Viral Inactivation in Bioprocessing

Viral inactivation is the first of usually two dedicated steps designed to enhance the safety of biotherapeutic products. During inactivation, any virus present within the pooled and semi-purified therapeutic suspension is intentionally destroyed or abruptly rendered non-pathogenic, usually by altering the environment around the virus to cause irreversible disruption and denaturation of the viral structure.

Typically, this is done by altering the environment around the virus to cause irreversible disruption and denaturation of the viral structure  – e.g., through chemical, physical, or even energetic methods. Virus removal is separate and distinct from viral inactivation as it is a complementary process that further enhances viral safety by eliminating or separating the virus from the protein(s) or products of interest.

Many biotherapeutic products contain or can become contaminated with viruses during production or processing. The pathogenicity and viral load or quantity of virus in these modalities need to be eliminated or cleared through a means of inactivation and removal to prevent harming the patient; neither process alone is typically sufficient. Multiple methods of viral inactivation and removal are available according to the specific characteristics of the virus and the type of biotherapeutic product. Many biotherapeutic processing workflows will use a combination of complementary methods to increase the spectrum of viruses which can be sufficiently eliminated.

Inactivation
Multiple methods of viral inactivation are possible. The most common are:

• Low pH based methods – especially for biotherapeutic products like monoclonal antibodies (mAbs)
• Solvent or surfactant-based methods – usually for polysaccharides

Less frequently used viral inactivation methods include pasteurization, dry heat and vapor heat - in particular for blood or serum based products.

Virus Removal
Well-documented methods of virus removal include precipitation, chromatography and nanofiltration.

The selection of methods to be employed for viral inactivation and removal depends on the size, type, and lability of the biotherapeutic product being prepared, the method(s) of purification which the manufacturer wishes to use, and the nature and titer of the viruses of concern. Drug Substance (DS) structure and function are equally important quality attributes along with viral safety and must be evaluated and maintained throughout. Any viral inactivation method, low pH or surfactant, benefits from precise and simultaneous control of multiple Critical Process Parameters (CPPs); a capability which is required to effectively understand the process itself and the effect on Drug Substance (DS). Chemical synthesis reactors enable mapping of a modern process and consistent operation within a defined parameter space, while also modeling for method transfer and scale-up.

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.

While the principal need for low pH viral inactivation studies in downstream bioprocessing is to define the scope of reagent addition and time needed for the process, characterization of process kinetics and the impact of compounding process parameters is also of importance and ultimately a requirement to ensure design of a viral inactivation process which is both robust and optimized. Yet, temperature monitoring and control is often an overlooked, and therefore, an uncontrolled parameter during process development studies for viral inactivation.

This oversight may be due in part to the use of pilot or even commercial manufacturing-scale systems which perform viral inactivation in hold or transfer vessels which might record but not control temperature. Representativeness of scale, in particular representativeness of mixing is often another parameter easily overlooked in low pH viral inactivation studies such as those performed via manual platforms controlled by magnetic stir plates. Lack of data capture for something as simple as mixing makes it impossible to verify if the assumed conditions of the study were correct and consistent. 

The low pH treatment for virus inactivation unit operation presents a potential risk for in-process product aggregation during downstream processing for a monoclonal antibody. Presented by Hiren D. Ardeshna of GlaxoSmithKline, this presentation discusses a full-factorial experimental design to investigate the effect of four process parameters:

• Low pH Endpoint
• Low pH Hold Time
• Low pH Titration Duration
• Neutralization Titration Duration

Solvent or detergent viral inactivation methods are commonly employed against enveloped viruses. Reagents used generally have negligible impact on the lability of the therapeutic protein or antibodies which are subject to the challenges of denaturation or deamidation possible with some low pH methods. Solvent or detergent treatment methods for viral inactivation have many of the same needs and drivers as low pH methods; principally to define the scope of reagent addition (in this case a solvent or detergent) and time needed for the process. As with low pH viral inactivation, conditions vary between immunoglobulins or other Drug Substance (DS) types. Therefore, studies must be undertaken for each molecule to characterize and validate the design space or the operational boundaries in which effective solvent or detergent viral inactivation can take place. These boundaries and the outcome of a viral inactivation process are similarly defined by Critical Process Parameters (CPPs) including temperature, protein content, solvent or detergent content, time at inactivation conditions, as well as mixing and effectiveness of solvent or detergent homogenization. Identifying and navigating these factors will positively affect product quality and quantity.

While detergent viral inactivation studies do not require the same manner of reagent titration as low pH studies, a design space of critical variables still needs to be evaluated. As with low pH, viral inactivation studies utilizing solvent or detergents traditionally characterize the process entirely manually. Reagent dosing and controls are similarly dependent on the accuracy and precision of highly skilled persons simultaneously performing multiple critical tasks. Solvent or detergent viral inactivation studies are likewise subject to natural variations, inaccuracies and challenges of reproducibility.

Viral inactivation methods relying on solvents or detergents generally require a further point of consideration not typically pertinent to low pH methods. Any added solvents or detergents must be removed from the Drug Substance (DS) or immunoglobulin solution and verified by an appropriate analytical method. Typically, solvents or detergents are removed via either chromatography or buffer exchange via tangential flow filtration. The removal of added solvents or detergents is somewhat analogous to purpose for reverse-pH titration from a low pH inactivation back to within an appropriate physiological or slightly basic range. At scale, buffer exchange or chromatographic methods would likely occur as continuous or semi-continuous unit operations as the material moves from a hold vessel through a column or other appropriate membrane for solvent or buffer exchange. In process development, the solvent or detergent viral inactivation is likely to occur separate and distinct from the following purification or removal step. As such, data or information continuity can be compounded by this practice.

Various vaccine products undergo viral inactivation, including toxoid, recombinant-protein, sub-unit, polysaccharide, and even a select few virus-like particle vaccines. Again, the selection of an appropriate viral inactivation method will consider the nature of the biotherapeutic product, as well as, the breadth of viruses which need to be effectively cleared.

Generally, strict consideration needs to be given to alternative methods or practices which prevent extraneous viral contamination for vaccine types such as live-attenuated viruses – where the biotherapeutic product is a viral particle. Specific methodologies for such products may include one or more nanofiltration or chromatographic processes for effective extraneous viral load reduction in the respective raw material sources. Inactivated or destroyed viruses may still undergo an appropriate low pH or solvent or detergent based viral inactivation process – as this may still maintain the desired immuno-stimulatory effect even if the viral product is denatured or otherwise disrupted.

Viral inactivation methods for oligonucleotide product or molecule types are not widely considered necessary. This is principally due to the nature of reagents, reaction conditions and processing methods which are consistently unfavorable to viruses. At present, many such oligonucleotide products in development and production are primarily purified, concentrated and formulated by various buffer exchanges via tangential flow filtration or by one of the various types of chromatographic separation.

With the need for data continuity and electronic batch records, chemical synthesis reactors function as a force multiplier in viral inactivation processes both by design and execution of experiments, and by facilitation of data capture and integrity. Orthogonal Process Analytical Technology (PAT) bridge multiple applications and workflows, such as viral inactivation with buffer exchange, and more accurately model process flows of large-scale operations.

View a live eDemo from your work or home office on your schedule.

• Press Play and walk away; iC software responds dynamically to conditions within the reactor. Consistently execute operations within the bounds of the viral inactivation protocol.
• Capture data from multiple sensors, including pH, conductivity, oxidative redox potential (ORP) and dissolved oxygen.
• Integrate peripheral units such as pumps or balances for gravimetric mass transfer.

In-situ FTIR and Raman Spectroscopy track critical bioprocess components throughout the upstream, downstream, conjugation and formulation unit operations.

Glucose and key metabolites can be monitored in upstream applications, while in downstream processes protein or vaccine concentration, stabilizers, excipients and impurities can all be monitored and controlled in real time without the delay or cost associated with offline analysis.

This enables fast determination of process endpoints, as well as, clear understanding of the impact of various process parameters on product quality and process efficiency.