Applications of statistical regression and modeling in fill-finish process development of structurally related proteins.

In order to increase efficiency and reduce cost, many biotechnology and pharmaceutical companies utilize platform approaches for discovery and development of structurally related therapeutic proteins. In the case of the monoclonal antibody modality, retention and reuse of prior development knowledge is especially useful to gauge risks, improve speed and reduce cost for developing similar molecules in the future. In this paper, we present two applications of statistical regression and modeling to help decision making during antibody drug product fill-finish process development. The applications are for estimating viscosity and filter capacity (Vmax) values. Experiments were performed to obtain relevant data sets of viscosity, protein concentration, density, and Vmax values for various candidate antibodies. Then, statistical models were developed and optimized to estimate viscosity and filtration Vmax values for new antibodies. Viscosity of protein formulations is an important physical property that impacts almost all manufacturing operations, as well as delivery or administration of drug products. Vmax is a critical parameter for filter size selection in manufacturing processes. Development and optimization of both models followed similar steps: identifying multicollinearity and interactions, removing unnecessary explanatory variables, performing appropriate data transformation, and evaluating different model options. We obtained 95% prediction limits for the mean and individual values from the models, and further verified the models by comparing predicted values with additional experimental data. These applications of statistical tools enabled leveraging prior knowledge for process development of new molecules belonging to the same class of structurally related proteins. Although the two specific models presented here may not be directly applicable for all proteins, the approach and methodology presented can be broadly useful for structurally related protein products during their development.

Mechanistic studies of glass vial breakage for frozen formulations. I. Vial breakage caused by crystallizable excipient mannitol.

The process of freeze-thaw not only subjects bioproducts to potentially destabilizing stress, but also imposes challenges to retain container integrity. Shipment and storage of frozen products in glass vials and thawing of the vials prior to use at clinics is a common situation. Vial integrity failure during freeze-thaw results in product loss and safety issues. Formulations of biomolecules often include crystallizable excipients, which can cause glass vial breakage during freeze-thaw operations. In this study, mannitol formulations served as models for mechanistic investigation of root causes for vial breakage. Several parameters and their impacts on vial breakage were investigated, including mannitol concentration (5% and 15%), different freeze-thaw conditions (fast, slow, and staging), fill configurations (varying fill volume/vial size ratio), and vial tray materials (plastic, stainless steel, corrugated cardboard, aluminum, and polyurethane foam). The results in this study were subjected to a statistical proportion test. The data showed that large fill volumes strongly correlated with higher percentage of vial cracks. Furthermore, the 15% mannitol was found to cause more breakage than 5% mannitol, especially with fast temperature gradient. Significantly more thawing vial breakage occurred in the fast compared to slow freeze-thaw with all types of vial trays. The freezing breakage was substantially lower than the thawing breakage using the fast temperature gradient, and the trend was reversed with the slow temperature gradient. An intermediate hold at -30 degrees C prior to further decrease in temperature proved to be a practical approach to minimize mannitol-induced vial breakage. Thermal mechanical analysis (TMA) and strain gage techniques were employed to gain mechanistic insights, and it was found that the primary causes for mannitol-induced vial breakage were partial crystallization during freezing and "secondary" crystallization of non-crystallized fraction during thawing. The strain on the vial's axial direction was significantly higher than the hoop direction, typically resulting in bottom lens of the vial coming off. Without a -30 degrees C hold, rapid volume expansions due to initial crystallization and secondary crystallization of mannitol were observed in TMA profiles, and these expansions were more apparent in 15% mannitol compared to 5% mannitol. With the introduction of a -30 degrees C hold step, abrupt expansions diminished in TMA profiles, suggesting that most of the mannitol crystallization occurred concurrently with ice solidification during the -30 degrees C holding step and, thus, secondary crystallization during thawing was minimal and the sudden expansion event was eliminated. Therefore, vial breakage during both freezing and thawing was reduced.

Mechanistic studies of glass vial breakage for frozen formulations. II. Vial breakage caused by amorphous protein formulations.

In an accompanying article we have described parameters that influence vial breakage in freeze-thaw operations when using crystalizable mannitol formulations, and further provided a practical approach to minimize the breakage in manufacturing settings. Using two diagnostic tools-thermal mechanical analysis (TMA) and strain gage, we investigated the mechanism of mannitol vial breakage and concluded that the breakage is related to sudden volume expansions in the frozen plug due to crystallization events. Glass vial breakage has also been observed with a number of frozen protein formulations consisting of only amorphous ingredients. Therefore, in this study, we applied the methodologies and learnings from the prior investigation to further explore the mechanism of vial breakage during freeze-thaw of amorphous protein products. It was found that temperature is a critical factor, as breakage typically occurred when the products were frozen to -70 degrees C, while freezing only to -30 degrees C resulted in negligible breakage. When freezing to -70 degrees C, increased protein concentration and higher fill volume induced more vial breakage, and the breakage occurred mostly during freezing. In contrast to the previous findings for crystallizable formulations, an intermediate staging step at -30 degrees C did not reduce breakage for amorphous protein formulations, and even slightly increased the breakage rate. The TMA profiles revealed substantially higher thermal contraction of frozen protein formulations when freezing below -30 degrees C, as compared to glass. Such thermal contraction of frozen protein formulations caused inward deformation of glass and subsequent rapid movement of glass when the frozen plug separates from the vial. Increasing protein concentration caused more significant inward glass deformation, and therefore a higher level of potential energy was released during the separation between the glass and frozen formulation, causing higher breakage rates. The thermal expansion during thawing generated moderate positive strain on glass and explained the thaw breakage occasionally observed. The mechanism of vial breakage during freeze-thaw of amorphous protein formulations is different compared to crystallizable formulations, and accordingly requires different approaches to reduce vial breakage in manufacturing. Storing and shipping at no lower than -30 degrees C effectively prevents breakage of amorphous protein solutions. If lower temperature such as -70 degrees C is unavoidable, the risk of breakage can be reduced by lowering fill volume.