Developing Lyophilized Formulations for Protein Biopharmaceuticals Containing Salt that Produce Placebos of Corresponding Appearance.

Obtaining an elegant finished pharmaceutical product remains a problem when salt concentration is high, protein concentration is low, and particularly when both conditions are combined. We propose a simple approach to develop a robust lyophilized formulation in the presence of salt and at low protein concentration. We combine this with a commercially viable lyophilization cycle that can serve as a starting point for many protein-based pharmaceutical products. In this manner the formulator scientist, even with little experience, can develop a robust and visually acceptable lyophilized product. In addition, this platform allows the production of placebo vials with no visual differences compared to the active product.

Caffeine as a Viscosity Reducer for Highly Concentrated Monoclonal Antibody Solutions.

Many monoclonal antibody (mAb) solutions exhibit high viscosity at elevated concentrations, which prevents manufacturing and injecting of concentrated mAb drug products at the small volumes needed for subcutaneous (SC) administration. Addition of excipients that interrupt intermolecular interactions is a common approach to reduce viscosity of high concentration mAb formulations. However, in some cases widely used excipients can fail to lower viscosity. Here, using infliximab and ipilimumab as model proteins, we show that caffeine effectively lowers the viscosity of both mAb formulations, whereas other common viscosity-reducing excipients, sodium chloride and arginine, do not. Furthermore, stability studies under accelerated conditions show that caffeine has no impact on stability of lyophilized infliximab or liquid ipilimumab formulations. In addition, presence of caffeine in the formulations does not affect in vitro bioactivities of infliximab or ipilimumab. Results from this study suggest that caffeine could be a useful viscosity reducing agent that complements other traditional excipients and provides viscosity reduction to a wider range of mAb drug products.

Lyophilized Drug Product Cake Appearance: What Is Acceptable?

Cake appearance is an important attribute of freeze-dried products, which may or may not be critical with respect to product quality (i.e., safety and efficacy). Striving for "uniform and elegant" cake appearance may continue to remain an important goal during the design and development of a lyophilized drug product. However, "sometimes" a non-ideal cake appearance has no impact on product quality and is an inherent characteristic of the product (due to formulation, drug product presentation, and freeze-drying process). This commentary provides a summary of challenges related to visual appearance testing of freeze-dried products, particularly on how to judge the criticality of cake appearance. Furthermore, a harmonized nomenclature and description for variations in cake appearance from the ideal expectation of uniform and elegant is provided, including representative images. Finally, a science and risk-based approach is discussed on establishing acceptance criteria for cake appearance.

Effects of pH and arginine on the solubility and stability of a therapeutic protein (Fibroblast Growth Factor 20): relationship between solubility and stability.

The purpose of this study was to dramatically enhance the solubility (> 400 fold) and stability of a therapeutic protein (Fibroblast Growth Factor 20) and to perform detailed biophysical characterization for the optimization of its formulation. The solubility of FGF-20 strongly depends on pH, arginine concentration and anions present in a buffer system. In the absence and presence of arginine, solubility was higher at lower pH (5 < or = pH < or = 6) and then decreased steadily with a minimum solubility at around pH 6.3 and plateaus at around pH 7.5 respectively. For a given pH, the protein was most soluble in arginine-sulfate. The solubility of FGF-20 increases with an increase in arginine-sulfate concentration for a given pH. However, a salting out effect was observed at higher arginine-sulfate concentration. Polysorbate-80 did not have any striking effect on solubility and no effect on thermal stability, but it significantly prevented the loss of protein under agitated conditions. Thermal stability of FGF-20 measured by DSC was increased with an increase in arginine-sulfate concentration (at least up to 0.5M). A sturdy dependence of thermal stability on pH was observed with about a 15 degrees C increase in T(m) (melting temperature) at pH 7.0 in comparison to pH 5.0. From the DSC data, approximate stability curves were generated and cold denaturation temperatures were predicted. Denaturant induced unfolding studies provided better insight of FGF-20 in different solution conditions in terms of structure and stability than the DSC data. An inverse relationship of solubility and thermal stability was observed in the pH range of 5.0 to 8.5 at a fixed arginine concentration and is consistent with Linderstrom-Lange's smeared model. A direct correlation between solubility and thermal stability was observed at different arginine concentrations for a fixed pH. The effect of arginine on the solubility and stability of FGF-20 was dominated by the preferential binding interaction.

Mapping of solution components, pH changes, protein stability and the elimination of protein precipitation during freeze-thawing of fibroblast growth factor 20.

This study discusses the effect of key factors like containers, buffers and the freeze (controlled vs. flash freezing) and thawing processes on the stability of a therapeutic protein fibroblast growth factor 20 (FGF-20). The freezing profiles monitored by 15 temperature probes located at different regions in a 2-L bottle during freezing can be grouped into three categories. A rapid drop in temperature was observed at the bottom followed by the top and middle center of the bottle. The freeze-thawing behavior in a 50 ml tube is considerably uniform, as expected. Among phosphate, HEPES (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid), citrate and histidine (each containing 0.5 M arginine-sulfate) buffer systems, a minimum pH change (0.4 pH unit vs. approximately 1.7 pH unit) was observed for the phosphate buffer system. Thawing in a 50 ml tube at room temperature standing resulted in a significant phase separation in citrate, histidine and HEPES buffers; however, phase separation was least in the phosphate buffer system. These phase separations were found to be temperature dependent. No effect of Polysorbate 80 on freeze-thawing of FGF-20 was observed. Significant concentration gradients in major buffer components and protein concentration were observed during freeze-thawing in a 2-L bottle. The segregation patterns of the various components were similar with the top and bottom layers containing lowest and highest concentrations, respectively. In the formulation buffer no pH gradient was formed, and the precipitation of FGF-20 during thawing at the top layer was related to an insufficient amount of arginine-sulfate and the precipitation at the bottom layer was due to a salting out effect. The precipitate generated during thawing goes into solution easily upon mixing whole solution of the bottle and the various gradient formations do not cause any irreversible change in structure, stability and isoform distribution of FGF-20. Comparison of slow freezing and flash freezing data suggests that the gradients in excipient and protein concentrations are mainly formed during thawing.

An RNA polymerase preparation from Methylobacterium extorquens AM1 capable of transcribing from a methylotrophic promoter.

RNA polymerase (RNAP) was purified from Methylobacterium extorquens AM1 cells grown on methanol or on succinate. The beta, beta', alpha and omega subunits were approximately the same size as those of Escherichia coli, and the identity of the omega subunit was confirmed by N-terminal sequence analysis. N-terminal sequence analysis suggested that two other polypeptides in the purified RNAP preparation might be sigma factors, a 40 kDa polypeptide that shared identity with sigma 32 homologues, and a 97 kDa polypeptide that shared identity with sigma 70 homologues in other bacteria. The 97 kDa polypeptide did not cross-react with antibody to E. coli sigma 70. The same complement of putative sigma factors was found in RNAP purified from M. extorquens AM1 grown on succinate and those grown on methanol, indicating that no major methanol-inducible sigma factor is present in this strain. Run-off assays showed that the purified RNAP was capable of initiating transcription specifically at the transcriptional start site of a methylotrophic gene, mxaF, which encodes the large subunit of methanol dehydrogenase and is found only in methylotrophic bacteria.