Compositions and methods for inactivating enveloped viruses during the manufacture of biologics

Abstract

The present invention provides a method of inactivating enveloped viruses during the production of a biologic product without harming the biologic product. The method comprises contacting cells containing the biologic product with at least one purified non-ionic detergent, wherein the detergent(s) lyses the cells to release the biologic product, thereby producing a lysate, and wherein the detergent(s) inactivates any enveloped viruses within the lysate. The detergent can be an ethoxylated alcohol, sorbiton ethoxylate, triblock copolymer and combinations thereof. Examples of biologic products include non-enveloped viruses and therapeutic recombinant proteins produced via lysis of mammalian or insect cells.

Description

COMPOSITIONS AND METHODS FOR INACTIVATING ENVELOPED VIRUSES DURING THE MANUFACTURE OF BIOLOGICSBACKGROUND OF THE INVENTION

[0001] Viral contamination poses a significant risk to biologies produced from mammalian cell culture or animal-derived sources8. Viruses have the potential to infiltrate the manufacturing process through multiple avenues, such as raw materials, contaminated cell lines, or failures in good manufacturing practices (GMP). Consequently, regulatory standards mandate the demonstration of sufficient viral clearance through the manufacturing process steps to mitigate this risk ’.

[0002] It is essential to show that the steps in biomanufacturing are effective in removing any potential viral contaminants. Depending on factors such as the product being made, the manufacturing process, and the types of viruses involved, achieving the desired level of viral clearance may involve employing a combination of methods, including two distinct approaches: viral inactivation (VI) and filtration4. Among the different techniques available, low pH and detergent-mediated viral inactivation (DMVI) methods have been commonly utilized in the production processes of therapeutic proteins, plasma-derived products, and adeno-associated virus (AAV)3’5’6.

[0003] However, there has not been any methods demonstrating virus clearance capability of a detergent in the presence of cell debris, cell culture media, or other intracellular components. That is, prior art processes do not include virus inactivation during cell lysis steps since the presence of cell culture media and cell debris impact viral inactivation efficiency. Also, the standard detergent can interact with cell culture media components, proteins and lipids present in the freestream thereby making processing difficult.

[0004] Thus, there is a need for methods by which to inactivate enveloped viruses even in the presence of cell debris and cell culture media while causing no harm to non-enveloped product viruses.SUMMARY OF THE PRESENT INVENTION

[0005] Ln one aspect, the present invention provides methods of inactivating enveloped viruses during the production of a biologic product. The method comprises contacting cells containing the biologic product with at least one purified non-ionic detergent, wherein the detergent(s) lyses the cells to release the biologic product, thereby producing a lysate, and wherein the detergent(s) inactivates any enveloped viruses within the lysate. The biologic product is not impacted by the detergents ). In one embodiment, the biologic product is a therapeutic recombinant protein.

[0006] In another aspect, the present invention provides methods of inactivating enveloped viruses during the production of a non-enveloped virus. The method comprises contacting cells containing the non-enveloped virus with at least one purified non-ionic detergent, wherein the detergent(s) lyses the cells to release the non-enveloped virus, thereby producing a lysate, and wherein the detergent(s) inactivates any enveloped viruses within the lysate. Non-enveloped viruses are not impacted by the detergent(s). In one embodiment, the non-enveloped virus is adeno-associated virus.

[0007] Examples of the detergents includes ethoxylated alcohol, sorbiton ethoxylate, triblock copolymer and combinations thereof. In some embodiments, the ethoxylated alcohol is TDA9, tergitol 15-S-9, TDA6 or combinations thereof. In some embodiments, the sorbiton ethoxylate is polysorbate 20, polysorbate 80, polysorbate 60 or combinations thereof. In some embodiments, the triblock copolymer is poloxamer 188, poloxamer 407, poloxamer 305, or combinations thereof. In some embodiments, the detergent is in a concentration range from about 0.01% (w / v) to about 5.0% (w / v). In one embodiment, the non-ionic detergent is in a concentration of about 0.5% (w / v). In one embodiment, the non-ionic detergent is TDA9 in a concentration of about 0.1% (w / v) and polysorbate 20 in a concentration of about 0.4% (w / v). In one embodiment, the non-ionic detergent comprises 0.1% TDA9, 0.40% Polysorbate 20, and 0.01% Poloxamer 188. In some embodiments, the detergent(s) further comprises a buffer including Tris, phosphate, histidine, citrate, acetate, and combinations thereof. In some embodiments, the biological product is exposed to the detergent(s) for about 1 minute to about 120 minutes at 2°C to 40°C. In some embodiments, the method takes place in the presence of cell debris, cell culture media,other intracellular components and other contaminants. In one embodiment, the cells are mammalian or insect cells. In one embodiment, the cells are HEK293 cells. In one embodiment, the detergent(s) is directly added to the cells in cell culture media or to a cell suspension.BRIEF DESCRIPTION OF THE FIGURES

[0008] FIG. 1 is a graph showing a Comparison of Virus Killing Kinetics for XMuLV with Detergent Compositions A and B. Composition A: 0.1% TDA9, 0.40 Polysorbate 20, 0.01 % Poloxamer 188 in crude cell lysate. Composition B: 1% TDA9, 4% Polysorbate 20, 0.1% Poloxamer 188 in crude cell lysate. The <1 , 30, and 60 min timepoint samples were analyzed by standard testing, and the 120 min one was analyzed by large volume platting assay.

[0009] FIG. 2 is a graph showing a Comparison of Virus Killing Kinetics for XMuLV with Detergent Compositions B and C. Composition B: 1% TDA9, 4% Polysorbate 20, 0.1% Poloxamer 188 in crude cell lysate. Composition C: 1% TDA9, 4% Polysorbate 20, 0.1% Poloxamer 188 in PBS. The <1, 30, and 60 min timepoint samples were analyzed by standard testing, and the 120 min one was analyzed by large volume platting assay.

[0010] FIG. 3 is a graph displays the comparison of Log Reduction Value (LRV) data for all three compositions. Samples taken at <1 , 30, and 60 minutes were analyzed using standard testing methods, while the 120-minute sample was assessed using a large volume plating assay. Results indicate that increasing detergent quantity results in immediate viral inactivation by rapidly disrupting viral structures, solubilizing their components, or denaturing essential viral proteins. However, beyond a certain incubation period, such as 30 minutes, no further impact of detergent concentration on viral inactivation is observed. Additionally, a matrix effect is noted, likely attributed to assay sensitivity and the lower limit of detection, contributing to variations in LRV.DETAILED DESCRIPTION OF THE PRESENT INVENTION

[0011] The present invention pertains to methods of inactivating enveloped viruses during the production of biologic products (i.e., biologies). In one aspect, the methods are utilized in the presence of cellular debris and / or other contaminants typically existing during the manufacture of biologies. Examples of biologies include non-enveloped viruses and therapeutic recombinant proteins (e.g., cytokines, growth factors, hormones), produced through the lysis of mammalian or insect cells.

[0012] In one embodiment, the method of inactivating enveloped viruses is used for the production of a non-enveloped virus biologic. The enveloped virus can be present in various process intermediates, such as, for example, cell culture media, crude cell lysate, harvested cell culture fluid and purification fractions. The method comprises contacting an enveloped virus with at least one detergent composition (i.e., ‘"detergent”) of the present invention, whereby the enveloped virus is inactivated. The detergent(s) can be directly added to the cells in cell culture media or to a cell suspension. The detergent(s) lyses the cells to release the non-enveloped virus, thereby producing a lysate. The detergent(s) inactivate any enveloped viruses within the lysate. The non-enveloped virus product is not impacted (i.e., not harmed) by the detergent(s). Examples of non-enveloped viruses include Adeno-associated Virus (AAV), Adenoviruses, Reovirus (i.e.. Sedoreoviridae) and Parvovirus.

[0013] For example, in the production of AAV vectors, a suitable host cell line, e.g., mammalian or insect cells (e.g., HEK293, SF9), is cultured. The host cells are then transfected with the plasmids of the AAV using a method such as calcium phosphate precipitation, lipofection, or electroporation. This transfection introduces the necessary genetic material into the host cells. Following transfection, the host cells begin to produce and assemble AAV particles. After a period of incubation (typically 48-72 hours), the cells and medium are collected. In prior art methods, the AAV particles are released by lysing the cells using methods like freeze-thaw cycles, enzymatic digestion, or sonication. The crude lysate containing AAV particles, cellular debris, and other contaminants is subjected to several purification steps. These steps may include: Centrifugation (to remove large cell debris); Density Gradient Centrifugation (e.g., using iodixanolor cesium chloride gradients to separate AAV particles based on density); Chromatography (e.g., affinity, ion exchange, or size-exclusion chromatography to further purify the AAV particles); Concentration and Buffer Exchange (i.e., the purified AAV particles are concentrated and often subjected to buffer exchange using methods such as ultrafiltration or dialysis).

[0014] However, in the present methods, the non-enveloped viruses (e.g., AAV particles) are released by lysing the cells by contacting the cells with the detergent(s) of the present invention. These detergent(s), in addition to lysing the cells to release the AAV particles, also inactivate any enveloped viruses without harming the AAV particles. In one embodiment, the cells are exposed to the detergent(s) for about 1 minute to about 120 minutes at 2°C to 40°C.

[0015] Examples of debris include cellular debris, cell culture media, and other contaminants and impurities. Cellular debris refers to the fragments and remnants of cells that remain after the cells have been lysed. Examples of cellular debris include cell membrane fragments (i.e., pieces of the phospholipid bilayer); organelles (i.e., intact or fragmented organelles, e.g., mitochondria, endoplasmic reticulum, Golgi apparatus, and lysosomes); cytoskeletal elements ( i.e.. remnants of the cell’s structural framework, e.g., actin, tubulin, and intermediate filaments); protein aggregates(i.e.. clumps of denatured or aggregated proteins); DNA and RNA fragments; and other cellular components: (i.e., various other macromolecules and small molecules that were part of the cell's cytoplasm or nucleus). Cell culture media can contain impurities such as, e.g., bacteria, viruses (e.g., enveloped viruses), antibiotics, plastics, heavy metals, serum proteins, reagents, dust, and organic compounds.

[0016] The non-ionic detergents of the present invention demonstrate high efficacy in inactivating enveloped viruses found in fluids, while not impacting non-enveloped viruses. Herein “fluid” refers to cell culture media, cell lysate, harvested cell culture fluid, filtrate, eluate, pool or process intermediates. In one embodiment, fluid refers to unclarified cell lysate. In one embodiment, fluid refers to media, water or any buffer matrix used in the manufacturing process of biologies obtained from living organism, such as non-enveloped viruses (e.g., AAV).

[0017] The detergents of the present invention include ethoxylated alcohol, sorbiton ethoxylate, triblock copolymers and combinations thereof. In one embodiment, the detergent is added to the cell lysate or cell culture in a concentration range from about 0.01% (w / v) to about 5.0% (w / v), more typically from about 0.5% (w / v) to about 3.0% (w / v).

[0018] Examples of suitable detergents include alcohol ethoxylates (e.g., TDA-9, TDA6, Tergitol 15-S-9), sorbiton ethoxylate (e.g.. Polysorbate 20, Polysorbate 60, Polysorbate 80), and triblock copolymer (e.g., Poloxamer 188, Poloxamer 407 and Poloxamer 305). The non-ionic detergents of the present invention possess surface activity characteristics crucial for damaging enveloped viral membranes, while not impacting non-enveloped viruses. Additionally, these detergents do not impact further processing of biologic products typically due to precipitations and flitrations.

[0019] In one embodiment, the non-ionic detergent is TDA9 in a concentration of about 0.08% (w / v) to about 0.3% (w / v) and polysorbate 20 in a concentration of about 0.2% (w / v) to about 0.5% (w / v), more typically, about 0.1% (w / v) of TDA9 and about 0.4% (w / v) of polysorbate 20.

[0020] In this specification, the terms "virus inactivation,” "viral inactivation,” “VI”, "killing virus," "inactivating virus," or analogous expressions denote the process of making a virus incapable of infecting cells, reproducing, spreading, or inducing disease.

[0021] Enveloped and non-enveloped viruses are differentiated primarily by their structural characteristics. Enveloped viruses have an outer lipid bilayer membrane, known as the viral envelope, which surrounds their protein capsid9. This envelope contains viral proteins and glycoproteins essential for the virus's ability to infect host cells10. The compositions of the present invention inactivate enveloped viruses by disrupting the lipid envelope. Enveloped viruses encompass a broad range of viral familiesl t,!2, including Retroviridae, Herpesviridae, Coronaviridae, Paramyxoviridae, Flaviviridae, Poxviridae, Filoviridae, Hepadnaviridae, Togaviridae, Orthomyxoviridae, Deltavirus, Paramyxoviridae, Rhabdoviridae, and Bunyaviridae.

[0022] Adeno-associated virus (AAV) is a small, non-enveloped virus categorized within the Parvoviridae family14. Its widespread adoption in gene therapy stems from its capacity to effectivelytransport genetic material to specific cells without eliciting substantial immune reactions or adverse health effects15 16, hr AAV manufacturing processes, viral inactivation measures are integrated to uphold the safety and effectiveness of the eventual product.

[0023] Conducting cell lysis and viral inactivation simultaneously pursuant to the present invention enhances productivity and product stability, saves operational time, reduces space requirements and improves overall process economics. In one embodiment, the detergent composition comprises about 0.08% to about 0.3% TDA9, about 0.2% to about 0.6% Polysorbate 20, and about 0.009% to about 0.03% Poloxamer 188. In one embodiment, the detergent composition comprises about 0.1 % TDA9, about 0.4% Polysorbate 20, and about 0.01% Poloxamer 188. HEK293 cells underw ent lysis using this detergent composition. Subsequently, the viral inactivation study was conducted within the resulting unclarified cell lysate to assess the detergent-treated cell lysate's ability to inactivate viruses.

[0024] The influence of the matrix on viral inactivation by the detergent(s) can be a critical factor to consider across diverse applications. The term "matrix" refers to the surrounding environment or medium in which the detergent and virus interact. This matrix can profoundly impact the efficacy of viral inactivation owing to its chemical composition, physical characteristics, and potential interactions with the detergent7. Various matrices, including biological fluids, cell lysates, or environmental samples, may harbor different proteins, lipids, cell debris, salts, and other constituents that can influence the behavior of both the detergent and the virus. For instance, proteins and lipids within the matrix might engage w'ith the detergent, modifying its capacity to disrupt viral membranes or impede viral proteins essential for infectivity'. Furthermore, salts or other ions present in the matrix could also influence. Understanding the impact of the matrix on viral inactivation induced by' detergent is essential for refining viral inactivation protocols across diverse applications, encompassing vaccine manufacturing, viral elimination in biopharmaceuticals, and disinfection protocols.

[0025] In some embodiments, the detergent composition, comprising of 1% TDA9, 4% Polysorbate 20, and 0.1% in unclarified HEK293 cell lysate or phosphate -buffered saline (PBS) or other matrices, with pH levels ranging from 3.0 to 9.0 and conductivity ranging from 0 to 200 mS / cm, is effective for virus inactivation.

[0026] Ethoxylated alcohols represent a category' of compounds produced through the chemical process known as ethoxylation, in which ethylene oxide molecules are introduced to alcohol molecules. This process yields ethoxylated compounds that possess both hydrophilic (waterattracting) and hydrophobic ( water-repelling) properties, making them versatile surfactants employed in various applications2^22. Widely utilized across industries including personal care products, detergents, cleaners, agricultural formulations, and pharmaceuticals, ethoxylate surfactants constitute the largest group of nonionic surfactants.

[0027] TDA-9, derived from isotridecyl alcohol and ethoxylated to an average of nine moles of ethylene oxide, is a biodegradable nonionic detergent. It exhibits rapid wetting properties, relatively minimal foaming, and effective detergency, serving as a versatile emulsifier, dispersant, and solubilizer. The removal of ethylene oxide from TDA-9 is essential due to the hazardous nature of ethylene oxide, known for its carcinogenic properties, necessitating extra precautions during handling. Additionally. TDA-9 acts as an environmentally friendly detergent, aligning with the Organization for Economic Co-operation and Development (OECD) guideline 301F for materials that are readily biodegradable2’. Its acute toxicity falls under category 2, eliminating the requirement for a warning label, with LC50 and EC50 values surpassing 1.00 and less than 100, respectively.

[0028] Poloxamers represent non-ionic tri-block copolymers comprising a hydrophobic core of polyoxypropylene (poly(propylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)). Poloxamer protects AAV by acting as a cell membrane stabilizer and facilitating membrane resealing. This property helps prevent the degradation of viral vectors induced by shear stress during processes such as cell lysis, viral inactivation, and purification. Additionally, Poloxamer’s hydrophilic and hydrophobic properties enable it to shield AAV particles from damage, preserving their integrity and functionality. The impact of Poloxamer on the viral inactivation solution's ability to lyse cells was examined within a pH range of 3.0 to 9.0. Remarkably, the inventors of this study observed that the inclusion of Poloxamer in the viral inactivation solution did not have an adverse effect on viral inactivation.

[0029] Polysorbate 20, a nonionic surfactant, significantly contributes to viral inactivation processes through various mechanisms. It disrupts lipid-based viral envelopes, leading to their destabilization and rupture, thus rendering the virus inactive. Additionally, Polysorbate 20 enhances the action of other detergent agents, improving the solubility and dispersal of viral particles in inactivation solutions. Acting as a stabilizing agent, it prevents protein aggregation during viral inactivation, ensuring that viral particles remain folly exposed to inactivating agents. Moreover, Polysorbate 20 reduces the surface tension of viral inactivation solutions, facilitating their uniform coverage of surfaces and enhancing penetration into hard-to-reach areas. Overall, the inclusion of Polysorbate 20 in viral inactivation processes enhances treatment efficacy by disrupting viral envelopes, boosting detergent action, preventing protein aggregation, and minimizing surface tension, ultimately leading to viral particle inactivation.

[0030] The detergents employed in this invention are notably pure and devoid of product-related impurities such as polyethylene glycol and free ethylene oxide, which could potentially impact the desired product adversely. The manufacturing process provides composition and impurity profde consistency per product specifications required for biopharmaceutical manufacturing processes. The detergents utilized herein exhibit low levels of both microbial contaminants and ethylene oxide. The reduction in microbial contamination during tire purification step of TDA-9 lessens tire burden on downstream processing to eliminate impurities like polyethylene glycol and free ethylene oxide during purification steps such as chromatography.

[0031] In one embodiment, a concentrated viral inactivation solution stock can be used for dilution to achieve the desired final concentration in the working solution. The concentration of the concentrated solution stock may range from approximately 1 to 500-fold, or about 5 to 200 times, of the intended use concentration.

[0032] Provided herein an efficient simultaneous approach for both cell lysis and viral inactivation utilizing a detergent solution containing TDA-9, Polysorbate 20, and Poloxamer at concentrations exceeding 0.01%, either in cell culture media, cell membrane rupture solution or appropriate buffer systems. A typical example of the upstream process for AAV vectors or other non-enveloped viruses entails concentrating cells into a slurry through centrifugation, followed by lysing them torelease viruses using detergent, freeze / thaw methods, or mechanical homogenization, and subsequently subjecting them to filtration. Subsequently, the cell lysate progresses through a sequence of downstream unit operations.

[0033] The downstream processing of viral vectors involves several key steps. Ini tially, nucleic acid removal is performed by treating lysates with endonucleases to reduce nucleic acid contaminants. Subsequently, solid removal is executed through centrifugation or microfiltration to eliminate cell fragments and debris. Following this, the clarified harvested cell culture fluid may undergo treatment with detergent before proceeding to chromatographic purification. Affinity chromatography is then employed to remove host cell proteins (HCPs) and any serum protein impurities. Additionally, techniques are applied to separate genome-containing infectious AAV viruses from empty, non- infectious capsids, such as cesium chloride gradient ultracentrifugation or ion-exchange chromatography3,31. Finally, a polishing step is conducted to further decrease HCPs or other low molecular weight contaminants using core-bead adsorbents.

[0034] Provided herein a method that involves utilizing detergent cocktails containing TDA-9, Polysorbate 20, and Poloxamer 188, at concentrations of at least 0.01%, in downstream process intermediates or appropriate buffers with pH ranging from 3 to 10 and conductivity between 0 and 200 mS / cm. This method facilitates an independent viral inactivation step as an integral part of the process, occurring either before initiating chromatography steps or between the chromatography steps.

[0035] The critical micelle concentration (CMC) for virus inactivation by detergent is a pivotal parameter determining the efficacy of viral inactivation while preserving the therapeutic properties of these biologies. At concentrations below the CMC, detergents exist as monomers and may not effectively disrupt viral lipid envelopes. However, once the CMC is exceeded, detergent fonn micelles, where hydrophobic tails aggregate inward, leaving hydrophilic heads exposed, enhancing then ability to solubilize lipids and effectively inactivate viruses28,32. Therefore, identifying and optimizing the detergent concentration in relation to the CMC is essential for achieving effective virus inactivation while preserving the therapeutic properties of plasma proteins or antibodies, ensuring the safety and efficacy of the final biologic products such as antibodies, plasma proteins or AAV. Tounderstand the phenomena, in some embodiments, the effectiveness of the active detergent concentration (TDA-9, Polysorbate 20, and Poloxamer 188) was investigated within the range of 0,5 to 5% (w / v) in unclarified cell culture media or PBS buffer to inactivate enveloped viruses.

[0036] Clearing residual detergent after vims clearance is imperative to prevent contamination, toxicity, and interference in subsequent processes. Residual detergent can compromise the accuracy of analyses, alter the structure or function of biological molecules, and introduce variability, thereby hindering reproducibility. Complete removal of detergent is pivotal for maintaining sample integrity, ensuring smooth progression of experiments, and adhering to regulatory standards. Typically, residual detergent removal is accomplished through diverse purification techniques like chromatography, filtration, or diafiltration, with the objective of eliminating detergent molecules while preserving the desired viral vectors or other target components33,34. This critical process ensures that the final product aligns with regulatory standards for pharmaceutical or biotechnological applications.

[0037] hi some embodiment, residual detergent from the post-treated samples can be eliminated through a single or combined series of typical purification unit operations, including affinity chromatography, ion-exchange chromatography, hydrophobic interaction chromatography, or similar chromatography methods, as well as centr ifugation or filtration procedures. For example, consider an in-use viral vector (VI) reagent containing less than 5% of an active detergent mixture comprising TDA-9, Polysorbate 20, and Poloxamer 188, present in process intermediates or buffer solutions. This detergent residue can effectively be removed using AAV affinity' chromatography, employing columns like Capto™ AVB, or AEX chromatography such as PolyQUAT. These chromatographic methods operate in bind and elute modes, enabling the selective capture and subsequent release of the target components while effectively clearing the detergent contaminants.

[0038] hi some embodiments, the composition may further include one or more of stabilizers. Examples of suitable stabilizers include sucrose, trehalose, dextrose, polyethylene glycol (PEG) of various chain lengths, sodium chloride (NaCI), and magnesium chloride (MgCh) at a concentration range of approximately 0.00 IM to about 2M. The chain length of PEG could vary from 100 to 10,000 monomer units.

[0039] In some embodiments, the detergents can further include a buffer in an amount sufficient to maintain pH at a range from about 3.0 to about 9.0. In one embodiment, the buffer can be Tris, phosphate, histidine, citrate, acetate, and combinations thereof.EXAMPLESMaterials and methodDetergent compositions

[0040] The compositions mainly consist of TDA-9, Polysorbate 20, and Poloxamer 188 in varying concentrations, and they have been tested in different matrices, such as HEK293 cell culture media and phosphate-buffered saline (PBS), likely to assess their effectiveness in killing viruses under different conditions. Table 1 summarizes the detergent compositions.Table 1. Detergent CompositionsComposition ID Formula MatrixComposition A 0.1% TDA9 Crude cell lysate0.40 Polysorbate 200.01% Poloxamer 188Composition B 1% TDA9 Crude cell lysate4% Polysorbate 200.1% Po loxamer 188Composition C 1% TDA9 Phosphate buffered saline4% Polysorbate 200.1% Po loxamer 188

[0041] Production of HEK 293 cellsHEK293 cells for the study were cultured in 200 mL BalanCD medium (Fujifilm) supplemented with 4 mM glutamine for 4 days in a 1 L non-baffled, vented shake flask at 125 rpm in a humidified 37 °C shaker incubator with 8% CO2. A sample was taken for viable cell count and analyzed using a Beckman Vi-Cell. The cell count was 5.3xl06cells / mL.

[0042] Lysis of HEK 293 cellsThe cells were divided into 2 x 80 mL volumes in non-baffled, vented 250 mL shake flasks and spiked with Composition A and Composition B, respectively. For the Composition A, 1.6 ml of cells were removed prior to cell lysis solution addition and replaced with 1.6 mL of Cell Lysis solution. Similarly for the flask with the Composition B spike, 16 mL of cells were removed prior to addition of the detergent. This was done as to keep the lysis solution concentration accurate. The flasks were placed in an incubator at 37 °C on a platform shaker for 2 hours at 125 rpm.Virus clearance studyModel virus

[0043] The model virus chosen for this study was Xenotropic Murine Leukemia Virus (XMuLV), an enveloped vims with a single-stranded RNA genome, measuring approximately 80-100 nm. Tire experiments utilized the PG-4 cell line and McCoy's growth media.Pre-study assessment

[0044] Before conducting the viral clearance studies, preliminary control experiments were conducted to assess the potential effects of viral clearance samples on the test systems utilized for virus quantification. The cytotoxicity of detergent treated samples to virus detector cells was examined at various dilutions. In eight-fold replicates, 100 uL of the test items were added to each well of a 96- well microtiter plate (MTP). The test items were initially undiluted and then serially diluted 1 :3 in cell culture medium. The MTP already contained the indicator cells suspended in 100 pL of cell culture medium. Subsequently, the cells were cultivated under appropriate conditions specified for the cell line being used. After at least 3 days all cultures were examined microscopically for cytotoxic effects. The evaluation of cytotoxicity was done by comparison of the cells incubated with the diluted pre-test samples and cells incubated hi standard medium.

[0045] The interference of detergent treated samples on each virus's ability' to infect the detector cells was also assessed. Serial threefold dilutions were performed with cell culture medium on the test items at a non-cytotoxic concentration. Subsequently, 100 uL aliquots of each dilution were added to 8 wells of a 96-well microtiter plate (MTP) containing indicator cells in 100 pL of cell culture medium per well. The cells were then cultured under appropriate conditions for the specific cell line.

[0046] Following an adequate incubation period, the MTPs underwent microscopic inspection to detect vims- induced changes in cell morphology. Any interference was monitored, and if the difference in viral titer determined in the non-toxic dilution was <0.5 loglO or within the 95% confidence limit compared to the viral titer determined in the cell culture medium, no interference was observed.

[0047] A quench assay was conducted to assess whether the virus inactivation process by detergent could be terminated by either diluting an inactivation sample with a cell culture medium, removing detergent from samples, or by adding a quench reagent. Pre-test samples underwent dilution with cell culture medium. The dilution level w'as determined based on the results of the cytotoxicity assay and served as the lowest pre-dilution for the process samples. Subsequently, at least 5 mL of the diluted test sample w as spiked with virus stock solution at a spike ratio of < 10%. Fifteen minutes after spiking, a sample was withdrawn and subjected to analysis for virus titer through endpoint titration.Detergent Treatment process

[0048] The procedure was carried out at 25 ± 1°C using a w'ater bath. Test samples were thawed at room temperature overnight if necessary. To ensure proper tempering of solutions, they were placed in the water bath before being utilized in the process. The temperature of the water bath was monitored using a calibrated thermometer. Prior to initiating the process and at each sampling interval, the temperature was measured to confirm that the inactivation process for the test samples remained within the range of 25 ± 1°C consistently throughout the procedure.

[0049] An aliquot of pre-tempered test samples containing detergent was spiked with virus stock solution at a spike ratio of 10% (v / v). The spiked samples w'ere stored at the process temperature of 25 ± 1°C for the entire duration of the process, which lasted 120 minutes, with continuous stirring. Samples were withdrawn at specified time intervals (<1, 30, 60, and 120 ± 1 minute) as outlined in the tables below; At each designated time point, a portion of the sample w'as withdrawn and immediately diluted with cell culture media to quench the detergent reaction.

[0050] For the control experiment, an aliquot of the pre-tempered test samples without detergent was spiked with vims stock solution at a spike ratio of 10% (v / v). Following mixing, a sample wrasextracted, referred to as "Load". The remaining volume was maintained at the process temperature of 25 ± 1°C with continuous stirring throughout the duration of the inactivation step. At the conclusion of the inactivation step, another sample was obtained, termed "Hold".TCID50 Infectivity Assay

[0051] The test samples were serially diluted in growth medium to enable determination of virus titers. These prepared samples were then applied onto indicator cells seeded in 96-well plates. In instances where large volume testing of the sample was conducted, the sample was applied onto multiple 96-well plates to enhance assay sensitivity. At the conclusion of the assay, wells were assessed for the presence or absence of cytopathic effect (CPE), and virus titers were calculated accordingly. The negative control consisted of indicator cells and growth medium without virus, while the positive control comprised indicator cells and virus, serially diluted to ascertain virus titer. For the assays to be deemed valid, the current virus titer needed to be within one logio of the reported titer, and no CPE should have been observed in the negative control.Detergent clearance

[0052] The examination of removing detergent from the samples treated with VI compositions involved utilizing AAV affinity chromatography (HiTrap Capto AVB column, 1 mL, P / N 17372212, Cytiva) and anion-exchange chromatography (BAKERBOND® PolyQLAT, 1 mL, S / N 6065-07, Avantor Inc.). In both instances, the materials containing 1% detergent were applied onto the column. Details regarding the process conditions for both chromatography methods are outlined in Table 2.Table 2. AAV affinity and AEX chromatography run parameters

[0053] An analytical technique utilizing High-performance liquid chromatography (HPLC) was developed and employed for the identification of the active detergent within the sample using Charged Aerosol Detection (CAD).Pre-study assessment

[0054] This test assesses whether the detergent treated cell lysate samples are toxic to the indicator cells used in the test system to detect viruses. If the substance is toxic to the cells, it might interfere with the accuracy of the viral infection assay by causing cell death or other adverse effects. On the other hand, the interference assay evaluates whether the substance interferes with the virus's ability to infect the indicator cells. This step ensures that any observed interference in viral infection is not due to cytotoxic effects of the substance on the cells. By diluting the substance to a non-toxic level, you can more accurately assess its specific effects on viral infection without confounding factors from cell death caused by toxicity. The cytotoxicity and interference data are plotted in Table 2.

[0055] In the investigation of detergent-induced virus inactivation, it’s crucial to establish methods for terminating the detergent's impact on the virus. Specifically, when assessing the kinetics of virus elimination, it's essential to understand how to cease the detergent's action after a specified duration of exposure. Determining the threshold of detergent inactivation involved assessing various dilutions of detergent treated sample to identify the optimal dilution at which the detergent's effect on virus elimination ceases. The data concerning detergent termination are also outlined in Table 3. Managingthe termination of detergent activity can pose challenges, particularly for certain detergents, when aiming for precise measurements of time-point samples to ascertain vims elimination kinetics.Table 3. Pre-study assessment for cytotoxicity, interference, and quenching detergent reaction.Simultaneous viral clearance and cell lysis

[0056] To investigate the effectiveness of detergent compositions in inactivating enveloped viruses during the cell lysis stage of AAV manufacturing, HEK293 cells were lysed using compositions A and B separately. Subsequently, a model virus, Xenotropic Murine Leukemia Virus (XMuLV), was introduced into the unclarified cell lysate. The experiments were executed at 25±1°C temperature. All the timepoint samples were diluted immediately to terminate the detergent’s activity. In parallel, hold control experiment was performed for each virus in the cell lysate without detergent (cell lysis by freeze-thaw) for the duration of the inactivating step. When an infectivity assay is used, a conservative approach was employed whereby the lesser of the logic total virus in the load and hold control was used to quantify vims and the Log Reduction Value (LRV) where applicable. Thetimepoint samples, <1 , 30, and 60 min were analyzed by standard testing method to observe the trend in virus killing ability of the detergent compositions. Large volume testing method was applied for 120 min sample to maximize the assay sensitivity with low lower limit of detection (LOD). The virus killing kinetics for composition A and B are represented in FIG. 1.

[0057] The LRV data for compositions A and B are depicted in Table 4. For composition A, there was no detectable virus present in the samples taken at the 60- and 120-minute time points. The higher efficiency observed in inactivating viruses at the 120-minute time point compared to the 60- minute time point can be attributed mainly to the relatively higher sensitivity and lower limit of detection (LOD) of the large volume platting assay used, as opposed to the standard testing method, hr the case of composition B, the absence of detectable virus in all samples taken at different time points suggests that it works rapidly to completely inactivate the virus, even within less than 1 minute. However, the relatively lower LRV observed is attributed to the necessity of a higher dilution, which impacted the assay sensitivity.Table 4. LRV values for the time point samples treated by composition A and B.Detergent composition Time points LRVComposition A 0.43 ± 0.39 3.18 ± 0.34 > 3.66 ± 0.29120> 5.34 ± 0.29Composition B < 1 min > 2.10 ± 0.2930 min > 2. 10 ± 0.2960 min > 2.10 ± 0.29120 min (L VP)* > 3.78 ± 0.29*LVP- Large volume platting assayEffect of matrix of viral inactivation capability

[0058] The impact of matrix on viral inactivation was investigated by adding the detergent composition to PBS buffer (Composition C), instead of cell culture media, as in Composition B. Composition B and C contained the same amount of detergent but in different matrices. The logreduction value (LRV) data for both compositions are presented in Table 5. Notably, for viral inactivation using composition C, no cell debris or other cellular components were present. Despite this, it was observed that the viral inactivation capability of both composition B and C was quite similar. These results suggest that the presence of cellular components does not significantly impact the efficiency of viral inactivation.Table 5. LRV values for the time point samples treated by composition A and B.Detergent composition Time points LRVComposition B > 2.10 ± 0.29 > 2.10 ± 0.29 > 2.10 ± 0.29120> 3.78 ± 0.29Composition C <1 min 1.31 ± 0.3830 min > 2.21 ± 0.2360 min > 2.21 ± 0.23120 min (LVP)* 4.37 ± 0.23*L VP- Large volume platting assay

[0059] FIG. 1 presents the results of a comparative analysis evaluating the virus inactivation efficacy of Composition B (1% TDA9, 4% Polysorbate 20, 0.1% Poloxamer 188) in crude cell lysate and Composition C (1% TDA9, 4% Polysorbate 20, 0.1% Poloxamer 188) in PBS. Samples from both compositions were collected at various timepoints, including less than 1, 30, 60, and 120 min. The samples taken at <1, 30, and 60 min were subjected to standard testing methods, while the sample at 120 minutes underwent analysis using a large volume plating assay. The study data demonstrate the effectiveness of the compositions disclosed in this application in inactivating virus under different environmental conditions over time.Clearance of residual detergent

[0060] The results of this study clearly indicated the outstanding ability of AAV affinity (HiTrapCapto AVB column) and AEX chromatography (BAKERBOND® PolyQUAT) to removedetergent from samples. In both chromatography methods, the detergent level in the eluate was detected to be below the limit of detection. The detergent removal data are presented in Table 6.Table 6. Detergent clearance by AAV affinity and AEX chromatography

[0061] Based on the study data regarding detergent clearance and considering the physicochemical properties of the detergents, it can be seen that the detergents were efficiently eliminated from samples using a variety of chromatography methods, such as affinity chromatography or other processes that utilize a bind-and-elute mode of operation.References: ICH Q5A (R2) - Quality Revision 2. Viral Safety Evaluation of Biotechnology Products Derived from Cell Lines of Human or Animal Origin. Food and Drug Administration (FDA) https: / / www.fda.gov / media / 163115 / download (2024). Singh, N. & Heldt, C. L. Challenges in downstream purification of gene therapy viral vectors. Curr Opin Chem Eng 35, 100780 (2022). Barone, P. W. et al. Viral contamination in biologic manufacture and implications for emerging therapies. Nat Biotechnol 38, 563-572 (2020). Cameron, R. & Smith, K. Virus clearance methods applied in bioprocessing operations: an overview of selected inactivation and removal methods. Pharm Bioprocess 2, 75-83 (2014). Shukla, A. A. & Aranha, H. Viral clearance for biopharmaceutical downstream processes. Pharm Bioprocess 3, 127-138 (2015). Feroz, H. et al. Assessing detergent-mediated virus inactivation, protein stability, and impurity clearance in biologies downstream processes. Biotechnol Bioeng 119, 1091-1104 (2022). Farcet, J. B., Karbiener, M., Zelger, L., Kindermann, J. & Kreil, T. R. Detergent-Mediated Virus Inactivation in Biotechnological Matrices: More than Just CMC. IntJ Mo! Sci 24, (2023). Ruppach, H. Viral safety for biotherapeutics and biosimilar. Drug Discovery Today: Technologies vol. 37 23-29 Preprint at https: / / doi.org / 10.1016 / j-ddtec.2020.08.001 (2020). Louten, J. Virus Structure and Classification, in Essential Human Virology 19-29 (Elsevier, 2016). doi:10.1016 / B978-0-12-800947-5.00002-8. Rheinemann, L. & Sundquist, W. I. Virus Budding, in Encyclopedia of Virology 519-528 (Elsevier, 2021). doi:10.1016 / B978-0-12-814515-9.00023-0. Gelderblom, H. R. Structure and Classification of Viruses. (1996). Rossmann, M. G. Structure of viruses: a short history. Q Rev Biophys 46, 133-180 (2013). Nims, R. & Plavsic, M. Identification of Worst-Case Model Viruses for Selected Viral Clearance Steps. BioProcessing Journal 13, 6-13 (2014). Kimura, T. et al. Production of adeno-associated virus vectors for in vitro and in vivo applications. Sci Rep 9, 13601 (2019). Naso, M. F., Tomkowicz, B., Perry, W. L. & Strohl, W. R. Adeno-Associated Virus (AAV) as a Vector for Gene Therapy. BioDrugs 31, 317-334 (2017). Carter, M., Essner, R., Goldstein, N. & Iyer, M. Gene Delivery Strategies, in Guide to Research Techniques in Neuroscience 245-258 (Elsevier, 2022). doi:10.1016 / B978-0-12-818646-6.00017-8. Jaber, T. & Tessarz, A. Viral Clearance Studies: Challenges and Beyond. BioPharm International 18,20- 21,33 (2022).Linke, D. Chapter 34 Detergents, in 603-617 (2009). doi:10.1016 / S0076-6879(09)63034-2. Conley, L. et al. Evaluation of eco-friendly zwitterionic detergents for enveloped virus inactivation; Evaluation of eco-friendly zwitterionic detergents for enveloped virus inactivation. Biotechnol. Bioeng 114, 813-820 (2017). Genova, C., Schoenkaes, U., Smith, D. & Stolz, M. Effect of hydrophobe structure on performance of alcohol ethoxylates. J Surfactants Deterg 6, 365-372 (2003). Li, Y. et al. Synthesis and Properties of Primary Alcohol Ethoxylates Using Different Catalytic Systems. ACS Omega 6, 29774-29780 (2021). Lukosek, M., Emmons-Burzynska, M., Alejski, K. & Szwach, I. Physicochemical Characterization of Ethoxylation Products of Fatty Acid Esters. Frontiers in Chemical Engineering 3, (2021). Takekoshi, S., Takano, K., Matoba, Y., Sato, M. & Tachibana, A. Investigation of OECD 301F ready biodegradability test to evaluate chemical fate in a realistic environment. J Pestic Sci 46, 143-151 (2021). G. Moloughney, J. & Weisleder, N. Poloxamer 188 (P188) as a Membrane Resealing Reagent in Biomedical Applications. Recent Pat Biotechnol 6, 200-211 (2012). Srivastava, A., Mallela, K. M. G., Deorkar, N. & Brophy, G. Manufacturing Challenges and Rational Formulation Development for AAV Viral Vectors. J Pharm Sci 110, 2609-2624 (2021). Patricio, M. I. et al. Inclusion of PF68 Surfactant Improves Stability of rAAV Titer when Passed through a Surgical Device Used in Retinal Gene Therapy. Mol Ther Methods Clin Dev 17, 99-106 (2020). Elveborg, S., Monteil, V. & Mirazimi, A. Methods of Inactivation of Highly Pathogenic Viruses for Molecular, Serology or Vaccine Development Purposes. Pathogens 11, 271 (2022). Farcet, J. B., Karbiener, M., Zelger, L., Kindermann, J. & Kreil, T. R. Detergent-Mediated Virus Inactivation in Biotechnological Matrices: More than Just CMC. Int J Mol Sci 24, (2023). Patterson, E. I. et al. Methods of Inactivation of SARS-CoV-2 for Downstream Biological Assays. J Infect Dis 222, 1462-1467 (2020). Dobrowsky, T., Gianni, D., Pieracci, J. & Suh, J. AAV manufacturing for clinical use: Insights on current challenges from the upstream process perspective. Carr Opin Biomed Eng 20, 100353 (2021). Keller, W. R. et al. Rational downstream development for adeno-associated virus full / empty capsid separation - A streamlined methodology based on high-throughput screening and mechanistic modeling. J Chromatogr A 1716, 464632 (2024). Farcet, J., Kindermann, J., Karbiener, M. & Kreil, T. R. Development of a Triton X-100 replacement for effective virus inactivation in biotechnology processes. Engineering Reports 1, (2019). Hebben, M. Downstream bioprocessing of AAV vectors: industrial challenges & regulatory requirements. Cell Gene Ther Insights 4, 131-146 (2018). Dias Florencio, G. et al. Simple downstream process based on detergent treatment improves yield and in vivo transduction efficacy of adeno-associated virus vectors. Mol Ther Methods Clin Dev 2, 15024 (2015).

Claims

CLAIMS:

1. A method of inactivating enveloped viruses during the production of a biologic product, wherein the biologic product is a non-enveloped virus, the method comprising: contacting cells containing the non-enveloped virus with at least one purified non-ionic detergent, wherein the detergent(s) lyses the cells to release the nonenveloped virus, thereby producing a lysate, and wherein the detergent(s) inactivates any enveloped viruses within the lysate, wherein the detergent(s) is selected from the group consisting of ethoxylated alcohol, sorbiton ethoxylate, triblock copolymer and combinations thereof, wherein the detergent is in a concentration range from about 0.01% (w / v) to about 5.0% (w / v); and wherein the non-enveloped virus is not impacted by the detergent(s).

2. The method according to Claim 1 wherein the non-enveloped virus is adeno-associated vims.

3. The method according to Claim 1 wherein the cells are mammalian or insect cells.

4. The method according to Claim 1 wherein the cells are HEK293 cells.

5. The method according to Claim 1 wherein the detergent(s) is directly added to the cells in cell culture media or to a cell suspension.

6. The method of Claim 1, wherein the ethoxylated alcohol is TDA9, tergitol 15-S-9, TDA6 or combinations thereof.

7. The method of Claim 1, wherein the sorbiton ethoxylate is polysorbate 20, polysorbate 80, polysorbate 60 or combinations thereof.

8. The method of Claim 1, wherein the triblock copolymer is poloxamer 188, poloxamer 407, poloxamer 305, or combinations thereof.

9. The method of Claim 1 , wherein the non-ionic detergent is in a concentration of about 0.5% (w / v).

10. The method of Claim 1, wherein the non-ionic detergent is TDA9 in a concentration of about 0.1% (w / v) and polysorbate 20 in a concentration of about 0.4% (w / v).

11. The method of Claim 1 , wherein the non-ionic detergent comprises 0.1% TDA9, 0.40% Polysorbate 20, and 0.01% Poloxamer 188.

12. The method of Claim 1 wherein the detergent(s) further comprise a buffer selected from the group consisting of Tris, phosphate, histidine, citrate, acetate, and combinations thereof.

13. The method according to Claim 1 wherein the biological product is exposed to the detergent(s) for about 1 minute to about 120 minutes at 2°C to 40°C.

14. The method according to Claim 1 wherein the method takes place in the presence of cell debris, cell culture media, other intracellular components and other contaminants15. A method of inactivating enveloped viruses during the production of a biologic product, the method comprising: contacting cells containing the biologic product with at least one purified non-ionic detergent, wherein the detergent(s) lyses the cells to release the biologic product, thereby producing a lysate, and wherein the detergent(s) inactivates any enveloped viruses within the lysate, wherein the detergent(s) is selected from the group consisting of ethoxylated alcohol, sorbiton ethoxylate, triblock copolymer and combinations thereof, wherein the detergent is in a concentration range from about 0.01% (w / v) to about 5.0% (w / v); and wherein the biologic product is not impacted by the detergent(s).

16. The method according to Claim 15 wherein the biologic product is a therapeutic recombinant protein.

Images