Influence of excipients in Protein A chromatography and virus inactivation.

The purification of monoclonal antibodies and Fc fusion proteins consist of several unit operations operated commonly as a platform approach, starting with Protein A chromatography. The first capture step, the following low pH virus inactivation, and subsequent ion exchange chromatography steps are mostly able to remove any impurities, like host cell proteins, aggregates, and viruses. The changes in pH and conductivity during these steps can lead to additional unwanted product species like aggregates. In this study, excipients with stabilizing abilities, like polyols, were used as buffer system additives to study their impact on several aspects during Protein A chromatography, low pH virus inactivation, and cation exchange chromatography. The results show that excipients, like PEG4000, influence antibody elution behavior, as well as host-cell protein elution behavior in a pH-gradient setup. Sugar excipients, like Sucrose, stabilize the antibody during low pH virus inactivation. All excipients tested show no negative impact on virus inactivation and dynamic binding capacity in a subsequent cation exchange chromatography step. This study indicates that excipients and, possibly excipient combinations, can have a beneficial effect on purification without harming subsequent downstream processing steps.

Lyophilization Cycle Design for Dual Chamber Cartridges and a Method for Online Process Control: The "DCC LyoMate" Procedure.

Freeze-drying process design is a challenging task that necessitates a profound understanding of the complex interrelation among critical process parameters (e.g., shelf temperature and chamber pressure), heat transfer characteristics of the involved materials (e.g., product containers and holder devices), and critical quality attributes of the product (e.g., collapse temperatures). The Dual Chamber Cartridge "(DCC) LyoMate" (from lyophilization and automated) is a manometric temperature measurement-based process control strategy that was developed within this study to streamline this complicated task. It was successfully applied using 5% sucrose formulations with 0.5 and 1 mL fill volumes. The system was further challenged using 2, 20, and 100 mg/mL monoclonal antibody formulations. The DCC LyoMate method did not only produce pharmaceutically acceptable cakes but was also able to maintain the desired product temperature irrespective of formulation and protein content. It enabled successful process design even at high protein concentrations and aided the design and online control of the lyophilization process for drying in DCCs within a single development run. Thus, it helps to reduce development cost and the DCC LyoMate can also be easily installed on every freeze-dryer capable of performing a manometric temperature measurement, without the need for hardware modification.

Evaluation of Different Holder Devices for Freeze-Drying in Dual-Chamber Cartridges With a Focus on Energy Transfer.

For freeze-drying in dual-chamber cartridges, a holder device to enable handling and safe positioning in the freeze-dryer is necessary. The aim of this study was to analyze 4 different types of holder devices and to define the best system based on energy transfer. The main criteria were drying homogeneity, ability to minimize the influence of atypical radiation on product temperatures, and heat transfer effectiveness. The shell holder reduced the influence of atypical radiation by almost 60% compared to a block system and yielded the most homogenous sublimation rates. Besides the most efficient heat transfer with values of 1.58E-4 ± 2.06E-6 cal/(s*cm2*K) at 60 mTorr to 3.63E-4 ± 1.85E-5 cal/(s*cm2*K) at 200 mTorr for Ktot, reaction times to shelf temperature changes were up to 4 times shorter compared to the other holder systems and even faster than for vials. The flexible holder provided a comparable shielding against atypical radiation as the shell but introduced a third barrier against energy transfer. Block and guardrail holder were the least efficient system tested. Hence, the shell holder provided the best radiation shielding, enhanced the transferability of the results to a larger scale, and improved the homogeneity between the dual-chamber cartridges.

Heat Transfer Analysis of an Optimized, Flexible Holder System for Freeze-Drying in Dual Chamber Cartridges Using Different State-of-the-Art PAT Tools.

The aim of this study was to determine the heat transfer characteristics of an optimized flexible holder device, using Tunable Diode Laser Absorption Spectroscopy, the Pressure Rise Test, and the gravimetric procedure. Two different controlled nucleation methods were tested, and an improved sublimation process, "preheated plate," was developed. Tunable Diode Laser Absorption Spectroscopy identified an initial sublimation burst phase. Accordingly, steady-state equations were adapted for the gravimetric procedure, to account for this initial non-steady-state period. The heat transfer coefficient, KDCC, describing the transfer from the holder to the DCC, was the only heat transfer coefficient showing a clear pressure dependence with values ranging from 3.81E-04 cal/(g·cm2·K) at 40 mTorr to 7.38E-04 cal/(g·cm2·K) at 200 mTorr. The heat transfer coefficient, Ktot, reflecting the overall energy transfer via the holder, increased by around 24% from 40 to 200 mTorr. This resulted in a pressure-independent sublimation rate of around 42 ± 1.06 mg/h over the whole pressure range. Hence, this pressure-dependent increase in energy transfer completely compensated the decrease in driving force of sublimation. The "flexible holder" shows a substantially reduced impact of atypical radiation, improved drying homogeneity, and ultimately a better transferability of the freeze-drying cycle for process optimization.

Energy transfer during freeze-drying in dual-chamber cartridges.

Freeze-drying essentially requires knowledge about the heat and mass transfer characteristics to assure product quality. Whereas this understanding has been created for freeze-drying in vials, only limited information is available for state-of-the-art multiple compartment container systems such as dual-chamber cartridges (DCCs). Therefore, the aim of this study was to investigate the heat transfer characteristics of this novel container format. Sublimation tests were carried out using pure water at 60, 100, 150, and 200 mTorr chamber pressure at a shelf temperature of 0°C. Custom-made aluminum blocks were used as holder systems. Two heat transfer coefficients could be identified: the coefficient characterizing heat transfer between shelf and block, KAl , and between block and cartridge, KDCC . KAl was dependent on all three modes of heat transfer: contact conduction, gas conduction, and radiation. For KDCC , contact conduction was negligible. Radiation strongly influenced the overall energy transfer as it is the major mode of heat transfer for KDCC and contributes up to 44% to KAl . A third coefficient, Ktot , was defined as an overall heat transfer coefficient. This knowledge about heat transfer enables a purposeful development and control of optimized lyophilization processes for this novel container system.