Identification of compendial nonionic detergents for the replacement of Triton X‐100 in bioprocessing

Abstract We have systematically investigated six compendial nonionic detergents as potential replacements for Triton ×‐100 in bioprocessing applications. Use of compendial raw materials in cGMP bioprocessing is advantageous for a variety of reasons including material specifications developed to meet stringent pharmaceutical product quality requirements, regulatory familiarity and comfort, and availability from vendors experienced supplying the biopharmaceutical industry. We first examine material properties of the detergents themselves including melting point and viscosity. Process performance and product contact in real‐world bioprocess applications are then investigated. Lastly, we test the detergents in virus inactivation (VI) experiments with recombinant proteins and adeno‐associated virus. Two of the detergents tested, PEG 9 Lauryl Ether and PEG 6 Caprylic/Capric Glycerides, showed favorable properties that make them attractive for use as potential Triton X‐100 replacements. Process performance testing indicated negligible impact of the detergents on product yield, purity, and activity compared to a control with no detergent. Importantly, both PEG 9 Lauryl Ether and PEG 6 Caprylic/Capric Glycerides demonstrated very fast VI kinetics with complete inactivation of XMuLV observed in less than 1 min at a target 1% detergent concentration. Potential advantages and disadvantages of both candidate detergents for use in cGMP bioprocessing are summarized and discussed.

room temperature. Further facilitating its ease of use in bioprocessing, Triton X-100 is available in compendial grades from high quality raw material vendors experienced supplying the pharma industry. Unfortunately, Triton X-100 has been identified as an environmental risk due to potential endocrine-disrupting properties of its degradation products.
This has led to its inclusion on the European Chemicals Agency's (ECHA) "candidate list of substances of very high concern (SVHC) for authorization" under the EU REACH regulation framework. 9 Thus, the industry has been searching for suitable replacements for Triton X-100. [10][11][12][13] Compendial monographs are documents maintained by private non-governmental standard setting bodies such as the United States Pharmacopeia (USP) or the European Pharmacopeia (PhEur). These documents contain chemical and testing information for many pharmaceutical inactive and active ingredients. 14 There are several advantages to use of compendial raw materials in cGMP bioprocessing. First, the specifications developed and reported in compendial monographs are typically designed to meet stringent pharmaceutical product quality requirements. Second, health authorities frequently participate in monograph development and the wide adoption of these standards results in regulatory familiarity and comfort with use of materials meeting them. Lastly, raw material vendors experienced in supplying compendial raw materials for biopharmaceutical manufacturing understand the strict quality and regulatory requirements of the industry, and expectations associated with a demanding quality audit.
In this work, we report the identification of several detergents with available compendial monographs as possible Triton X-100 replacements in cGMP bioprocessing applications. We first evaluate physical properties of the candidate detergents, impact on product quality, and process performance for an example protein biotherapeutic. Virus inactivation (VI) studies with protein biotherapeutics are then presented under a range of realistic bioprocess conditions and compared to Triton X-100 as a control. Lastly, we demonstrate use of one of the detergents for cell lysis and inactivation of an enveloped virus in an adenoassociated virus (AAV) biotherapeutic process intermediate.

| Buffer reagents and detergents
Chemicals used for buffer preparation were obtained from Millipore-

| Viscosity measurements
Absolute detergent viscosities as a function of temperature were determined using an mVROC viscometer and associated control software purchased from Rheosense (San Ramon, CA, USA) and integrated Thermo Cube temperature control unit from Solid State Cooling Systems (Wappingers Falls, NY, USA).

| Turbidity measurements
Turbidity of diluted detergent and protein solutions were measured using an Orion Aquafast turbidity meter from Thermo Scientific. Solutions were gently mixed end-over-end briefly to achieve a homogeneous solution and avoid foaming.
1 ml/min with a mobile phase consisting of 100 mM sodium phosphate, 100 mM sodium sulfate, pH 6.8. Samples (250 μg) were applied to the column neat and the elution profile was monitored at 280 nm using the system spectrophotometer. Aggregate levels were determined as a ratio of peak areas of the early-eluting aggregate peak(s) and the monomer peak.

| Protein bioactivity assay
Protein activity was measured using an in-house cell based assay. Bioactivity was measured relative to a reference standard before and after the treatment with detergent to assess the impact of detergent treatment on the protein activity.

| AAV generation and purification
An AAV6 cell paste slurry was generated using standard cell culture techniques and stored at À80 C. Capto AVB resin was sourced from Cytiva. C0SP filters were obtained from Millipore-Sigma. Sartopore 2    Lastly, it was observed that one of the detergents, G767, showed a marked increase in turbidity when concentrations dropped below 1% (w/w) in water. Figure 2, which shows percent detergent in water versus turbidity at room temperature, illustrates this behavior. As is also shown in Figure 2, it was possible to shift the concentration onset of turbidity to lower concentration by the addition of PS80 in a ratio of 10:1 G767:PS80 avoiding a "cliff effect" where solutions may become turbid near the 1% (w/w) threshold. Increasing ratios of PS80 in G767 solution lowered the concentration at which turbidity was no longer observed, and a 2:1 G767:PS80 ratio completely inhibited turbidity formation in the concentration range evaluated. Preparations of 10% and 1% (w/w) L9 and 10% (w/w) G767 in deionized water were also examined at low-temperature conditions in a 2-8 C refrigerator and remained completely homogeneous without any observable phase separation over a period of approximately 1 month.

| Product contact and process performance
Prolonged detergent contact in HCCF containing BisAb was assessed to understand the potential impact to a realistic biopharmaceutical and MS40 detergents at 2% concentration produced substantial increases in solution turbidity after 1 h of incubation, compared to the control condition which was not spiked with detergent. G767 at 1% concentration also produced distinct increase in turbidity, while a 2% concentration did not form turbidity during the 24-hour incubation.
The turbidity formation observed at lower concentrations of G767 in HCCF followed the behavior outlined in Figure 2, and addition of PS80 similarly prevented the turbidity formation in HCCF containing BisAb. TX100, L9, S20, and P35CO detergents were not found to increase HCCF solution turbidity over a 24 h hold at room temperature.
As displayed in Table 2, prolonged incubation in HCCF did not impact affinity chromatography process performance or BisAb product quality relative to a no detergent control for all detergents evaluated. Affinity chromatography product yields were consistent with control conditions, which were not spiked with detergents, and product quality by SEC-HPLC showed consistent monomer purity. The consistent chromatographic behavior we observed may be expected due to lack of significant non-specific binding of non-ionic detergents to typical agarose-based mAb affinity media, but impact to performance needs to be further evaluated for other separation media.
While the impact of detergent on 0.2 μm filtration throughput and product recovery was not specifically evaluated in this study, the load materials were passed through 0.2 μm polyethersulfone membranes prior to affinity chromatography process performance and product quality studies referenced in Table 2. No obvious impact to filtration or process performance was observed. Additionally, no impact on BisAb bioactivity was observed after prolonged contact with detergents. However, further consideration for MS40 was abandoned due to the relatively high increase in turbidity observed during product contact.

| Virus inactivation studies
A summary of the virus inactivation studies performed with the BisAb and XMuLV as a model enveloped virus is provided in Table 3. Figure 3 shows results of VI kinetics for detergents for which slow or no inactivation was observed. These were MS25, S20, and P35CO, which were not considered further, and PS80, which was evaluated to confirm its lack of significant virus inactivation capability alone. In contrast to this, two detergents showed exceptionally fast inactivation kinetics, these were L9 and G767. Figure 4 shows inactivation curves for L9 and displays the capability of L9 to provide fast inactivation in two distinct protein load materials. L9 achieved complete virus inactivation to below the limit of detection (LOD), represented as greater than 4.0 LRV in BisAb HCCF, and greater than 3.8 LRV in BisFusion HCCF, within 1 min at 1% concentration. It was also tested with the BisFusion protein at 0.1% and similarly achieved virus inactivation to below the LOD within 1 min. These results clearly demonstrate L9 robustly achieves virus inactivation with very fast kinetics. As such, it has high potential to replace Triton X-100 for this bioprocessing application. Figure 5 shows VI studies performed with G767, or G767 and PS80. As is shown in Figure 5, G767 at 1% concentration with no PS80 showed very rapid inactivation kinetics, achieving greater than 4.0 LRV within 1 min. As is also shown in Figure 5, when 0.5% PS80 is added in addition to 1% G767, VI kinetics slow considerably, achieving complete inactivation to below the LOD only after 60 min. This result was unexpected and suggests PS80 is actually protecting the virus in some fashion given G767 was maintained constant at 1% con-  Virus inactivation using L9 detergent was performed in AAV at 1% detergent concentration as shown in Figure 6. As can be seen in the figure and similar to prior observations with recombinant proteins, complete inactivation to below the LOD is achieved within 1 min.

| AAV experiments
Inactivation was also tested at 0.1% detergent concentration with AAV and the results are shown in supplemental material Figure S2. At this concentration 1 of the 2 replicates achieved complete inactivation between 1 and 30 min. Nonetheless, the results indicate L9 is very robust from a VI perspective at the target concentration of 1%, and exhibits fast kinetics even at 1/10 the target concentration. Data provided in Table S1 in the supplemental materials also shows that AAV titer and infectivity is comparable when purified material is spiked with

| CONCLUSIONS AND RECOMMENDATIONS
We have systematically investigated six compendial detergents as potential replacements for Triton X-100 in bioprocessing applications.
Two of them, L9 and G767, were identified as having favorable properties and good potential for this purpose. Both showed very fast virus inactivation kinetics, and L9 was also shown to be suitable for cell lysis in an AAV application. The other four detergents investigated in this study were excluded from further consideration due to slow VI kinetics as this was a critical requirement for our applications.
Both L9 and G767 have advantages and disadvantages, which are summarized in Table 5. Overall, we would give a minor advantage to L9, provided the necessary equipment is available either to gently warm the pure detergent slightly above room temperature to facilitate handling in a liquid state, or thoroughly mix and pump the viscous semi-solid consistency observed at room temperature. While we were unable to obtain a viscosity measurement for L9 below 25 C using the Rheosense instrument, suggesting the viscosity is above 100,000 cP, in our lab it was nonetheless possible to effectively transfer it below this temperature using a conventional peristaltic pump (see supplemental Video S1 shot at 22 C for additional details). For room temperature handling of pure L9, we would recommend a heavy duty mixer with a high torque motor and high viscosity impeller such as an anchor or VISCO JET be used to first homogenize the material as it appears to separate into regions of low and high density on standing. To pump L9 at room temperature following thorough mixing, a heavy-duty positive displacement pump suitable for very high viscosities such as a peristaltic or progressive cavity pump is recommended.

ACKNOWLEDGMENTS
We thank Weimin Chen and Diana Zhang-Hulsey for sample analysis.
Dominique WuDunn assisted with preparation of the supplemental video. We owe a special thanks to William Wang for his support and review of this manuscript.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.