Serguei Tchessalov

Impact of sucrose level on storage stability of proteins in freeze-dried solids: II. Correlation of aggregation rate with protein structure and molecular mobility*

The purpose of this study is to investigate the impact of sucrose level on storage stability of dried proteins and thus better understand the mechanism of protein stabilization by disaccharides in lyophilized protein products. Five proteins were freeze dried with different amounts of sucrose, and protein aggregation was quantified using Size Exclusion Chromatography. Protein secondary structure was monitored by FTIR. The global mobility was studied using Thermal Activity Monitor (TAM), and fast local dynamics with a timescale of nanoseconds was characterized by neutron backscattering. The density of the protein formulations was measured with a gas pycnometer. The physical stability of the proteins increased monotonically with an increasing content of sucrose over the entire range of compositions studied. Both FTIR structure and structural relaxation time from TAM achieved maxima at about 1:1 mass ratio for most proteins studied. Therefore, protein stabilization by sugar cannot be completely explained by global dynamics and FTIR structure throughout the whole range of compositions. On the other hand, both the fast local mobility and free volume obtained from density decreased monotonically with an increased level of sucrose in the formulations, and thus the local dynamics and free volume correlate well with protein storage stability.

Next Generation Drying Technologies for Pharmaceutical Applications

Drying is a commonly used technique for improving the product stability of biotherapeutics. Typically, drying is accomplished through freeze-drying, as evidenced by the availability of several lyophilized products on the market. There are, however, a number of drawbacks to lyophilization, including the lengthy process time required for drying, low energy efficiency, high cost of purchasing and maintaining the equipment, and sensitivity of the product to freezing and various other processing-related stresses. These limitations have led to the search for next-generation drying methods that can be applied to biotherapeutics. Several alternative drying methods are reviewed herein, with particular emphasis on methods that are commonly employed outside of the biopharmaceutical industry including spray drying, convective drying, vacuum drying, microwave drying, and combinations thereof. Although some of the technologies have already been implemented for processing biotherapeutics, others are still at an early stage of feasibility assessment. An overview of each method is presented, detailing the comparison to lyophilization, examining the advantages and disadvantages of each technology, and evaluating the potential of each to be utilized for drying biotherapeutic products.

Impact of Sucrose level on Storage Stability of Proteins in Freeze-Dried Solids: I. Correlation of Protein-Sugar Interaction With Native Structure Preservation

The purpose of this study is to investigate protein-sugar interactions in dried protein solids as a function of sucrose level using water sorption isotherm data and secondary structure information from Fourier transform infrared (FTIR) spectroscopy. Three IgG1 fusion proteins and two cytokines were freeze-dried with sucrose at different sucrose/protein mass ratios. The water monolayer of the colyophilized sucrose/protein samples, as determined by BET analysis of water sorption data, was found to be lower than that expected based on additive contributions of pure protein and pure sucrose. This negative deviation suggests the presence of a solid-state interaction between protein and sucrose that reduces the availability of total water-binding sites. The difference in water monolayer between colyophilized and a physical mixture of protein and sucrose reached a maximum value at sucrose/protein mass ratio of 1/1 for these proteins, suggesting saturation of the protein-sugar interaction at this ratio. In addition, for four proteins studied, the normalized peak height of the major band in the FTIR spectra reached a plateau at about a 1/1 mass ratio. Therefore, it appears that there is a coupling between the preservation of protein secondary structure and the protein-sugar interaction as measured by water sorption isotherms.

The Impact of Protein Concentration on Mannitol and Sodium Chloride Crystallinity and Polymorphism Upon Lyophilization

The effect of protein concentration, in the range of 0-26 mg/mL for two Fc-fusion proteins, on the crystallinity and polymorphism of mannitol and sodium chloride in a lyophilized model formulation was examined. Mannitol hydrate levels were quantified based on moisture data and correlated to the X-ray diffraction peak area. In all formulation conditions, sodium chloride did not crystallize in samples with >44% total amorphous content. As protein concentration increased through the range of 1-5 mg/mL prior to lyophilization, beta-mannitol decreased in amount, becoming undetectable at protein concentrations above 5 mg/mL. Conversely, delta-mannitol increased as a function of protein concentration, reaching a maximum level at approximately 5 mg/mL protein. Above 10 mg/mL protein, mannitol crystallization was increasingly inhibited. Sucrose control vials showed higher levels of mannitol hydrate than either model protein. Both proteins behaved comparably with respect to mannitol crystallinity and polymorphism despite significant differences in molecular weight. Because of the differences between protein and sucrose control samples, protein concentration must be taken into consideration when assessing the lyophilization of mannitol containing solutions.

Recommended Best Practices for Process Monitoring Instrumentation in Pharmaceutical Freeze Drying—2017

Recommended best practices in monitoring of product status during pharmaceutical freeze drying are presented, focusing on methods that apply to both laboratory and production scale. With respect to product temperature measurement, sources of uncertainty associated with any type of measurement probe are discussed, as well as important differences between the two most common types of temperature-measuring instruments—thermocouples and resistance temperature detectors (RTD). Two types of pressure transducers are discussed—thermal conductivity-type gauges and capacitance manometers, with the Pirani gauge being the thermal conductivity-type gauge of choice. It is recommended that both types of pressure gauge be used on both the product chamber and the condenser for freeze dryers with an external condenser, and the reasoning for this recommendation is discussed. Developing technology for process monitoring worthy of further investigation is also briefly reviewed, including wireless product temperature monitoring, tunable diode laser absorption spectroscopy at manufacturing scale, heat flux measurement, and mass spectrometry as process monitoring tools.

Drying technologies for biopharmaceutical applications: Recent developments and future direction

ABSTRACT Drying is a commonly used processing technique for the manufacture of biotherapeutic products. Removal of water provides numerous benefits, including ease of handling and storage, reduction in transportation costs, and improved stability, to name a few. Typically, drying is accomplished through freeze-drying, as evidenced by the availability of several lyophilized products on the market. There are, however, several drawbacks to lyophilization, including the lengthy process time required for drying, low energy efficiency, and the high cost of purchasing and maintaining the equipment. Furthermore, lyophilization is a batch process and may be challenging to adapt and implement within a continuous manufacturing process scheme. These limitations have led to the search for next-generation drying technologies that can be applied to the manufacture of biotherapeutic products. Several alternative drying methods are reviewed herein with particular emphasis on the advantages and disadvantages of each technology in comparison to lyophilization and the potential of each to be utilized for drying biotherapeutic compounds.

Lyophilization: Process Design, Robustness, and Risk Management

This chapter briefly reviews the formulation literature with a focus on defining formulations that can support aggressive lyophilization. Each step in the lyophilization process is reviewed with considerations for scale-up with an emphasis on mathematical modeling (enabled by equipment characterization) and application of process analytical technologies, when possible. A scientific approach to process robustness relying upon grouping of process parameters to provide worst case conditions is described, and the consequences of grouping these parameters is discussed. Process robustness, assessed in this fashion, can then support the definition of the process design space and inform risk management activities.