Interfacial Stresses During Drug Substance Purification Processes

Abstract

Recombinant proteins, such as monoclonal antibodies for therapeutic use, are typically expressed in cultured cells and either secreted into the media or obtained from cell culture by disrupting the cells. Isolation of the protein of interest from cultured cells involves several disruptive steps and buffer matrix changes, where interfacial adsorption and interfacial stress may occur. After clarification and isolation, recombinant proteins undergo purification via a capture chromatography step (e.g., affinity chromatography for monoclonal antibodies), low pH viral inactivation, polishing purification steps (e.g., ion-exchange or hydrophobic interaction chromatography), virus filtration, and concentration/buffer exchange steps. During purification, various chromatographic columns are selected based on different interfacial, electrostatic, or hydrophobic interactions between proteins and adsorbents or the stationary phase to separate the target recombinant proteins from other impurities. Affinity chromatography is often the first step, where purification is accomplished by binding via specific interactions between the protein and the stationary phase, followed by elution through the disruption of these interactions by changes in the mobile phase. The affinity step is followed by subsequent column purification polishing steps to further increase the purity of the target protein and remove host cell proteins, residual DNA, and aggregates. Hydrophobic interaction chromatography (HIC) is frequently utilized in protein purification polishing steps and characterization, where the protein hydrophobicity drives interaction with a stationary phase that contains a hydrophobic ligand, such as a butyl-, octyl-, or phenyl group. On the other hand, ion-exchange chromatography is driven by the electrostatic interaction between proteins and the adsorbents (ligand-immobilized chromatographic resins). For each technique, an understanding of the interfacial interaction of proteins with adsorbents will facilitate the column and condition selection in separation. In addition to chromatographic purification steps, Multiple filtration steps are included in a given manufacturing scheme. Filter selection is critical, as certain molecules are more likely to adsorb on the filter material than others since the filter composition may influence the adsorption behavior. Typically at the last step of the manufacturing process, an ultrafiltration/diafiltration (UF/DF) unit operation is used to concentrate the protein to its target concentration and place it in the final formulation buffer. The UF/DF process is also a dedicated step for small molecule clearance to clear excipients and salt species from previous chromatography steps. In the UF/DF process, the molecule is exposed to interfacial and shear stress, along with other mechanical effects like cavitation, as it is recirculated over the course of several hours. Furthermore, the buffer matrix undergoes changes as the previous buffer species are cleared and the final formulation buffer is added. Understanding the interfacial behavior of the molecule through these unit operations can influence the purification process and allow development of a robust drug substance manufacturing scheme. This chapter provides an overview on different potential sources of interfacial stress, including a focus on the UF/DF step, which very often is the highest risk step for shear stress leading to potential reduction in product quality attributes. The impact of next-generation biomanufacturing processes on interfacial stress is also briefly discussed, and optimal configurations to minimize exposure to interfacial stress are provided.