Process development of protein A chromatography, cation-exchange chromatography and finishing ultrafiltration for the purification of murine monoclonal antibodies

Scalability and Impact of Freeze/Thaw Conditions on the Drug Substance Quality of

Monoclonal Antibody Formulations

William Rayfield1, Heera Khan1, Sunitha Kandula1, Matt Flamm2, Nihal Tugcu1

1Process Development and Engineering, BioProcess R&D, Merck Research Laboratories, Kenilworth, NJ.

2Informatics Information Technology, Merck Research Laboratories Information Technology,

West Point, PA.
Abstract: Biopharmaceutical production often involves an extended storage of bulk protein prior to drug product fill. A majority of biologics undergo numerous degradative pathways at 2-8°C over long time periods, so it is essential to ensure that the product quality is maintained during the storage period. In addition, when shipping biologics in the liquids phase, the shear and agitation stress that can occur during transport can potentially be detrimental to product stability1. Thus, it is important to store the bulk drug substance at subzero temperatures to retard the degradation kinetics and extend the shelf life. The freezing of bulk protein at subzero temperatures can be performed in an uncontrolled or controlled manner. Uncontrolled freezing/thawing of bulk protein at high concentration (>25 g/l) in conventional large storage containers (>2L) can lead to cryoconcentration which excludes solute molecules, leading to zones of higher protein and excipient concentrations that can reach nine times higher than the initial protein concentration. Cryoconcentration can result in changes in the formulation environment via the Donnan effect which can cause a negative impact on product quality (high molecular weight species, analytical ion-exchange chromatography profile, etc). This paper will discuss the impact of freeze and thaw on monoclonal antibody drug substance quality at multiple scales over various timeframes using different storage container types. Commonly used storage temperature ranges (-20°C, -40°C, -
70°C) were evaluated. The study investigated the modeling of both controlled and uncontrolled freezing for scale-up from lab scale to production scale. The information obtained in this study is critical for designing freezing/thawing conditions for a manufacturing process.

1 S. Singh, P. Kolhe, W. Wang, S. Nema. Large Scale Freezing of Biologics. BioProcess International, Vol

7, No. 10, Nov 2009, pp. 34-42.