Search

The assessment of protein stability requires the use of many sophisticated analytical techniques. Lyophilization procedures, commonly used to improve the stability profile of protein products, may potentiate undesirable protein degradation. The potential effects of lyophilization on proteins may include denaturation, decreased potency, aggregation, oxidation, and deamidation. Methods such as high-performance size-exclusion chromatography (HPSEC), peptide mapping, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and high-performance ion-exchange chromatography (HPIEC) may be used to evaluate these effects as well as to predict the long-term stability of the product. Typical degradation mechanisms of protein products and examples of the methods used for their analysis are described. In addition, methods for monitoring residual moisture levels in lyophilized proteins are presented with emphasis on their relative advantages and disadvantages.

The Center for Biologics Evaluation and Research has changed its regulations pertaining to residual moisture in freeze-dried biological products as published in Title 21 of the Code of Federal Regulations for Food and Drugs. The new regulation requires that each lot of dried product be tested for residual moisture and meet and not exceed established limits as specified by an approved method on file in the product license application. The gravimetric or loss-on-drying method is no longer listed as the required method; the 1.0% moisture limit is no longer specifically stated in the regulation. These revisions were made to bring the regulation into line with changes in residual moisture testing methods and the results obtained when new testing methods were applied to the determination of residual moisture. This is illustrated with data for Measles Virus Vaccine Live and Haemophilus b Polysaccharide Vaccine using final container residual moisture test results obtained by the gravimetric, coulometric Karl Fischer, thermogravimetric and thermogravimetric/mass spectrometric methods. Guidelines for the determination of residual moisture in dried biological products have been issued to describe residual moisture test methods and procedures used to set product residual moisture limits. For most products levels of residual moisture should be low, usually from less than 1.0% to 3.0%, so that the viability, immunologic potency and therefore the stability of the product is not compromised over time.

Gamma interferon from genetic recombination (IFN) has been found to have an optimal pH at about 7. An increase in IFN concentration may cause a decrease in solution clarity. A proper selection of isotonizing agent, as well as the addition of sugars, is effective in improving the clarity. The amount of IFN adsorbed on filter membranes varies with the membrane materials: cellulose acetate adsorbs much IFN, 2-fluorovinylidene is the next, followed by polysulfon, and polycarbonate adsorbs it least of all materials tested. Stainless steel adsorbs little IFN, and the level can be lowered even more by electropolishing. Silicone coating can decrease the amount adsorbed to about 1 microgram per vial of 10 ml. The effect of pressure given to the IFN solution during filtration is negligible. Transfer of IFN solution through pipings of conventional shape may result in partial deactivation by bubbling. At around pH 7, a lower pH of IFN solution causes a higher moisture level of the freeze-dried product. Moisture levels up to 3% have no effect on IFN stability. Upon reconstitution of freeze-dried IFN by vigorous shaking with distilled water, filtration of the solution may become difficult because polymers might have been formed during vigorous shaking. The addition of L-cysteine, maltose, and human serum albumin, has been found to be as effective in preventing such unfavorable reactions. Fatty acids in human serum albumin, which is effective in stabilizing IFN, has been found to participate in preventing denaturation of human serum albumin upon freezing and freeze-drying; however, the denaturation prevention mechanisms are not clear yet.