Recommended Best Practices for Lyophilization Validation 2021 Part II: Process Qualification and Continued Process Verification.

This work describes the lyophilization process validation and consists of two parts. Part one (Part I: Process Design and Modeling) focuses on the process design and is described in the previous paper, while the current paper is devoted to process qualification and continued process verification. The goal of the study is to show the cutting edge of lyophilization validation based on the integrated community-based opinion and the industrial perspective. This study presents best practices for batch size determination and includes the effect of batch size on drying time, process parameters selection strategies, and batch size overage to compensate for losses during production. It also includes sampling strategies to demonstrate batch uniformity as well as the use of statistical models to ensure adequate sampling. Based on the LyoHUB member organizations survey, the best practices in determining the number of PPQ runs are developed including the bracketing approach with minimum and maximum loads. Standard practice around CQA and CPP selection is outlined and shows the advantages of using control charts and run charts for process trending and quality control. The case studies demonstrating the validation strategy for monoclonal antibody and the impact of the loading process on the lyophilization cycle and product quality as well as the special case of lyophilization for dual-chamber cartridge system are chosen to illustrate the process validation. The standard practices in the validation of the lyophilization process, special lyophilization processes, and their impact on the validation strategy are discussed.

Investigation of design space for freeze-drying: use of modeling for primary drying segment of a freeze-drying cycle.

In this work, we explore the idea of using mathematical models to build design space for the primary drying portion of freeze-drying process. We start by defining design space for freeze-drying, followed by defining critical quality attributes and critical process parameters. Then using mathematical model, we build an insilico design space. Input parameters to the model (heat transfer coefficient and mass transfer resistance) were obtained from separate experimental runs. Two lyophilization runs are conducted to verify the model predictions. This confirmation of the model predictions with experimental results added to the confidence in the insilico design space. This simple step-by-step approach allowed us to minimize the number of experimental runs (preliminary runs to calculate heat transfer coefficient and mass transfer resistance plus two additional experimental runs to verify model predictions) required to define the design space. The established design space can then be used to understand the influence of critical process parameters on the critical quality attributes for all future cycles.

The study of amorphous phase separation in a model polymer phase-separating system using Raman microscopy and a low-temperature stage: effect of cooling rate and nucleation temperature.

The freezing process is a source of product instability in many freeze-dried pharmaceuticals. During freezing, the solute is concentrated in the interstitial spaces between ice crystals, and phase separation may occur, with likely negative stability implications. Phase separation may involve crystallization but may also occur in completely amorphous systems even though there is little direct evidence to support this speculation in protein formulation applications. Previous work in our laboratory focused on the application of a novel Raman mapping technique to the study of amorphous phase separation in freeze-dried polymer systems. We report here the application of a similar Raman mapping technique to frozen systems, using a low-temperature stage. We study the impact of nucleation temperature and thermal history on phase separation using a model 1:1 polyvinylpyrrolidone:dextran phase separating system. Although cooling rate and nucleation temperature had a small effect on the extent of phase separation, it was clear that the large latent heat of crystallization controls the thermal history and propensity for phase separation in practical applications. The results suggest that phase separation can be somewhat controlled by minimizing fill depth and controlling nucleation temperature.

Effect of hydration on the secondary structure of lyophilized proteins as measured by fourier transform infrared (FTIR) spectroscopy.

The impact of hydration on the secondary structure of proteins using FTIR spectroscopy was investigated. Alternative sampling techniques were investigated since KBr pelletization of hydrated proteins is not recommended. Spectra of lyophilized dry proteins were collected in transmission mode by palletizing, mulling, and in ATR mode. Spectra for hydrated proteins were collected in mulls and in ATR mode. Spectra for reconstituted solutions were collected in transmission mode. Spectra of Protein-sucrose colyophilized mixtures were collected in KBr pellets and in ATR mode. Pure proteins underwent significant change in structure upon lyophilization, reforming upon reconstitution. ATR spectra differed from transmission spectra in peak intensity and position, suggesting a more nativelike structure even after correction for refractive index dispersion. No significant differences were found between KBr pellet and mull spectra. Colyophilization with sucrose led to protection of structure. The effect of hydration on the structure was protein dependent, ranging from loss of native structure (IgG) to partial reformation of native structure (BSA). It is concluded that spectra collected in different modes are not directly comparable and caution must be exercised in interpreting the data. Contrary to general view, the secondary structure of proteins in a hydrated state was not equivalent to that in solution.

Impact of critical process and formulation parameters affecting in-process stability of lactate dehydrogenase during the secondary drying stage of lyophilization: a mini freeze dryer study.

The stresses during the secondary-drying stage of lyophilization were investigated using a controlled humidity mini-freeze-dryer [Luthra S, Obert J-P, Kalonia DS, Pikal MJ. 2007. Investigation of drying stresses on proteins during lyophilization: Differentiation between primary and secondary-drying stresses on lactate dehydrogenase using a humidity controlled mini freeze-dryer. J Pharm Sci 96: 61-70.]. Lactate dehydrogenase (LDH), was formulated in: (1) Tween 80, (2) citrate buffer, and (3) both Tween 80 and citrate buffer. Protein activity recovery was measured as a function of relative humidity (RH), product temperature, and drying duration. Studies were also conducted with different concentrations of sucrose, sorbitol, and poly (vinyl pyrrolidone) (PVP). LDH stability was affected to a small extent by RH and significantly by drying temperature and duration. Complete stabilization of LDH was observed when lyophilized with sucrose and PVP but only a partial stabilization was observed with sorbitol. The mini-freeze-dryer enabled studying the process parameters independently, unlike a conventional study where these effects are generally convoluted. The results suggest that the stability of the protein is a function of the dynamics of the system during lyophilization. The origin of the stabilization effect of sucrose, which could, in principle, be attributed both to direct interaction with the protein or vitrification of the protein was elucidated using lyoprotectants that can either hydrogen bond well with the protein (sorbitol) or form a good glass (PVP). It appears both effects are required for complete stabilization of the protein.

Simultaneous measurement of water desorption isotherm and heats of water desorption of proteins using perfusion isothermal microcalorimetry.

The purpose of this work was to study protein-water interactions using a perfusion isothermal calorimetry method by simultaneously measuring the water (de)sorption isotherm and heats of desorption (DeltaH(desorption)). Lysozyme, bovine serum albumin (BSA), and a monoclonal immunoglobulin (IgG) were studied. Desorption isotherms and DeltaH(desorption) were calculated using data from two perfusion systems, which measured heat flow resulting from interaction of water vapor with the protein sample and with pure water, respectively. The desorption isotherms calculated from the calorimetry were in good agreement with the gravimetric data. The average DeltaH(desorption) at high hydration was 54.6 kJ/mol and decreased (approaching heat of water evaporation) with desorption and passed through a minimum at protein specific water content, below which it increased again reaching 59.0 kJ/mol at the lowest hydration levels. The difference between the DeltaH(desorption) above the minimum and heat of water evaporation has been attributed to conformational changes in the protein. This conclusion is supported with data for lysozyme in which a dynamic glass like transition has been observed at the water content of the minimum in the calorimetric enthalpy data at 293 K. This work establishes perfusion calorimetry as a rapid and controlled method to study the thermodynamics of protein-water interaction.

Investigation of drying stresses on proteins during lyophilization: differentiation between primary and secondary-drying stresses on lactate dehydrogenase using a humidity controlled mini freeze-dryer.

This article describes the design, performance testing, and application of a controlled humidity mini-freeze-dryer in studying the physical stability of lactate dehydrogenase during lyophilization. Performance evaluation of the mini-freeze-dryer was conducted with tests, namely water sublimation, radiation heat exchange, lowest achievable temperature, and leak testing. Protein stability studies were conducted by comparing protein activity at various stages of lyophilization with the initial activity. The shelf and condenser temperature were stable at <-40 degrees C, wall temperature was within 2 degrees C of the shelf temperature, and the leak rate was small. The chamber pressure was controlled by the ice on the condenser and the product temperature during sublimation was equal to the shelf temperature. Addition of Tween 80 prevented activity loss in solution and after freeze-thaw. No activity loss was observed after primary-drying even in absence of lyoprotectants and with collapse of cake structure. Five percent (w/w) sucrose concentration was required to achieve full stabilization. In conclusion, performance testing established that the mini-freeze-dryer was suitable for mechanistic freeze-drying studies. Secondary-drying was the critical step for protein stability. The concentration of sucrose required to stabilize the protein completely was several orders of magnitude higher than that required to satisfy the direct interaction requirement of the protein.