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Stability issues for protein-based in vitro diagnostic products
Patrick E. Guire
Maintaining the stability of dry and liquid protein reagents can require significant experimentation during the product development phase of a new immunoassay.
Diagnostic immunoassay products are based on proteins, a class of biochemical macromolecules named after the Greek mythical fast-change artist, Proteus. Like their namesake, proteins have the ability to change their shape, which can lead to loss of activity. The specific-binding and catalytic functions of protein reagents, such as antibodies, lectins, and enzymes, are quite sensitive to the potentially detrimental effects of preparation, storage, and handling.
This article discusses the various issues in protein stability that are of concern to the manufacturers of IVD products. These issues may be broken down into those concerning reagents that will be dried and those concerning reagents kept in solution.
Drying and/or freezing steps have the potential to severely denature proteins unless the proteins are properly protected. The correct drying methods and storage conditions are vital to the shelf life of dried components. Liquid stabilization is affected by a larger number of variables. Temperature, microbial contamination, and pH level or buffer can all affect stability.
Additionally, this article will discuss why performance problems should not be automatically attributed to stability issues. Factors in the production protocol may be the culprits.
The Nature of Protein
Proteins are flexible polymeric molecules with functional three-dimensional catalytic or binding sites.1 The spatial arrangement of amino acids in the catalytic and/or binding site is dependent upon the interaction of these amino acids with one another, the solvent, and solute molecules.2–4
Protein molecules comprise both hydrophilic and hydrophobic amino acids. This composition results in the protein's spontaneous folding in aqueous solution, with the more hydrophobic amino acids clustered in "dry pockets" inside a globular molecule and the more polar amino acids concentrated on the hydrated exterior.5,6
Other crosslinking interaction forces include electrostatic, H-bond, van der Waals, chelation, and covalent (e.g., disulfide bonds). Several of these interaction forces are strongly sensitive to the effects of phase changes (freezing and drying).
Problems with Dry Stabilization
While most specific-binding proteins function in the aqueous state in nature, the dry or frozen state is much preferred for stable storage. In these relatively immobile states, the frequency of collision with harmful cosolutes, such as proteases or oxidants, is immensely reduced. However, removal of solvent from protein molecules through drying—or through other phase changes such as precipitation and freezing—puts stress on the functional conformation of proteins. These phase changes can expose the hydrophobic amino acids normally buried within the molecule. Such exposure increases the proteins' association with other molecules and with hydrophobic surfaces, resulting in denaturation of the protein molecules.
Figure 1. Monoclonal antibody room temperature stability study.
Drying steps include air-drying or freeze-drying of, for example, conjugates in vials or antibody-coated plastic or membrane material. One hazard to avoid is unintentional drying between steps, which can result from working with too large a batch of coated product (plates or tubes) at one time or working in an extremely dry environment.
Inclusion of compatible solutes in the drying solution to prevent denaturation is an effective approach to stabilizing proteins for storage. These solutes typically contain hydrophilic groups, which stabilize the functional conformation of the protein molecules by burying the hydrophobic amino acids within. Therefore, it is very important to apply a stabilizing solution before the removal of solvent has promoted protein-protein and especially protein-surface interactions.
Use of Commercial Stabilizers
Commercial stabilizers are available to optimize shelf life. In one experiment, 96-well polystyrene plates (Immulon 2; Dynex Technologies Inc.; Chantilly, VA) were coated with monoclonal or polyclonal antibodies. They were then stabilized with one of the following: Stabilguard biomolecule stabilizer (SurModics Inc.; Eden Prairie, MN), Stabilcoat immunoassay stabilizer (SurModics Inc.), Superblock blocking buffer (Pierce Chemical Co.; Rockford, IL), or bovine serum albumin (BSA). Phosphate-buffered saline (PBS) was included as a negative control. The plates were then dried and stored in sealed foil pouches with desiccant. After storage at room temperature, the monoclonal plates were subjected to a sandwich enzyme immunoassay (EIA). A direct-binding assay with horseradish peroxidase (HRP) as the antigen was used for the polyclonal plates.
Figure 2. Polyclonal antibody room temperature stability study.
After 18 months storage at room temperature, all the samples, except those with BSA as the stabilizing agent, had good activity recovery in the range of 70 to 85% (see Figure 1). More pronounced differences were seen with the polyclonal antibody (see Figure 2). However, the 50% activity loss with Superblock seems to have occurred during the drying step, leaving an activity level fairly stable to storage thereafter. The fact that this activity loss was seen with the polyclonal but not the monoclonal antibody illustrates the possibility that testing multiple products may be necessary to find the stabilizer that works best for each system.
Effect of Drying Methods
Both drying methods and storage conditions are critical to maintaining stability. Membrane applications generally require very fast drying or lyophilization to retain optimum performance and activity. For coated plates, tubes, or beads, drying rapidly is not as important as ensuring that the components are completely dry. All dried products need to be stored in an airtight container with desiccant for maximum shelf life.
Following are data from a study of the effect of different drying methods on antibody activity after storage at room temperature for 6 and 12 months (see Figure 3). For the comparison of the effects of drying methods, 96-well plates were incubated with monoclonal antirabbit antibody and washed. Stabilguard biomolecule stabilizer and Superblock blocking buffer were added to separate sections of the same plate and, after incubation, removed by aspiration. The plates were then dried by the following commonly available methods:
- Four hours in a dry room atmosphere (16% relative humidity) at room temperature (20–24°C).
- Four hours inside a sealed plastic bag containing desiccant (23% relative humidity) at room temperature.
- Two hours in a drying oven (26% relative humidity) at 37°C.
Figure 3. Percent of activity retained by monoclonal antirabbit antibody samples prepared using either Stabilguard or Superblock, dried using one of four techniques (including an undried control), and stored at room temperature for 6 and 12 months.
The plates were then placed in sealed foil pouches with desiccant and stored for long-term stability assessment at room temperature. The activity recovery was measured in a sandwich EIA using rabbit antibody as the analyte and goat antirabbit antibody-HRP as the second antibody. The standard or 100% activity value was that found with plates freshly prepared (i.e., not dried) at the time of the assay.
While the differences among the methods were not profound, the drying room method consistently gave the best results. Had the long-term storage pouch not contained desiccant, greater activity loss of the negative control would most likely have been observed.
Problems with Solution Stabilization
While aqueous solution is the natural functioning state for antibodies, enzymes, and other diagnostic proteins, this is not their state of greatest stability. Numerous factors may affect the functional activity of proteins while they are in solution:
- The protein molecules become more flexible and prone to conformational changes.
- The frequency of collision with other molecules and/or the container surface increases.
- The possibility of microbial contamination increases.
- Sensitivity to elevated temperatures and low protein concentrations is heightened.
- The proteins become more susceptible to oxidation.
The Importance of Proper pH Level
The functional conformations of protein molecules are stabilized by interactions with ionic cosolutes, through electrostatic and/or chelate bridging within the molecule.7,8 The sensitivity of catalytic proteins (enzymes) to pH or hydrogen ion concentration is especially well known, leading to the almost universal use of buffer cosolutes to maintain the desired pH of the enzyme solution. Perhaps less widely recognized is the sensitivity of the enzyme to the chemical character of the buffer ion.
Figure 4. Effect of pH on stability. Solutions tested were HEPES (a), citrate (b), MOPS (c), glycine (d), Tris (e), and saline (f), all mixed 1:1 in Stabilzyme Select conjugate stabilizer and stored at 37°C. Each graph shows the percentage of activity retained by the test solution compared to a control stored at 4°C.
Following are data from a stability study of an enzyme-antibody conjugate in aqueous solution containing Stabilzyme Select conjugate stabilizer (SurModics Inc.) in various buffers and saline at different pH values. The data were obtained with a direct EIA using goat antirabbit antibody-HRP conjugate on freshly prepared rabbit antibody plates.
Five different pH values were tested. Some of the pH values tested were notably outside the useful range for a specific buffer, but all were included to directly compare the effect of different buffer molecules. The five buffers examined were:
- citrate
- glycine
- N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES)
- 3-(N-morpholino)propanesulfonic acid (MOPS)
- Trizma Base (tris[hydroxymethyl]-aminomethane) (Tris).
The buffers were prepared at a 0.1-mole concentration, divided, and adjusted to the five different pH levels. They and the saline were then mixed 1:1 with a preparation of buffer-free Stabilzyme Select. The pH was tested again and adjusted if necessary. The conjugate was diluted 1:20,000 in these solutions before storage. The conjugate solutions were stored at 4°C and 37°C for 30 days.
The solutions were equilibrated to room temperature before testing of EIA activity. The pH was also tested at each time point. Most solutions remained within 0.2 units of the original pH. However, at 37°C, citrate, glycine, and saline showed a greater decrease in pH over time when the original pH levels were higher than 7.0. Retained activity values from conjugate solutions stored at 37°C are expressed as the percentage of the respective activities at 4°C (see Figure 4).
The recovered EIA activity of the antibody-HRP conjugate differs significantly among solutions containing different buffer ions as well as among solutions with different pH levels for each buffer. HEPES provided useful stability only at the lowest pH (6.0) and citrate at none of the pH values. The citrate's pH dependence was low; however, poor stability might result from its competition, by virtue of its chelating activity, with the conjugate for required polyvalent cations. The other solutions effectively stabilized the conjugate at activity recovery rates of 75% or better over 30 days, although not at all pH levels.
MOPS exhibited highest retained activity at pH levels between 6 and 7. Glycine, Tris, and saline performed best at pH levels between 6 and 7.7. Surprisingly, saline in Stabilzyme Select provided stability as good as that of all the buffers at pH 6 to 7.7 and better at pH 8.5. This study demonstrates the importance of selecting both the appropriate buffer and the optimal pH for an assay system.
Additional Sources of Stability Problems
Protein sources, purification methods, and conjugation chemistries can affect the activity and stability of protein reagents. For example, the source of alkaline phosphatase can have a profound effect on conjugate stability. Separation of the desired protein(s) from active harmful enzymes (e.g., hydrolases, oxidases) in the source material is of major importance to storage stability in solution. The removal or inactivation of these contaminating enzymes, and the viable microbial cells that produce them, is necessary for solution stability.
Numerous chemical procedures have been demonstrated for forming covalent bonds between the enzyme and the antibody molecule in conjugate preparation. Some of the procedures are much less destabilizing for certain enzyme and antibody combinations than are others. If an enzyme conjugate is losing EIA activity but retaining enzyme activity, electing an alternative conjugation chemistry may resolve the stability issue.
Other Performance Issues
Occasionally, issues that are assumed to be stability problems are actually deficiencies caused by other variables. The problem may be something as obvious as a dilution error. Manufacturers must reject container materials that could potentially adsorb and/or inactivate proteins, such as raw polystyrene, polysulfone, polycarbonate, or glass. Preferable container materials would be polyethylene or polypropylene. Colored enzymes and other protein solutions containing oxidative metal ions should not be exposed to light. Poor control of temperature for both the storage and the enzyme assay stages can be a significant cause of variability. Careless timing of the enzyme activity measurement stage of the assay can also be a factor.9 As mentioned earlier, allowing coated wells, tubes, or beads to dry between steps can also cause variability.
Conclusion
Antibodies, enzymes, and other water-soluble proteins represent a major portion of the materials used in diagnostic immunoassays. To safeguard the shelf life and accuracy of these diagnostic tests, the proteins must be kept stable and viable.
Drying has the potential to severely denature proteins unless they are properly protected. The manner in which the protein-coated components are dried and stored is also very important. Storage in an airtight, desiccated environment helps to ensure long-term stability.
With liquid storage, the number of potentially destructive variables increases. Simple aspects, such as changes in pH level or buffer, can make significant differences in the stability of proteins in solution.
Liquid stabilization methods effective for one product may not be effective for another. Commercially available stabilizers address specific stabilization concerns. Testing a number of these products may identify a highly effective stabilizer for a specific need.
Other factors in the production protocol may affect performance. These factors include source of materials, dilution and timing errors, inappropriate temperature and humidity, exposure to light, and container materials. Finally, elementary as it may seem, when using a commercially produced stabilizing agent, one must be sure to follow package directions.
References
1. A Sali, E Shakhnovich, and Martin Karplus, "How Does a Protein Fold?" Nature 369 (1994): 248–251.
2. IM Klotz, "Protein Conformation: Autoplastic and Alloplastic Effects," Archives of Biochemistry and Biophysics 116 (1966): 92–96.
3. T Asakura, K Adachi, and E Schwartz, "Stabilizing Effect of Various Organic Solvents on Protein," Journal of Biological Chemistry 253 (1978): 6423–6425.
4. VP Torchilin, "Enzyme Stabilization without Carriers," Enzyme and Microbiological Technology 1 (1979): 74–82.
5. MI Kanehisa and TY Tsong, "Local Hydrophobicity Stabilizes Secondary Structures in Proteins," Biopolymers 19 (1980): 1617–1628.
6. SK Burley and GA Petsko, "Aromatic-Aromatic Interaction: A Mechanism of Protein Structure Stabilization," Science 229 (1985): 23–28.
7. FH Arnold and J-H Zhang, "Metal-Medicated Protein Stabilization," Trends in Biotechnology 12 (1994): 189–192.
8. EC Dawson, JDH Homan, and BK VanWeemen, Stabilization of Peroxidase, U.S. Pat. 4,228,240, October 14, 1980.
9. HU Bergmeyer ed., Methods of Enzymatic Analysis, 3rd ed. (Deerfield Beach, FL: Verlag Chemie, 1983).
Patrick E. Guire, PhD, is senior vice president, chief scientific officer, and a founder of SurModics Inc. (Eden Prairie, MN).
