Development of Freeze and Thaw Processes for Bulk Biologics in Disposable Bags
PAT
Kin Ho, Serguei Tchessalov, Angela Kantor, Nicholas Warne Drug Product Development, Wyeth Biopharma
Summary
Disposable technology for freezing protein based drug substances is a new approach designed to enable short or long term storage of drug substances in a convenient, cost effective container. Three case studies focus on the development of freeze-thaw cycles in disposable bags using cryopreservation systems. Development starts from the production system, and then scales down to the lab system for protein assessment. In all three cases, freeze-thaw cycles were developed for the lab system either to match a product-specific production scale temperature trace, or to bracket defined minimum and maximum production volumes (loads). For a high concentration protein study, a model protein
was used in cycle development in an attempt to eliminate an observed super cooling phenomenon.
Introduction
Frozen drug substance storage is a conservative and generally preferred method, when extended storage is needed, due to the benefits of increasing product stability, extending shelf life, and decreasing potential for microbial growth. Drug substance can be frozen, then thawed and finished according to market demands, or in the case of clinical drug product, to supply potentially lengthy early clinical trials. However, freeze-thaw stresses may arise during these processes by cold and heat denaturation, by freeze concentration (cryo-concentration), and by interaction of solutes with the ice-liquid interface [1-4]. These stresses may cause precipitation of buffer resulting in pH shifts [5, 6], concentration of salt to destabilize protein [7], and protein partial unfolding [8], all of which could lead to activity loss.
Minimizing these stresses to achieve maximum product stability should be the primary objective when developing a freeze-thaw process. Additionally, freezing and thawing rates are also important factors that can impact protein stability and activity, and require attention to detail in defining the freezing and thawing processes [9, 10].
Freeze-thaw, when coupled with disposable bags, provides some advantages over the traditional rigid container system including operational flexibility and a reduction in capital requirements. Frozen bags can be conveniently stored in a choice of freezers: chest, upright, and walk-in. A single use bag system can be designed to provide a shorter freezing distance for a given volume than commercially available rigid containers, thereby significantly reducing the resistance to
heat transfer [11]. Handling and transport configurations are available to facilitate shipping. Additionally, pre-sterilized bag systems offer aseptic connections, and eliminate the need for hard-piped in-house sterilization then further reducing capital as well as operating and maintenance costs. Single use systems could virtually eliminate the risks of cross contamination, particularly in multiple product facilities. The use of disposables combined with freeze-thaw production technology is therefore gaining attention.
Developing a robust freeze-thaw process to maintain product stability is essential to product preservation and storage. Unlike freeze-drying, seemingly little attention is given to the development of freezethaw processes. For freeze-drying scale-up, it is important to maintain equivalent product temperature profiles or similar product 'thermal history' [12], and equivalent drying rates [13]. It is believed that a similar product temperature profile or drying rate would yield the same product characteristics and consequently the same product stability.
This principle can be applied to freeze-thaw process scale-up or scale-down. In a recent publication, P. Shamlou [14] demonstrated that geometry of the freezing container is a key parameter, and it could be designed to optimize the freeze-thaw operation by minimizing cryo-concentration and freeze time. Rectangular container geometry can be manipulated in such a way that the freezing rate is determined predominantly by one-dimensional heat conduction, or by temperature. Linear scale-up is then based on equivalency of temperature-time profile (equivalency of cryo-concentration). This equivalency concept should be applied to both freezing and thawing steps.
Considerations for Freeze-Thaw Cycle Development
Programmed parameters including temperature, time and mixing speed during thawing affect the outcome of the freeze-thaw operation. During freezing, the set point temperature and processing time yield the resulting product temperature profile. When performed properly, mixing during thawing minimizes the effect of cryo-concentration caused by freezing and promotes a homogeneous product upon thaw. All of these parameters should be considered in the freeze-thaw
development plan.
...To view the complete article, please login or subscribe if you don't already have an account.