Low pH viral inactivation of biotherapeutic products is known to be inﬂuenced by pH, time, temperature, protein content and solute or buffer content. Many viruses are irreversibly denatured and effectively destroyed at pH 5.0–5.5. Depending on the scope of viruses targeted for inactivation and clearance, this range might be sufficient. However, several enveloped viruses are only effectively inactivated at pH range 3.5–4.
Many mAb biotherapeutic products require especially broad spectrum clearance of multiple virus types, such that, a 'low' pH target of 3.5–4 is commonly practiced (figure A). However, prolonged exposure to this pH range can also damage or inactivate some biotherapeutic products, specifically proteins or enzymes such as blood proteins, insulin and others (figure B). With prolonged exposure to pH stress, proteins and enzymes are subject to significant deamidation, denaturation and aggregation. Immunoglobulin solutions (including both IgG and IgM mAbs) are typically less susceptible than other proteins or enzymes at pH 3.5–5.5 – though they remain susceptible to various degrees. Following sufficient time at viral inactivating conditions the infectious viral burden should be effectively minimized, however, residual viral particles, debris or other content will not yet have been physically removed (figure C).
For immunoglobulin mAb products, low pH is the most frequently used method for viral inactivation as it is relatively simple, maintains a small footprint and typically requires little intervention nor additional steps to remove, unlike surfactants or other solvents. Yet, the appropriate and optimal conditions vary between molecules as well as the required spectrum of viral clearance. Therefore, studies must be undertaken for each molecule to characterize and validate the design space or the operational boundaries in which effective viral inactivation can take place. These boundaries and the outcome of a viral inactivation process are typically defined by all, or at least a selection, of the variables or Critical Process Parameters (CPPs) that influence viral inactivation outcome and therefore Drug Substance (DS) quality. Identifying and navigating these factors will positively affect product quality and quantity.
Traditionally, low pH viral inactivation studies are performed with a set volume and concentration of the immunoglobulin solution in a vessel such as a beaker with magnetic stirring. As most of the study material will use immunoglobulin solutions with a starting pH that is near physiological conditions, viral inactivation studies will seek to elucidate the reagent addition parameters. Typically, a manual titration is performed by using a burette or pipetting while intermittently recording the pH measurement. Following the completion of a prescribed time and other parameter-hold at low pH conditions sufficient to inactivate targeted virus content, the Drug Substance (DS) or immunoglobulin solution will be reverse-titrated from the low pH range to within an appropriate physiological or slightly basic range. This serves as the completion of the viral inactivation by low pH hold. Still, throughout the low pH titration study for viral inactivation, sample extraction is required for offline analysis to document various quality attributes such as aggregation or deamidation through methods such as Size Exchange Chromatography (SEC). Although precision is possible from skilled scientists, the viral inactivation process is typically laborious and suffers from the natural variations, inaccuracies and challenges of reproducibility of any manual process.