WITHDRAWN: for Stärtzel, P. "The Application of Amino Acids in Freeze Dried Protein Formulations".

This article has been withdrawn due to an agreement between the authors, the Journal's Editorial Office, and the PDA. We sincerely apologize for any inconvenience this may cause.

Arginine as an Excipient for Protein Freeze-Drying: A Mini Review.

Successful development of marketable freeze-dried protein formulations requires adequate stabilization of the active biopharmaceutical ingredient. The choice of a stabilizer must therefore be based on sound knowledge of the physical and chemical properties of the excipients and specific needs of the protein component. Amino acids, such as arginine, have exhibit cryo- and lyoprotective effects similar to those of sugars and polymers and may therefore be considered to be an alternative approach to these established formulation strategies. The chemical structure and physicochemical characteristics of arginine are unique among amino acids and can provide additional benefits to freeze-dried protein formulations with regard to liquid and solid-state stability. This mini review provides a brief summary of research focused on the application of arginine in freeze-dried protein pharmaceuticals, including a discussion of its basic physical and chemical attributes as well as thermal behavior in the frozen and solid states. Mechanisms contributing to solid-state stabilization by arginine are discussed in the context of available stability studies on arginine-containing protein formulations. This mini review seeks to deepen the understanding of the opportunities and challenges associated with arginine-based preparations for freeze-dried protein pharmaceuticals.

The Application of Amino Acids in Freeze Dried Protein Formulations.

The development of freeze dried proteins as a market formulation poses a significant challenge to formulation scientists. The choice of a suitable stabilizer to prevent protein degradation during the process is essential and based on sound knowledge of available excipients and their stabilizing properties. Amino acids have been found to exhibit lyo- and cryoprotective effects similar to those of established stabilizers, such as sugars and/or polymers, but offer a greater diversity of chemical structures and physicochemical properties. Their ability to prevent protein aggregation in the liquid and solid states, as well as their beneficial physicochemical properties in frozen and dried state, may render them a new generation of protein stabilizers for freeze drying. The objective of this review article is to provide a summary of research focused on the application of amino acids in freeze dried protein formulations. In addition to the general physical and chemical properties of proteinogenic amino acids, their thermal properties in the frozen and solid states that are relevant to the freeze drying process are discussed. A review of available stability studies illustrating the application of amino acids to protein formulation design and a discussion of currently known mechanisms contributing to solid-state protein stability in amino acid-based systems are also taken into account.

Mannitol/l-Arginine-Based Formulation Systems for Freeze Drying of Protein Pharmaceuticals: Effect of the l-Arginine Counter Ion and Formulation Composition on the Formulation Properties and the Physical State of Mannitol.

Previous studies have shown that protein storage stability in freeze-dried l-arginine-based systems improved in the presence of chloride ions. However, chloride ions reduced the glass transition temperature of the freeze concentrate (Tg') and made freeze drying more challenging. In this study, l-arginine was freeze dried with mannitol to obtain partially crystalline solids that can be freeze dried in a fast process and result in elegant cakes. We characterized the effect of different l-arginine counter ions on physicochemical properties of mannitol compared with mannitol/sucrose systems. Thermal properties of formulations with different compositions were correlated to thermal history during freeze drying and to physicochemical properties (cake appearance, residual moisture, reconstitution time, crystallinity). Partially crystalline solids were obtained even at the highest l-arginine level (mannitol:l-arginine of 2:1) used in this study. All l-arginine-containing formulations yielded elegant cakes. Only cakes containing l-arginine chloride and succinate showed a surface "crust" formed by phase separation. X-ray powder diffraction showed that inhibition of mannitol crystallization was stronger for l-arginine compared with sucrose and varied with the type of l-arginine counter ion. The counter ion affected mannitol polymorphism and higher levels of mannitol hemi-hydrate were obtained at high levels of l-arginine chloride.

Freeze-Drying of L-Arginine/Sucrose-Based Protein Formulations, Part 2: Optimization of Formulation Design and Freeze-Drying Process Conditions for an L-Arginine Chloride-Based Protein Formulation System.

We recently reported that the presence of chloride counter ions in freeze-dried l-arginine/sucrose formulations provided the greatest protein stability, but led to low collapse temperatures and glass transition temperatures of the freeze concentrates. The objectives of this study were to identify l-arginine chloride-based formulations and optimize freeze-drying process conditions to deliver a freeze-dried product with good physical quality attributes (including cake appearance, residual moisture, and reconstitution time). Additional properties were tested such as thermal properties, cake microstructure, and protein physical stability. Excipient concentrations were varied with and without a model protein (bovine serum albumin, BSA). Formulations were frozen with and without annealing or with and without controlled nucleation. Primary drying was conducted at high and low shelf temperature. Cakes with least defects and optimum physical attributes were achieved when protein to excipient ratios were high. Controlled nucleation led to elegant cakes for most systems at a low shelf temperature. Replacing BSA by a monoclonal antibody showed that protein (physical) stability was slightly improved under stress storage temperature (i.e., 40°C) in the presence of a low concentration of l-arginine in a sucrose-based formulation. At higher l-arginine concentrations, cake defects increased. Using optimized formulation design, addition of l-arginine chloride to a sucrose-based formulation provided elegant cakes and benefits for protein stability.

Freeze drying of L-arginine/sucrose-based protein formulations, part I: influence of formulation and arginine counter ion on the critical formulation temperature, product performance and protein stability.

The objective of this study was to investigate product performance of freeze dried l-arginine/sucrose-based formulations under variation of excipient weight ratios, l-arginine counter ions and formulation pH as a matrix to stabilize a therapeutic monoclonal antibody (MAb) during freeze drying and shelf life. Protein and placebo formulations were lyophilized at aggressive primary drying conditions and key attributes of the freeze dried solids were correlated to their thermal properties and critical formulation temperature. Stability (physical) during processing and long-term storage of the MAb in different formulations was assessed by SE-HPLC. Thermal properties of the mixtures were greatly affected by the type of l-arginine counter ion. High glass transition temperatures were achieved by adding multivalent acids, whereas the temperature values significantly decreased in the presence of chloride ions. All mixtures were stable during freeze drying, but storage stability varied for the different preparations and counter ions. For l-arginine-based formulations, the protein was most stable in the presence of chloride ion, showing no obvious correlation to estimated global mobility of the glass. Besides drying behavior and thermal properties of the freeze dried solids, the counter ion of l-arginine must be considered relevant for protein shelf life stability.

Application of process analytical technology for monitoring freeze-drying of an amorphous protein formulation: use of complementary tools for real-time product temperature measurements and endpoint detection.

Process analytical technology (PAT) and quality by design have gained importance in all areas of pharmaceutical development and manufacturing. One important method for monitoring of critical product attributes and process optimization in laboratory scale freeze-drying is manometric temperature measurement (MTM). A drawback of this innovative technology is that problems are encountered when processing high-concentrated amorphous materials, particularly protein formulations. In this study, a model solution of bovine serum albumin and sucrose was lyophilized at both conservative and aggressive primary drying conditions. Different temperature sensors were employed to monitor product temperatures. The residual moisture content at primary drying endpoints as indicated by temperature sensors and batch PAT methods was quantified from extracted sample vials. The data from temperature probes were then used to recalculate critical product parameters, and the results were compared with MTM data. The drying endpoints indicated by the temperature sensors were not suitable for endpoint indication, in contrast to the batch methods endpoints. The accuracy of MTM Pice data was found to be influenced by water reabsorption. Recalculation of Rp and Pice values based on data from temperature sensors and weighed vials was possible. Overall, extensive information about critical product parameters could be obtained using data from complementary PAT tools.

Robustness testing in pharmaceutical freeze-drying: inter-relation of process conditions and product quality attributes studied for a vaccine formulation.

CONTEXT: The recent US Food and Drug Administration (FDA) legislation has introduced the evaluation of the Design Space of critical process parameters in manufacturing processes. In freeze-drying, a "formulation" is expected to be robust when minor deviations of the product temperature do not negatively affect the final product quality attributes. OBJECTIVE: To evaluate "formulation" robustness by investigating the effect of elevated product temperature on product quality using a bacterial vaccine solution. MATERIALS AND METHODS: The vaccine solution was characterized by freeze-dry microscopy to determine the critical formulation temperature. A conservative cycle was developed using the SMART™ mode of a Lyostar II freeze dryer. Product temperature was elevated to imitate intermediate and aggressive cycle conditions. The final product was analyzed using X-ray powder diffraction (XRPD), scanning electron microscopy (SEM), Karl Fischer, and modulated differential scanning calorimetry (MDSC), and the life cell count (LCC) during accelerated stability testing. RESULTS: The cakes processed at intermediate and aggressive conditions displayed larger pores with microcollapse of walls and stronger loss in LCC than the conservatively processed product, especially during stability testing. For all process conditions, a loss of the majority of cells was observed during storage. CONCLUSION: For freeze-drying of life bacterial vaccine solutions, the product temperature profile during primary drying appeared to be inter-related to product quality attributes.