Markus Mattern

Formulation of Proteins in Vacuum-Dried Glasses. II. Process and Storage Stability in Sugar-Free Amino Acid Systems

The purpose of this research was to investigate the freeze- and vacuum-drying behavior of L-amino acids of current/potential use as adjuvants for formulating proteins. The analytical methods used were wide-angle x-ray diffraction, differential scanning calorimetry, and scanning electron microscopy. Protein analysis was performed either as an activity assay (lactate dehydrogenase [LDH]) or by size-exclusion chromatography (granulocyte colony-stimulating factor [rhG-CSF]). After samples were freeze-dried, only the four basic amino acids (arginine, lysine, histidine, and citrulline) formed amorphous solids, which, however, were partially crystalline. The remaining amino acids all formed fully crystalline solids. After samples were vacuum-dried, (20 degrees C, 0.1 mbar, 1 ml fill volume in 2-ml vials) fully crystalline solids were formed by all of the amino acids. For arginine, the addition of either HCl, H3PO4, or H2SO4 sufficient to form the respective salt produced amorphous solids after vacuum-drying, but they had high residual water contents and low glass transition temperatures (Tg). Addition of phenylalanine to arginine base inhibited crystallization of the latter at low concentrations during vacuum-drying procedure, leading to formation of a pure rubbery solid. At higher concentrations the phenylalanine crystallized, producing dry products with glass transition temperatures of > 60 degrees C. The process and storage stability of LDH and rhG-CSF in the vacuum-dried phenylalanine/arginine glasses was greatly improved at temperatures up to 40 degrees C compared with the unprotected proteins. Uptake of moisture during storage was, however, a complicating factor, reducing Tg, promoting crystallization, and leading to decreased protein stability. The PO4 salt of arginine produced especially high glass transition temperatures after it was vacuum-dried. These sugar-free amino acid formulations thus are potential stabilizes for proteins.

Formulation of proteins in vacuum-dried glasses. I: Improved vacuum-drying of sugars using crystallising amino acids

Abstract Aqueous solutions of maltose or sucrose containing phenylalanine, tryptophan or arginine were vacuum-dried in 2 ml glass vials (fill-volume = 1 ml) at 20 °C over 24 h at pressures down to 0.1 Pa. The dried products were examined using DSC, X-ray scattering and SEM. The inactivation of rhG-CSF and LDH on storage at 4, 30, 40 and 50 °C was determined. Pure maltose or sucrose remained in the rubbery state at room temperature, since their water contents could not be reduced below ≈ 6% w/w under the conditions used. The inclusion of phenylalanine reduced the residual water to ≈ 1% giving glass transitions of maltose of > 80 °C. X-ray diffraction and SEM showed that the phenylalanine crystallises during vacuum-drying as a network structure, which becomes coated with a thin film of the amorphous sugar. The small diffusional pathlength within the sugar film produces rapid drying at 20 °C and Tg climbs to > 80 °C in 12 h. rhG-CSF was stable in these glasses at 40 °C for at least 1 year. LDH was less stable, but still much better than the raw enzyme.

Integrated process performance assessment considering uncertainty in biopharmaceutical manufacturing operations

Abstract In this work, an integrated and systematic methodology is presented for the assessment of process performance—e.g., run time and cost—of biopharmaceutical drug product manufacturing. The methodology is represented as an IDEF0 activity model that rigorously defines the interconnections of information and activities with incorporating Good Manufacturing Practice, the quality standard of the pharmaceutical industry. Monte Carlo simulation is used to account for operational uncertainty of the process, and global sensitivity analysis is conducted to identify operations with large impacts on the process performance. The introduction of feasibility indicator permits the integration of industrial know-how in the methodology, and increases its applicability at any level of the shop floor. An industrial case study was conducted on the performance assessment of the cleaning and sterilization processes, where the methodology was applied to identify a set of improvement potentials.