Protein adsorption, the nonspecific binding of protein molecules to solid surfaces, has always complicated the development as well as the commercialization of protein therapeutics. During research and development, proteins bind to different lab consumables such as microplates, storage tubes, pipette tips, and centrifuge tubes. During commercialization, proteins may bind to the primary container (glass or plastic vial) storing them.
In commercial packaging applications, protein adsorption issues can be offset. The amount of protein that will be adsorbed is calculated, and additional protein in that amount is added to compensate for the anticipated loss. This expedient, however, means additional cost, which is borne by the customers.
In research and development applications, protein adsorption issues are less straightforward. Accounting and compensating for protein adsorption is difficult because researchers are usually working with scarce samples and are looking for unknown target proteins or markers that are rare and present in very low quantities.
While the process of adsorption has been extensively studied, it is still an enigma. The process is complex, the result of the interplay of multiple factors.1 These factors, however, can be grouped into three categories: protein properties, labware surface qualities, and buffer formulation parameters (Table 1).2,3
Most studies have shown that the adsorption process is mainly driven by hydrophobic and electrostatic interactions between proteins and the solid surfaces they interact with. If these interactions could be eliminated or even just decreased, then protein adsorption would be decreased and, consequently, protein recovery would be increased, leading to lower operational costs, more accurate results, and a higher probability of finding the therapeutic hit protein.
To counter hydrophobic and electrostatic interactions, many of the new low-protein-binding consumables and packaging containers add a hydrophilic layer. This hydrophilic layer can be obtained by employing 1) a hydrophilic coating such as siliconization or different mold release agents; 2) hydrophilic copolymer blends; or 3) plasma treatment technology to modify plastic surfaces from hydrophobic to hydrophilic.
The biggest disadvantage of a hydrophilic coating or the use of a copolymer is the interacting and leaching of the hydrophilic species into solution in present of commonly used chromatography solvents . With plasma treatment technology, the lab consumable surface is modified at a molecular level to give a hydrophilic functionality without creating any species that could leach into solution and interfere with the sample.
Below are important considerations in planning experiments involving proteins and bio-analytical assays:
1. Know your protein: The amino acid composition as well as the three-dimensional structure of the protein will play a vital role in the adsorption process. In general, larger proteins have more sites of contact with the surface, so these proteins tend to bind more strongly to the surface than do smaller proteins. Protein with high hydrophobic amino acid content or a high degree of hydrophobicity will tend to bind to the surface more strongly than do most hydrophilic proteins.
2. Know the effect of your buffer solution on the lab consumable: Low pH value, detergent, and solvents may interact with lab consumables and increase the amount of leachates that enter solution, as well as the rate of leaching from the polymer into solution. The resulting leachates may bind to the protein and interfere with its catalytic efficiency, and may also interfere with the analytical tests being performed.
3. Research the lab consumable you are using: Make sure lab consumables are made from high-quality plastic resins that contain low amounts of additives. Such resins are used to make chromatography-approved labware, the use of which can lessen the problem of leachable and extractable species. Another consideration is topography, which refers to surface texture. Greater texture exposes more surface area for interaction with proteins. Finally, heterogeneity should be assessed. Heterogeneity refers to nonuniform surface characteristics, which can give rise to domains that interact differently with proteins.
Table 1. Factors Affecting the Protein Adsorption Process
- Amino acid composition (hydrophilic vs. hydrophobic )
- Amino acid surface distribution
- Molecular size of the protein
- Tertiary structure of the protein in solution
- Protein stability in solution
Labware Surface Qualities
- Chemical composition of the lab ware
- The amount of additives and leachable in the plastic manufacturing process
- The hydrophobicity of the surface
- Interfacial energy
- Electron donor and acceptor potentials
- Steric influences
Buffer Formulation Parameters
- pH value
- Buffer type
- Ionic strength
1. Tan, J.S., and P.A. Martic. 1990. Protein adsorption and conformational change on small polymer particles. J. Colloid Interface Sci. 136: 415–431.
2. Wang, K., C. Zhou, Y. Hong, and X. Zhang. 2012. A review of protein adsorption on bioceramics. John Wiley & Sons. Hoboken, NJ
3. Dee K.C., D.A. Puleo, and R. Bizios. 2002. Tissue–biomaterial interactions. John Wiley & Sons. Hoboken, NJ. In press.