A Case Study of Developing Analytical Methods - A contract manufacturer used seven analytical methods while developing an early-phase protein therapeutic in six short months. - BioPharm International


A Case Study of Developing Analytical Methods
A contract manufacturer used seven analytical methods while developing an early-phase protein therapeutic in six short months.

BioPharm International

Initial work using an anion-exchange method used a Dionex ProPac PA-1 column with 20 mM Tris pH 7.3. The protein was eluted with an increasing gradient of sodium chloride in Tris. The peak shape was significantly improved compared to that of the cation-exchange method, however, charge variants eluting after the main peak were still not well resolved. The same mobile phase (Tris) was used with the Dionex weak-anion-exchange (WAX)-10 column. We observed a greater number of charge variant peaks and improved resolution. The mobile phase pH was adjusted to 8.0, and the gradient was adjusted to improve baseline shape and resolution. The WAX method resulted in resolution of a greater number of charge variants than the cation-exchange methods. Ultimately, seven charge-variant peaks were resolved from the main peak. Most related substances in the WAX method eluted on the backside of the main peak, indicating that they were acid charge variants, most likely the result of deamidation.

Figure 2. WAX Analysis of Solution Stability Study Evaluating Buffer pH. Solution samples were held at room temperature for two weeks. Higher buffer pH resulted in a significant increase in undesirable acidic charge variants eluting after the main peak.
STABILITY TESTING The WAX method was applied during development to evaluate sample stability. Single-excipient formulations were freeze-dried and analyzed after reconstitution. A significant drop in main peak purity was observed in samples freeze-dried with lactose as a bulking agent when compared to other single-excipient formulations and a frozen control. Near the end of development, the WAX method was used to select a suitable buffer pH range for acceptable pharmaceutical processing. Solution samples ranging in pH from 5.99 to 9.63 were stored at room temperature and colder conditions. They were analyzed at 5, 7, and 14 days. A significant increase in acidic charge variants was observed in high pH samples held at room temperature for two weeks (Figure 2). The detection of charge variants indicated that the buffer pH should be kept below pH 8.5 for all process steps, and that if a higher pH buffer was required, the solution should be held at refrigerated temperatures.

GEL ELECTROPHORESIS Formulation development could not progress until an initial method to evaluate product quality was available. Although not required as a release method, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was the first technique established because of the simplicity of method development. SDS-PAGE separates proteins based on size6 and can be used to detect impurities, truncation, or aggregation. All materials used for SDS-PAGE were purchased from Invitrogen.

Figure 3. SDS-PAGE of Freeze-dried Samples with only Mannitol. A significant amount of dimer was observed in the accelerated stability sample (1) when compared to the sample stored at -20C (2). The discovery of aggregation in samples containing only mannitol resulted in the subsequent addition of sucrose to the formulation to eliminate the aggregation.
SDS-PAGE was used to analyze all early formulation development samples and was invaluable in identifying formulations prone to aggregation. In particular, a significant dimer band was observed in samples freeze-dried with only mannitol and stored at 37∞C for two weeks (Figure 3). Dimer was not observed in frozen controls or frozen solutions of the same formulation. Also, dimer was not observed in samples freeze-dried with only sucrose, or with mixtures of sucrose and mannitol.

Like IEF, SDS-PAGE required little development time, but it was not an ideal product release method. The technique was useful for comparing the purity of samples within one analysis, but intermediate precision was generally poor. An analysis of the purity results by densitometry was subjective because streaks, bubbles, and background must be excluded from the purity calculation. In addition, the limit of quantitation (LOQ) was high. While not experimentally determined, the LOQ can be estimated from linearity data to be approximately 60 g/mL or 10 percent aggregates (proteins stuck together but still soluble). Aggregates are undesirable because there are concerns that they can cause immunogenic reactions in patients.

SDS-PAGE was optimized during initial method development. We loaded samples at 0.5, 1, 2, 3, 4, and 5 g to determine optimum loading conditions. The gel was scanned with a desktop scanner and analyzed by gel analysis software. We then plotted the amount of protein loaded against optical density to determine the linear range of staining, which was from 0.5 to 3g with a 0.999 coefficient of determination.

SIZE-EXCLUSION CHROMATOGRAPHY An SEC method was developed for final release testing. SEC separates proteins on the basis of differences in molecular size using a porous matrix packed into a chromatographic column.7 Larger molecules are excluded from pores and are eluted prior to smaller molecules.

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