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

Freeze–Thaw QbD Approach

Figure 6. DoE surface response representation of the interferon dimer reduction expressed as a percentage of a reference freezing–thawing procedure under non-controlled conditions (as explained in the text). For each experimental condition dimer, content was measured and divided by the dimers content of the reference.
The impact of freeze and thaw on different molecules, an IFN, an Fc-fusion, and two MAbs was investigated using a DoE approach as outlined in Table 1. Two parameters, i.e., freezing time and thawing time, that are known to have a major damaging impact on proteins were studied.4,9,10 The formation of high molecular weight (HMW) species, which has been described as the most probable protein-degradation pathway during freeze–thaw cycles was then monitored by SE-HPLC.4,5

Figure 7. Temperature profile of the 2 h/2 h freeze–thaw condition (solid line) and kinetic study of the interferon dimer conversion during incubation at high temperature (triangle).
The amount of these multimeric forms (i.e., dimers for IFN and aggregates for the Fc-fusion and MAbs) generated during freezing or thawing was compared to the HMW species content generated during a standard freezing procedure that used a simple and uncontrolled freeze–thaw system, which requires >10 h for freezing and >10 h for thawing. The difference expressed in percentage of multimeric content between the reference procedure and the freeze–thaw conditions of the DoE, was then used as response for the DoE. The output of the study carried out on the IFN is summarized in Figure 6, which describes the DoE results modeled and analyzed by the Minitab 15.1 software. The R2 value of 97.2% indicates the high validity of the model. The P value for freezing time was 0.067, higher than the typically chosen a-level of 0.05, indicating that freezing rate does not significantly affect the reduction of a dimer compared with the uncontrolled system. On the other hand, the P value of the thawing time was 0.001, well below the a-level, indicating that the thawing rate has a strong impact on protein quality and that a dimer content reduction has to be expected. Figure 6 clearly points out that a maximal dimer content reduction, up to 18%, can be achieved for very short thawing times of <3 h. However, despite this strong decrease in dimer content, the observed value was still above specification limits.

Figure 8. Kinetic study of interferon (IFN) formulated with a protecting agent. Solid line: temperature profile of the incubation. Square: IFN dimer conversion during incubation at high temperature.
When the product is thawed using the fastest condition, a thawing plateau of 2 h and incubated at a higher temperature (>29 °C) for an additional few hours, up to 56% of the formed dimers were converted to the more active monomeric form (Figure 7). These findings clearly indicate that dimers are preferentially formed during freezing and that they are then partially converted into monomers during thawing. The conversion rate is visibly temperature dependent, and the higher the temperature, the faster the dimer conversion.

Figure 9. Fc-fusion protein aggregate content measured for each DoE condition. Ref. represents the initial sample before any freeze–thaw cycles.
In an additional experiment, the IFN was formulated in the presence of a protecting agent aimed at limiting the dimer formation during freezing, and a fast freeze–thaw cycle (with a thawing plateau of 2 h) was applied. After complete thawing, the sample was immediately incubated at +7 °C for a few hours to stop any conversion activity and the dimer content was measured. A conversion kinetic study was then started by incubating the sample at +29 °C during a period of 6.5 h. The results are summarized in Figure 8 and indicate that almost complete conversion is obtained after only 2.5 h incubation. The obtained value of <1% of dimer content aligns with the specification in use for this product.

Figure 10. Celsius FFT system: a single-use bag encapsulated inside a protective polymeric shell.
As previously mentioned, a similar approach was carried out on an Fc-fusion protein and two additional MAbs. However, in these cases, no significant difference in terms of aggregate content was observed (Figure 9). It means that a well-controlled freezing procedure such as the Celsius system might not be necessary, and a simpler and cheaper system such as the Flexible Freeze Thaw containers (Celsius FFT) may be more appropriate. The Celsius FFT concept combines a flexible container with a semi-rigid polymeric protective shell (Figure 10). The contribution of the protective shell is predominant in the absorption of stresses resulting from processing or handling conditions (i.e., protection against impact and vibration). At low temperatures, the physical properties of plastic materials may change sufficiently to introduce brittleness that can reduce the capacity of the bag to absorb shocks leading to possible bag, port, tubings, or connector breakage. The FFT system is designed for freezing and thawing protein solutions in conventional and commercially available equipment (e.g., laboratory and walk-in freezer, cold room, temperature controlled cabinet, or water bath), facilitating its implementation in existing facilities and eliminating the high capital costs associated with specialized technologies.

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