Safe Freeze–Thaw of Protein Drug Products: A QbD Approach - Apply a DoE strategy to test several formulations in parallel. - BioPharm International


Safe Freeze–Thaw of Protein Drug Products: A QbD Approach
Apply a DoE strategy to test several formulations in parallel.

BioPharm International


Design of Experiment (DoE) Approach

Table 1. Two factors, freezing and thawing times, were studied for their damaging effects on proteins
To define the freeze–thaw space for each studied molecule, a two-factors, two-levels face centered composite surface response was applied as described in Table 1. The two factors investigated were freezing and thawing rates and the response was the aggregates content as determined by size exclusion high throughput liquid chromatography (SE-HPLC). These screening conditions were generated by Minitab factorial designs to identify significant main effects and interactions among the two variables.

This approach was applied to four different molecules: an interferon (IFN), two monoclonal antibodies (MAbs), and an Fc-fusion protein. To increase the reliability of the statistical output, each condition was performed twice.

Freeze and Thaw Runs

Freeze and thaw cycles were run using the Celsius S3 system and the 30-mL Celsius-Pak bags equipped with a thermocouple, allowing aseptic temperature measurements during freeze–thaw operations.

Figure 1. A) Laboratory-scale Celsius S3 system B) Celsius-Pak insertion inside the S3 module.
The Celsius S3 system is a laboratory–scale tool specifically designed for scale-up and scale-down freeze–thaw studies. The system, shown in Figure 1A, includes a freeze–thaw module, an orbital mixer, a temperature control unit, and a data-acquisition system for temperature control and recording. The 30-mL single-use bags, filled to the nominal volume with protein samples, were placed in the Celsius S3 module between a pair of heat-exchange plates within which circulates a heat-transfer fluid (HTF) as shown in Figure 1B. This setup reproduces the freezing and thawing conditions encountered at large scale because it uses the same freezing distance and the same material of construction as the production-scale Celsius-Paks. This configuration allows for a controlled freezing process based on bidirectional crystal growth along the general direction of the heat flow.9

The thermocouple used to monitor the temperature of the sample was located 1 cm below the liquid level at the last point to freeze (LPTF) of the container (Figure 4). Then, by monitoring the temperature of the product and that of the HTF, it was possible to generate the typical temperature profiles of the freezing and thawing processes for each molecule and condition studied.

Figure 2. Temperature profile of the freezing phase for the best case point (2 h freezing and 2 h thawing) generated with the S3 system. Solid line: interferon; dashed line: set point; dotted line: heat-transfer fluid temperature.
All freezing and thawing parameters of the Celsius S3 system were defined using the Cryopilot 5.0.1 software. This custom-built software allows control and monitoring of the sample, the HTF temperatures, and the mixer unit. To generate the necessary freezing and thawing times required by the DoE listed in Table 1, a freeze–thaw program was created for each studied condition by changing the temperature set point of the HTF during time as represented by the dashed line in Figure 2.

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