.2.
The bags and glass containers were filled with the protein solutions and stored at 5C and 37C. Sample containers were removed from storage and analyzed at regular time intervals (4 hours, 1 day, 3 days, 1 week and 1 month).
Conclusions. Low binding levels of the model proteins were measured on polymeric surfaces and on the borosilicate glass surface. The maximum amount of protein adsorbed (g/cm2) over the storage period for each container is summarized in Table 2. The adsorption level of the two proteins to tested plastic films was low.
The maximum level of protein potentially adsorbed onto Stedim bags filled at nominal capacity at 5C is summarized in Table 3 and illustrated for Flexboy bags in Figure 2.
These values were obtained for the bags with the greatest surface-to-volume ratio for each bag range, which represent a worst-case compared to large volume bags.
The extrapolation of the results obtained with the Stedim product range gives a maximum level of adsorption of 0.14% of the initial protein content, which is low and not significant according to the ICH Harmonized Tripartite Guideline Q1A titled Stability Testing of New Drug Substances and Products.