Exploring Viral Clearance for AAV Products: A Study of Detergent Lysis and Chromatography StepsExploring Viral Clearance for AAV Products: A Study of Detergent Lysis and Chromatography Steps

In 1999, the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) introduced the ICH Q5A(R1) guideline, which covers the viral safety of biotechnology products derived from cell lines of human and/or animal origins. The recommendations focus on biologics derived from in vitro cell cultures such as interferons, monoclonal antibodies (mAbs), and other recombinant-DNA–derived therapeutics. Recommendations for inactivated vaccines — live vaccines containing self-replicating agents — and genetically engineered live vectors were excluded from the document’s scope (1). Over the subsequent decades, the number of platforms using viral vectors to deliver gene therapies for treating a range of clinical indications has multiplied. A few examples of approved and clinical-stage viral-based therapies include Gendicine (recombinant human p53 adenovirus) for non–small-cell lung cancer, Roctavian (valoctocogene roxaparvovec) for hemophilia A, Zolgensma (onasemnogene abeparvovec) for spinal muscular atrophy, and Luxturna (voretigene neparvovec) for retinal dystrophy. The latter three products all use adenoassociated-virus (AAV) delivery systems: serotypes 5, 9, and 2, respectively. Viral vectors also have been used for some COVID-19 vaccines, such as AstraZeneca’s ChAdOx1 nCoV-19 and Ad26.COV2.S vaccines (2–5).

Figure 1: Process steps of the InAAVate viral-safety and manufacturing platform; HEK293 = human embryonic kidney 293, SU = single use.

Figure 1: Process steps of the InAAVate viral-safety and manufacturing platform; HEK293 = human embryonic kidney 293, SU = single use.

Made effective in June 2024, ICH Q5A(R2) clarifies viral-safety guidelines for genetically engineered viral vectors by adding a section devoted to viral-vector–derived products. The guideline calls for comprehensive virus testing and assessments of virus removal and inactivation during biomanufacturing. Risk-based approaches should be followed to demonstrate viral safety of final products, and associated viral safety assessments should be part of the initial process development through investigational new drug (IND) and subsequent marketing applications (6).

Viral-risk mitigation in biomanufacturing relies on three pillars: prevention, detection, and removal. Developers can prevent adventitious viruses from entering viral-vector manufacturing processes through careful selection and testing of raw materials, such as cell lines and media. Physical barriers and closed systems also reduce risks of viral contamination. Testing at different process steps for viral contaminants is vital to ensuring the safety of final products. The type and extent of such tests vary depending on the production mode of a given viral vector and initial risk assessment but should include, at minimum, characterization of a master and working cell bank (MCB, WCB), virus stocks, bulk harvest, and drug substance (DS). The most important risk-mitigation tool is the cumulative viral-clearance capability of upstream inactivation kinetics and the downstream purification process, as determined by viral-clearance studies (6–12).

The Viral-Safety Platform

Figure 1 shows an overview of the Oxford Biomedica (OXB) InAAVate platform and its viral-control elements. The platform uses a transient transfection production system with human embryonic kidney 293 (HEK293) cells and helper plasmids (13). Control strategies address the prevention of viral entry through cell lines and raw-material control, as well as viral testing.

Cell-Bank Viral Safety Testing: The teams performed several adventitious-virus tests on samples from a HEK293 cell bank. In vitro tests were performed in HEK293, Vero, and MRC-5 cell lines; in vivo assays used adult mice and embryonated hen eggs. Samples were screened for standard pathogens such as hepatitis viruses A, B, and C; human immunodeficiency virus 1 and 2; (HIV-1, HIV-2); human T-lymphotropic viruses 1 and 2 (HTLV-1, HTLV-2); and bovine and porcine viral agents. Test results were documented as part of the HEK293 MCB specification. Release depended on confirmation that materials were free of viral contaminants.

Raw-Material Screen: Transfection plasmids were grown in Escherichia coli and tested for mycoplasma contamination. Media used for growth and transfection of suspension-adapted HEK293 cells were chemically defined and free of proteins and sera.

Plasmid-Based Transient Transfection: Drug developers use different methods to produce recombinant AAV (rAAV) vectors; such vectors bring unique considerations regarding initial viral-risk assessments (14–16). For example, AAV processes that use stable producer cell lines with adenovirus 5 (Ad 5) will be required to show clearance of those helper viruses (17, 18). Production platforms that use transient plasmid-transfection systems without helper viruses involve less risk but still must be assessed for clearance with model viruses that represent a breadth of relevant biophysical traits, such as size, genome type, and envelope (7, 8, 19–21).

Operational Controls: Single-use closed systems throughout manufacturing provide barriers to lessen viral-contamination risks by reducing batch-to-batch virus carryover from inadequate cleaning. Closed systems — such as single-use mixers, manifolded filling assemblies, and molded sample collection — provide physical barriers against the entry of viral contaminants.

In-Process Testing and Release Specification: Routine bioburden samples were taken from preinoculation media, postinoculation cell cultures, posttransfection, prelysis, postclarification, affinity capture, anion-exchange (AEX) polishing and drug-substance steps. Prelysis and preharvest samples were taken from bioreactors and assayed for mycoplasma and adventitious agents. The DS release panel tested for residual host-cell DNA (hcDNA), plasmid DNA (pDNA), helper-plasmid DNA (hpDNA), and adventitious agents.

Viral-Clearance Study

OXB partnered with Texcell North America to evaluate the InAAVate platform’s ability to remove and/or inactivate two model viruses (Table 1). The team assessed three unit operations and spiked either xenotropic murine leukemia virus (XMuLV) or minute virus of mice (MVM) into load material at the start of each process step, then quantified remaining amounts. For inactivation, XMuLV was spiked into prelysed culture harvest followed by treatment with a detergent-based lysis buffer. For capture, MVM was spiked into an affinity-chromatography load. MVM also was spiked into the load of an AEX-chromatography process, which served as a polishing step. All load material used for spiking came from aliquots of process intermediates generated from a 50-L pilot-scale run.

Table 1: Model viruses used in the viral-clearance study.

Table 1: Model viruses used in the viral-clearance study.

XMuLV was chosen as a model RNA virus for several reasons: It is larger than AAV and, as an enveloped virus, is more susceptible to inactivation by commonly used cell-lysis detergents (e.g., Tween 20, Triton X-100, and Deviron C16 products). MVM was chosen because it belongs to the same family as AAV and thus shares similar physicochemical characteristics, such as resistance to detergent treatment. And because MVM’s size is similar than that of AAV, use of small-pore retentive viral filtration could result in product-recovery issues.

Materials and Methods

Generation of AAV9 Material: AAV9 process intermediates used in our study were produced in a 50-L bioreactor and purified using OXB’s InAAVate platform for that serotype. Scaled-down versions of each production-scale unit operation were used during the viral-clearance study.

Chromatography Resins: Prepacked Repligen columns were used for both chromatography steps in this study, along with POROS CaptureSelect AAV9 resin for the affinity capture and POROS 50 HQ resin for the AEX polishing unit operation (Thermo Fisher Scientific).

In a preliminary evaluation of test materials, we determined potential effects of viral-clearance samples on the test systems used to quantify the model viruses. Toxicity, interference, and hold-control testing assessed effects of process materials on infectivity assays; potential effects to test systems were mitigated by sample dilution as necessary. For virus inactivation by detergent treatment, we performed an additional evaluation (threshold of inactivation). The threshold of inactivation enabled us to determine the dilution of detergent-treated material required to terminate virus inactivation.

Experimental Design: Centerpoint conditions were selected as operating parameters to help us understand the viral-clearance capability of the InAAVate platform. For chromatography steps, our centerpoint settings included column:load ratios, residence times, elution pH, and conductivities, all of which can affect viral clearance. Although the use of worst-case conditions on operating parameters was not in the scope of this study, note that both chromatography steps were run at cycle 2 (cycle 1 being a control run with an AAV load without a virus spike).

Virus Quantitation: Texcell personnel used validated infectivity assays to quantify virus titers for XMuLV and MVM. Virus titers of samples spiked with XMuLV were determined with a plaque-forming infectivity assay based on direct quantitation of plaque-forming units (PFUs). MVM process samples were evaluated using a 50% tissue culture infectious dose (TCID₅₀) assay and determined in a quantal assay, in which measured virus titers were based on detection of infected cells by observation of cytopathic effects. Log-reduction values (LRVs) were calculated using one of the following equations:

LRV = (lesser of log₁₀ total virus in load and hold control) – (log₁₀ total virus in timepoint sample)

LRV = (lesser of log₁₀ total virus in load and hold control) – (log₁₀ total virus in product sample)

Detergent Treatment: Cell cultures were spiked with XMuLV and treated with lysis-buffer–containing detergent to test enveloped-virus inactivation. A spiked-culture sample was assayed to determine virus titer before detergent treatment. An aliquot of the load was held for the duration of the inactivation process and assayed. The remaining volume of the spiked test article was treated with lysis-buffer–containing detergent and held at 37.0 ± 1.0 °C for 120 minutes. Samples of the spiked inactivation load were taken at three timepoints to demonstrate inactivation kinetics over time.

Affinity and AEX Chromatography: Load materials for affinity and AEX columns were spiked with MVM to evaluate nonenveloped-virus removal during each chromatography step. Load samples were assayed to determine virus titer at the start of chromatography. An aliquot of the load, designated as the hold control, was held for the duration of the chromatography process and assayed for virus titer. Affinity and AEX eluates also were sampled and assayed to determine virus titers at the end of each chromatography step. No process intermediates (e.g., flow-through, wash, or strip) were analyzed for virus removal.

Results and Discussion

Among the three strategies for mitigating viral risk, virus removal has the most significant role in reducing potential viral contamination of final therapeutic products. A viral-clearance capability demonstration evaluates the effectiveness of upstream and downstream unit operations to inactivate or remove spiked-in model viruses. Traditional approaches used for mAbs and other recombinant proteins — e.g., heat application, low-pH hold, and detergent exposure — have minimal effects on nonenveloped viral vectors that share characteristics with AAVs. Small-pore (20-nm) viral filters used in antibody production processes remove parvoviruses but also target AAVs and therefore decrease yield. Large-pore (35–70-nm) viral filters can remove larger viruses while allowing AAVs to pass but cannot filter small adventitious agents. Thus, adding chromatography for surface-charge modulation to separate AAVs from similar-sized adventitious viral agents could be the key to demonstrating robust viral clearance (11, 22, 23). Bsed on data from previous experiments, we designed our study to assess the effects of detergent treatment and inactivation kinetics (for XMuLV) with cumulative chromatography clearance (for MVM).

Detergent treatment with lysis buffer resulted in an LRV of >4.67 log₁₀ for XMuLV with near-immediate inactivation. Along with calculated LRVs, Figure 2 shows log₁₀ values of total virus present in samples as a function of time. In under one minute and throughout the time course investigated in the study, virus titer in the treated sample was <1.50 PFU/mL (assay detection limit). That corresponded to <2.48 log₁₀ of total virus for XMuLV and indicated rapid viral inactivation with an LRV of >4.67 log₁₀ that persisted over the two-hour timeframe.

Figure 2: Xenotropic murine leukemia virus (XMuLV) inactivation by detergent treatment; LRV = log-reduction value.

Figure 2: Xenotropic murine leukemia virus (XMuLV) inactivation by detergent treatment; LRV = log-reduction value.

Chromatographic Clearance: Figure 3 illustrates the log₁₀ value of total virus present in the affinity load and product samples and LRV obtained for the affinity unit operation. Affinity chromatography was an effective virus-removal step for MVM as demonstrated by an LRV of 4.6 log₁₀ using manufacturing centerpoint conditions. We did not expect the resin beads to bind to MVM due to their ligands’ high selectivity to AAV9 capsids. Because MVM is resistant to detergent inactivation, it is important to demonstrate effective removal through affinity-based chromatography (23, 24). The percentage of spiked MVM particles present in the affinity product compared with the spiked affinity load was negligible (Table 2).

Figure 3: Minute virus of mice (MVM) removal by affinity chromatography; LRV = log-reduction value.

Figure 3: Minute virus of mice (MVM) removal by affinity chromatography; LRV = log-reduction value.

^{Table 2: Percentage of minute virus of mice (MVM) in affinity product compared with spiked affinity load;}

^TCID50 ^{= 50% tissue culture infectious dose.}

Figure 4 compares elution peaks from the blank and spike runs, showing no extra peaks or substantial differences in chromatographic profiles. Elution and strip peak areas were comparable, as A260:A280 ratios for blank and spiked runs. Such results accord with previous studies. For instance, Winkler et al. observed that model viruses (MVM and XMuLV specifically) showed no affinity toward resin base beads or functionalized ligands (POROS CaptureSelect AAVX); the ability of AAV serotypes to bind to the functionalized ligand was uncompromised in the presence of model viruses, and the majority of MVM particles were present in affinity capture flow-through (25).

Figure 4: Affinity elution chromatogram comparison with and without minute virus of mice (MVM) spike.

Figure 4: Affinity elution chromatogram comparison with and without minute virus of mice (MVM) spike.

Figure 5 shows the log₁₀ value of total virus present in the AEX load and product samples, with the LRV for the AEX step. AEX chromatography was also an effective virus removal step for MVM, as demonstrated by an LRV of 4.38 log₁₀ at manufacturing centerpoint conditions. That bind–elute AEX chromatography step provided DNA-containing capsid enrichment by exploiting small charge differences between empty and full AAV capsids. The resolution between capsid populations partially depends on the concentration of process-related impurities in load materials. An LRV of >4 log₁₀ for MVM, without modifications to platform process parameters, demonstrates a lack of interference on resin performance from MVM at the tested concentration. In the affinity step, the percentage of spiked MVM particles present in the AEX product compared with the spiked AEX load was negligible (Table 3).

Figure 5: Minute virus of mice (MVM) removal by anion-exchange (AEX) chromatography; LRV = log-reduction value.

Figure 5: Minute virus of mice (MVM) removal by anion-exchange (AEX) chromatography; LRV = log-reduction value.

^{Table 3: Percentage of minute virus of mice (MVM) in anion-exchange (AEX) eluant compared with spiked AEX load;}

^TCID50 ^{= 50% tissue culture infectious dose}

Figure 6 overlays the blank and spiked AEX elution peaks. As with the affinity step, no extra peaks or differences in chromatographic profiles were observed. The AEX polishing step’s ability to remove MVM may be tied to the specific pH levels and conductivities used to target full AAV9 capsids based on charge profiles. Platform conditions for the separation of empty and full capsids did not coelute the majority of MVM particles despite the similar physicochemical properties of the two viruses. Those observations support findings published by Winkler et al., in which the majority of MVM particles were present in the AEX strip (CIMmultus QA column, Sartorius) (25).

Figure 6: Anion-exchange (AEX) elution chromatogram comparison with and without minute virus of mice (MVM) spike.

Figure 6: Anion-exchange (AEX) elution chromatogram comparison with and without minute virus of mice (MVM) spike.

Evolving Platform and Future Regulatory Outlook

Table 4 lists viral-clearance values obtained with the InAAVate platform using virus inactivation and removal. Typically, LRVs below 1 log₁₀ fall within assay variability and therefore are considered negligible with no contribution to overall clearance. LRVs of 1–3 log₁₀ are considered supportive steps and contribute acceptably, but LRVs >4 log₁₀ demonstrate robust viral clearance across the particular unit operation being evaluated (26, 27).

Table 4: Total log-reduction values (LRVs) for nonenveloped xenotropic murine leukemia virus (XMuLV) and enveloped minute virus of mice (MVM); NA = not applicable.

Table 4: Total log-reduction values (LRVs) for nonenveloped xenotropic murine leukemia virus (XMuLV) and enveloped minute virus of mice (MVM); NA = not applicable.

Virus inactivation by detergent treatment was shown to be effective, as evidenced by an LRV of >4.67 log₁₀ for XMuLV. An overall reduction factor of 9.07 log₁₀ of MVM for the manufacturing process highlights the effectiveness of our platform at manufacturing centerpoint conditions for an AAV9 serotype. A similar LRV of >4 log₁₀ was obtained for each chromatographic step in the Winker et al. study for the MVM model virus at platform centerpoint conditions across POROS CaptureSelect AAVX affinity resin and CIMmultus QA monolith AEX column for an AAV8 serotype (25). OXB’s platform control strategy, viral-clearance data, and overall viral-risk assessment highlight a built-in viral safety factor greater than the industry-accepted target of 6 log₁₀ LRV for processes without endogenous viruses (24).

Acknowledgments

We thank everyone who assisted with this study. Guang Yang, Iraj Ghazi, Qi Zhang, and Richard Gilmore contributed through planning and material generation. Ashish Sharma, Luke Mustich, Thomas Thiers, Bill Kerns, and Akunna Iheanacho contributed to the manuscript.

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Corresponding author Ashish Sharma is gene therapy and biologics process development leader; Luke Mustich is a senior research associate; Thomas Thiers, Guang Yang, PhD, and Iraj Ghazi, PhD, are scientists; Qi Zhang is an upstream scientist; and Richard Gilmore is a senior scientist in upstream process development, all at Oxford Biomedica, LLC, One Patriots Park, Bedford, MA 01730. Bill Kerns is a study director, and Akunna Iheanacho, PhD, is chief operating officer, both at Texcell North America, Inc., 4998 International Boulevard, Frederick, MD 21703.