Protein and solute distribution in drug substance containers during frozen storage and post‐thawing: A tool to understand and define freezing–thawing parameters in biotechnology process development

Abstract

Active pharmaceutical ingredient for biotechnology-based drugs, commonly known as drug substance (DS), is often stored frozen for longer shelf-life. Freezing DS enhances stability by slowing down reaction rates that lead to protein instability, minimizes the risk of microbial growth, and eliminates the risk of transport-related stress. High density polyethylene bottles are commonly used for storing monoclonal antibody DS due to good mechanical stress/strain resistant properties even at low temperatures. Despite the aforementioned advantages for frozen storage of DS, this is not devoid of risks. Proteins are known to undergo ice-water surface denaturation, cryoconcentration, and cold denaturation during freezing. A systematic investigation was performed to better understand the protein and solute distribution along with potential of aggregate formation during freeze and thaw process. A significant solute and protein concentration gradient was observed for both frozen and thawed DS bottles. In case of thawed DS, cryoconcentration was localized in the bottom layer and a linear increase in concentration as a function of liquid depth was observed. On the other hand, for frozen DS, a "bell shaped" cryoconcentration distribution was observed between the bottom layers and centre position. A cryoconcentration of almost three-fold was observed for frozen DS in the most concentrated part when freezing was conducted at -20 and -40 °C and 2.5-fold cryoconcentration was observed in the thawed DS before mixing. The information obtained in this study is critical to design freeze thaw experiments, storage condition determination, and process improvement in manufacturing environment.

Aggregates in monoclonal antibody manufacturing processes

“…Freezeconcentration can cause phase separation of excipients and loss of native protein structure during subsequent drying (Heller et al, 1997). All these stresses might cause protein aggregation as reported for antibody freeze-thawing with decreasing pH, which correlated well with T m (protein melting temperature) values (Kohle and Badkar, 2010;Kueltzo et al, 2008;Manning et al, 2010). Partial unfolding of proteins at the ice-freeze concentrate interface was found to be one mechanism of aggregates formation during freezing (Chang et al, 1996;Eckhardt et al, 1991;Strambini and Gabellieri, 1996).…”

Protein freeze concentration and micro-segregation analysed in a temperature-controlled freeze container

“…After incubation for 12 h at 4 °C in the dark, 2.2 ml of 0.5 M NH 4 Cl solution was added and incubation continued for 2 h. Unbound FITC was removed and buffer exchanged to 50 mM potassium phosphate (pH 7.5) using Vivaspin ultrafiltration. The molar FITC/BSA ratio ( F / P ) was determined with the relationships: $\frac{F}{P}=\frac{A_{495}}{A_{280}-(0.35\times A_{495})/0.614}\times C$ $C=0.876=\frac{66,433}{389\times 195}$66,433 and 389 are BSA and FITC molecular weight, respectively; 195 is absorption E 0.1% of bound FITC at 490 nm, (0.35 ×  A 495 ) is a correction factor for FITC absorbance at 280 nm [11], and 0.614 is BSA absorption at 280 nm at 1.0 mg ml −1 . Absorbances measured ( A ) are given with wavelength in subscript.…”

Forced Degradation of Therapeutic Proteins

“…During freezing and thawing, proteins are exposed to a combination of stress factors,62 including interfacial stresses (protein/ice surface),63–66 temperature fluctuations,67 cryoconcentration,64 crystallization of excipients,68–70 phase separation,71,72 and pH shifts 65,73–78. The relative importance of each of these factors with respect to protein instability depends on the protein, the formulation, and the freeze–thaw process, and thus needs to be evaluated on a case‐by‐case basis.…”

“…Freezing of larger volumes as typically applied to storage of drug substance may require several hours72,82,83 and may result in a heterogeneous frozen product with respect to protein and excipient concentration, pH, and osmolarity. To elucidate such cryoconcentration effects, one can collect samples from various positions in the container of the frozen formulation, or of the liquid formulation directly after quiescent thawing, prior to homogenizing 68,72,75. In most cases, a significant increase in protein and excipient concentration toward the middle and the bottom of the container has been found in larger containers 68,72,75.…”