Viral clearance by maintaining low pH

Abstract

A method for viral clearance using low pH hold based on statistical experimental design is provided. To characterize the impact of a low pH hold step on viral inactivation, several factors are evaluated, including pH conditions, conductivity conditions, protein type, temperature, acid titrant, spike timing, and post-spike filtration. In addition to the effect of pH on viral inactivation, increasing ionic strength via manipulation of conductivity may be an important component affecting viral inactivation kinetics.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 023,154, filed May 11, 2020, which is incorporated herein by reference.

[0002] The present invention relates generally to a method for inactivating virus particles in a protein sample using a low pH hold step. A statistical experimental design incorporating several factors is used to evaluate and characterize the effect of a low pH hold step on virus inactivation. [Background technology]

[0003] background Biological products are susceptible to contamination from bacteria, fungi, and viruses, both endogenous and exogenous (from external sources). Viral clearance, e.g., viral inactivation, is a critical step during the manufacturing of biopharmaceutical products made using mammalian cell lines. Global health authorities require viral clearance assessment for manufacturing biological or biotechnology products because viral contamination can be amplified during mammalian cell culture growth. Effective viral clearance studies are an important part of process validation and are crucial for ensuring drug safety. Viral contamination can also affect raw materials, cell culture processes, bioreactors, and downstream purification processes.

[0004] Viral validation studies are designed to document selected operating conditions on product quality to ensure viral safety. Experimental designs for viral clearance studies include characterization of manufacturing processes to improve understanding of processing conditions and identify critical development factors to justify the selection of worst-case conditions. Viral inactivation or removal steps include pH treatment, heat treatment, solvent / detergent treatment, filtration, or chromatography. The mechanism of viral inactivation for low pH incubation involves pH-based chemical reactions that cause irreversible denaturation of viral surface glycoproteins or disruption of the lipid envelope.

[0005] The pH conditions adapted to the manufacturing process of biopharmaceutical products may not be effective for virus inactivation. However, the pH required for virus inactivation may differ significantly from the pH range used in other manufacturing conditions. Using low pH incubation to achieve effective virus inactivation in protein samples is challenging in the case of biopharmaceutical manufacturing because exposure of biopharmaceutical products to low pH may alter the quality or stability of the protein. It can be appreciated that there is a need for methods to evaluate and characterize the impact of low pH hold steps for virus inactivation during biopharmaceutical manufacturing. These methods are necessary to provide an effective and robust experimental design to ensure virus inactivation for the planning of manufacturing processes such as purification processes. Summary of the Invention

[0006] overview This application provides a method for viral clearance using low pH hold based on a statistical experimental design that incorporates several factors to evaluate and characterize the impact of a low pH hold step on viral inactivation. Statistically designed experiments are used to evaluate the effects of pH conditions, ionic strength conditions, protein isotype, temperature, acid titrant, spike timing, and post-spike filtration on viral inactivation. Using these methods, for example, by manipulating the ionic strength of the low-pH starting material, one can predict effective clearance when the viral inactivation step is performed in the pH range of approximately 3.60 to 3.90.

[0007] The present disclosure provides methods for purifying peptides or proteins, such as antibodies, from a sample containing one or more impurities, including viral particles. In some exemplary embodiments, the methods of the present application include adjusting the ionic strength conditions of a sample, adjusting the pH conditions of the sample to an acidic pH, and then maintaining the sample at the ionic strength and pH conditions for at least about 15 minutes to inactivate a quantity of viral particles, wherein the sample contains one or more impurities, including viral particles. In one aspect, the amount of viral particle inactivation is at least about 3 LRF (log reduction) using the methods of the present application. In one aspect, the amount of viral particle inactivation is at least about 4 LRF using the methods of the present application.

[0008] In one embodiment, the pH conditions of the sample in the method of the present application are about pH 3.90 or less. In one embodiment, the pH conditions of the sample are in the range of about pH 3.60 to about pH 3.90. In another embodiment, the pH conditions of the sample are in the range of about pH 3.65 to about pH 3.80. In another embodiment, the peptide or protein in the sample is an antibody produced in a host cell. In yet another embodiment, the sample in the method of the present application is maintained at the ionic strength and pH conditions of the sample for at least about 30 minutes to inactivate the amount of virus particles. In one embodiment, the sample is maintained at the ionic strength and pH conditions of the sample for about 15 to about 30 minutes to inactivate the amount of virus particles.

[0009] In one embodiment, the method of the present application further comprises optimizing the inactivation of the quantity of viral particles by performing a D-optimal experimental design. In another embodiment, the D-optimal experimental design evaluates the following factors: pH conditions of the sample, and salt concentration added to the sample. In one embodiment, the D-optimal experimental design further evaluates the following factors: type of peptide or protein, temperature of the sample, acid titrant for adjusting pH conditions of the sample, spike timing for spiking viral particles into the sample, or presence of post-spike filtration.

[0010] In one embodiment, the sample in the method of the present application is an eluate from Protein A chromatography. In another embodiment, the ionic strength of the sample is adjusted using sodium chloride, and the concentration of sodium chloride is in the range of about 1 mM to about 100 mM, about 1 mM to about 500 mM, about 25 mM, about 50 mM, about 72 mM, about 82 mM, about 100 mM, about 125 mM, about 150 mM, about 175 mM, or about 200 mM. In one embodiment, the pH condition of the sample is adjusted using phosphoric acid or glycine HCl. In another embodiment, the peptide or protein in the sample of the present method is an antibody having an IgG1 isotype or an IgG4 isotype. In one embodiment, the peptide or protein is a monoclonal antibody or a bispecific antibody. In yet another embodiment, the peptide or protein is an antibody, an antibody fragment, an Fab region of an antibody, an antibody-drug conjugate, a fusion protein, a protein pharmaceutical product, or a drug.

[0011] The present disclosure provides a method for producing a preparation comprising a protein of interest and a reduced amount of viral particles from a sample having the protein of interest and infectious viral particles. In some exemplary embodiments, the method for producing a preparation comprising a protein of interest and a reduced amount of viral particles from a sample having the protein of interest and infectious viral particles comprises subjecting the sample to pH and ionic strength conditions of greater than about pH 3.60 by adding a salt having a concentration of up to about 100 mM, and maintaining the sample at the pH and ionic strength conditions for a period of time appropriate to produce a preparation comprising the protein of interest and a reduced amount of infectious viral particles.

[0012] In one embodiment, the concentration of the protein of interest in the sample is less than about 25 g / L.

[0013] In one aspect, a suitable period of time is about 15 minutes, about 20 minutes, about 25 minutes, or about 30 minutes.

[0014] In one embodiment, the method reduces the amount of infectious viral particles from a sample by about 3 LRF (log reduction). In another embodiment, the method reduces the amount of infectious viral particles from a sample by about 4 LRF.

[0015] In one embodiment, the pH conditions of the sample are greater than about pH 3.70. In another embodiment, the pH conditions of the sample are greater than about pH 3.80. In yet another embodiment, the pH conditions of the sample are greater than about pH 3.90. In yet another embodiment, the pH conditions of the sample are greater than about pH 4.0.

[0016] In one embodiment, the pH conditions of the sample are in the range of about pH 3.60 to about pH 4.0. In another embodiment, the pH conditions of the sample are in the range of about pH 3.70 to about pH 4.0. In yet another embodiment, the pH conditions of the sample are in the range of about pH 3.80 to about pH 4.0.

[0017] Exemplary sources of a "sample" can include affinity chromatography, such as Protein A eluate; the sample can be obtained from the flow-through fraction of an ion exchange chromatography procedure, from a strip of an ion exchange column, or from other sources during purification steps known to those skilled in the art from which a sample can be obtained. In one aspect of this embodiment, the sample is the eluate from Protein A chromatography.

[0018] In one embodiment, the ionic strength of the sample is adjusted using the addition of sodium chloride, the concentration of sodium chloride being in the range of about 1 mM to about 200 mM.

[0019] In one aspect, the ionic strength conditions are adjusted by using sodium chloride at a concentration greater than about 50 mM. In another aspect of this embodiment, the concentration is greater than about 100 mM.

[0020] In one embodiment, the pH condition of the sample is adjusted using phosphoric acid or glycine HCl.

[0021] These and other aspects of the present invention will be better appreciated and understood when considered in conjunction with the following description and the accompanying drawings. The following description, while indicating various embodiments and numerous specific details thereof, is given by way of illustration and not by way of limitation. Many substitutions, modifications, additions, or rearrangements may be made within the scope of the present invention. [Brief explanation of the drawings]

[0022] [Figure 1A] The Adjustment, Spike, and Reconditioning method is shown, and according to an exemplary embodiment, the sample is adjusted / titrated to the target pH, then spiked with virus stock at approximately pH 7.2, followed by readjusting the pH of the sample to the target pH before holding at the desired temperature for the remainder of the pH hold. Timing begins at the time of the spike. [Figure 1B] The spike adjustment method is shown, and according to an exemplary embodiment, the sample is first spiked with a virus stock solution and then adjusted / titrated to the target pH. Timing begins when the target pH of the sample is reached. [Figure 2A] 1 shows a scatter plot matrix and multivariate correlations of a D-optimal model design to investigate the effect of several factors associated with a low pH hold step for viral inactivation, according to an exemplary embodiment. [Figure 2B] A neutral control performed for each monoclonal antibody salt condition is shown, the purpose of which is to ensure that the measured viral activity is the result of chemical inactivation at low pH, according to an exemplary embodiment. [Figure 3A] 1 shows the inactivation kinetics of X-MuLV at a target pH of 3.65, according to an exemplary embodiment. According to an exemplary embodiment, an LRF curve at a target pH of 3.65 was obtained by plotting LRF values ​​against time points. [Figure 3B] 1 shows the inactivation kinetics of X-MuLV at a target pH of 3.73, according to an exemplary embodiment. According to an exemplary embodiment, an LRF curve at a target pH of 3.73 was obtained by plotting LRF values ​​against time points. [Figure 3C] 1 shows the inactivation kinetics of X-MuLV at a target pH of 3.80, according to an exemplary embodiment. According to an exemplary embodiment, an LRF curve at a target pH of 3.80 was obtained by plotting LRF values ​​against time points. [Figure 4A] 1 shows the inactivation kinetics of X-MuLV at 0 mM NaCl at various target pH conditions, e.g., about pH 3.65, pH 3.73, or pH 3.80, according to an exemplary embodiment. According to an exemplary embodiment, LRF curves were obtained by plotting LRF values ​​against time points for various target pH conditions. [Figure 4B] 1 shows the inactivation kinetics of X-MuLV at 50 mM NaCl at various target pH conditions, e.g., about pH 3.65, pH 3.73, or pH 3.80, according to an exemplary embodiment. According to an exemplary embodiment, LRF curves were obtained by plotting LRF values ​​against time points for various target pH conditions. [Figure 4C]1 shows the inactivation kinetics of X-MuLV at 100 mM NaCl at various target pH conditions, e.g., about pH 3.65, pH 3.73, or pH 3.80, according to an exemplary embodiment. According to an exemplary embodiment, LRF curves were obtained by plotting LRF values ​​against time points for various target pH conditions. [Figure 4D] 1 shows a predicted profiler including parameter estimates for evaluating operating conditions containing about 25 mM NaCl at about pH 3.70-3.75, according to an exemplary embodiment. [Figure 4E] 1 shows a predicted profiler including parameter estimates for evaluating operating conditions containing about 50 mM NaCl at about pH 3.70-3.75, according to an exemplary embodiment. [Figure 4F] Figure 1 shows a predicted profiler including parameter estimates for evaluating operating conditions containing approximately 80 mM NaCl at approximately pH 3.70-3.75, according to an exemplary embodiment. The effect size of post-spike filtration was minimal and was not included in the overall predicted profiler. [Figure 5A] 10 shows actual versus predicted values ​​of a multivariate linear regression model for LRF for the 15 minute time point according to an exemplary embodiment. [Figure 5B] 10 shows actual versus predicted values ​​of a multivariate linear regression model for LRF for the 30 minute time point according to an exemplary embodiment. [Figure 6] 1 shows a predictive profiler created to optimize the model to achieve greater than 4 LRF after both the 15 and 30 minute time points, according to an exemplary embodiment. According to an exemplary embodiment, the predictive profiler represented X-MuLV inactivation as a function of the salient factors evaluated in the D-optimal DoE. [Figure 7] 1 shows a predicted profiler including parameter estimates for several existing operating conditions at approximately pH 3.70-3.75 for low pH retention, according to an exemplary embodiment. The effect size of post-spike filtration was minimal and was not included in the overall predicted profiler. [Figure 8]Figure 1 shows a predicted profiler including parameter estimates for operating conditions rescaled to approximately pH 3.65-3.70 for low pH retention, according to an exemplary embodiment. The effect size of post-spike filtration was minimal and was not included in the overall predicted profiler. [Figure 9] According to an exemplary embodiment, effect sizes for evaluated factors including NaCl, pH, acid titrant, temperature, protein type (mAb, monoclonal antibody), spike timing and combinations thereof for protein types including IgG1 and IgG2, and LRF of X-MuLV at 30 minutes based on retrospective data are shown. [Figure 10] According to an exemplary embodiment, effect sizes for evaluated factors including NaCl, pH, acid titrant, temperature, protein type (mAb, monoclonal antibody), spike timing and combinations thereof, and retroviral LRF for various temperature conditions based on retrospective industrial data are shown. [Figure 11] According to an exemplary embodiment, effect sizes are shown for evaluated factors including NaCl, pH, acid titrant, temperature, protein type (mAb, monoclonal antibody), spike timing and combinations thereof, and spike / adjust time for two methods. DETAILED DESCRIPTION OF THE INVENTION

[0023] Detailed Description Viral clearance is critical for manufacturing biopharmaceutical products, especially those made using mammalian cell lines such as Chinese hamster ovary (CHO) cells. Ensuring viral clearance is crucial when planning purification processes. A typical workflow for studying viral clearance in a manufacturing process involves spiking a sample load with virus, running the process in a scale-down experiment that mimics the large-scale step, and recording the ability to remove the spiked virus. Viral inactivation or removal steps include pH treatment, heat treatment, solvent / detergent treatment, filtration, or chromatography. Evaluation of viral clearance should include demonstrating removal of a specific model virus for retrovirus-like particles specific to the genome of CHO cells (Anderson et al., Endogenous origin of defective retrovirus-like particles from a recombinant Chinese hamster ovary cell line, Virology 181(1):305-311, 1991). Retroviruses are enveloped RNA viruses that propagate in host cells using reverse transcriptase to generate DNA from their RNA genome. The generated DNA is then integrated into the host genome for replication. Xenotropic murine leukemia virus (X-MuLV) can be used as a model virus to evaluate viral inactivation in pharmaceutical proteins derived from CHO cells. MuLV is a retrovirus with a positive single-stranded sense RNA that replicates via reverse transcription. MuLV can induce leukemia in inoculated mice.

[0024] Viral reduction or viral clearance refers to the difference in total or infectious viral load between an input and output sample after a specific process step, such as a chromatography step. Viral reduction capacity is expressed as the log reduction value (LRV) or log (log 10The clearance rate may be defined as the viral load reduction factor (LRF). The reduction rate is calculated based on the total viral load before and after the clearance step is applied. Viral validation studies can be performed to document the clearance of known viruses associated with the product and to estimate the effectiveness of the process to remove potential adventitious viral contaminants by characterizing the ability of the process to remove nonspecific model viruses.

[0025] This application provides statistically designed experiments that can be used to evaluate and characterize the effect of a low pH hold step on virus inactivation, including evaluation of several factors such as protein isotype, pH conditions, temperature, acid titrant, ionic strength conditions, spike timing, or post-spike filtration. These methods can be used to predict effective clearance when the virus inactivation step is performed at a pH range of approximately 3.60 to 3.90 by manipulating ionic strength through increasing the conductivity of the low-pH starting material. Models generated from the experiments can be used to predict the clearance of viruses such as X-MuLV over a range of process conditions at various time points, such as approximately 15 and approximately 30 minutes. In some exemplary embodiments, in addition to the effect of pH conditions on virus inactivation, increasing the conductivity of the starting solution can increase virus inactivation. For example, increasing sodium chloride (NaCl) concentration can be an important component influencing virus inactivation kinetics to achieve greater virus inactivation. In one aspect, the present application provides the advantage of robust and effective inactivation of viruses, such as greater than about 4 LRF for inactivation of X-MuLV, which can be achieved through an increase in the ionic strength of the low pH starting material.

[0026] In accordance with ICH Q5A(R1) (Viral Safety Assessment of Biotechnology Products Derived from Cell Lines of Human or Animal Origin. International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use. Current Step 4 version, September 23, 1999), downstream purification processes for biopharmaceutical products are developed to ensure the removal and / or inactivation of endogenous or adventitious viral contaminants. A typical downstream purification process can incorporate several orthogonal virus removal steps, including one dedicated step for inactivating enveloped viruses. Low pH incubation can be used to inactivate enveloped viruses, such as by irreversible denaturation of the capsid (Brorson et al., Bracketed generic inactivation of rodent retroviruses by low pH treatment for monoclonal antibodies and recombinant proteins, Biotechnol Bioeng 82(3):321-329, 2003). Filtration is a size-based removal method that can be used to remove both enveloped and non-enveloped viruses (Lute et al., Phage passage after extended processing in small-virus-retentive filters, Biotechnol Appl Biochem 47(Pt 3):141-151, 2007).Chromatography steps can be used to purify biologics with the potential to provide viral reduction for viral clearance, such as the use of protein A chromatography (Bach et al., Clearance of the rodent retrovirus, XMuLV, by protein A chromatography, Biotechnol Bioeng 112(4):743-750, 2015) or anion exchange chromatography (Strauss et al., Anion exchange chromatography provides a robust, predictable process to ensure viral safety of biotechnology products, Biotechnol Bioeng 102(1):168-175, 2009a).

[0027] Low-pH viral inactivation is commonly performed after a Protein A capture step to inactivate viruses during the purification of biopharmaceutical products such as monoclonal antibodies. The product eluted from Protein A chromatography is typically at low pH, which is further acidified and held for at least 30 minutes for viral inactivation. The mechanism of viral inactivation is primarily via pH-based chemical reactions that cause irreversible denaturation of viral surface glycoproteins or disruption of the lipid envelope (Brorson et al., supra). Once the pH hold is complete, the product is neutralized and proceeds to further downstream processing. The low-pH step has been observed to reliably generate large enveloped viruses with a 4LRF (Miesegaes et al., Analysis of viral clearance unit operations for monoclonal antibodies. Biotech Bioeng. 2019;106:238-246). Previous studies have shown that ionic strength can affect the inactivation kinetics of X-MuLV. Experimental conditions with increased ionic strength, such as higher buffer concentrations or higher protein concentrations with weak acid titration, can correlate with higher LRV at pH 3.7 and 3.8 (Chinniah et al., Characterization of operating parameters for XMuLV inactivation by low pH treatment, Biotechnol Prog, 2016. Jan-Feb 32, (1), 89-97, doi:10.1002 / btpr.2183. Epub 2015 Nov 5).

[0028] Studies have been conducted to understand viral clearance by low pH maintenance. These studies support a pH-based chemical reaction that is influenced by pH, time, and temperature (Brorson et al.). The inactivation step is robust, leading Brorson et al. to report a 4.6 log reduction in X-MuLV. 10To consistently achieve clearances above this level, a "lumped comprehensive clearance" method has been developed in which low-pH inactivation is performed at a pH of 3.8 or less and at a temperature of 14°C or greater, with a hold time of at least 30 minutes. According to ASTM E2888-12, the American Society for Testing and Materials (ASTM) further reduced the operating space by requiring a low pH hold of 3.6 or less and a temperature of 15°C or greater to achieve a 5.0 LRF or greater (ASTM E2888-12: Standard Practice for Process for Inactivation of Rodent Retrovirus by pH. West Conshohocken, PA: ASTM International; 2012. http: / / www.astm.org). Furthermore, according to ASTM E2888-12, the hold time is 30 minutes or greater.

[0029] Over time, guidance has been revised to decrease the pH of the hold to achieve a greater LRF. To claim the comprehensive clearance provided by ASTM E2888-12, the protein of interest must conform to the comprehensive type defined by that document. Effective viral inactivation can be particularly challenging for biopharmaceutical products such as monoclonal antibodies, because exposure to low pH can alter the protein's quality or stability. Health authorities expect low-pH hold to be validated under worst-case conditions, indicating that retroviral inactivation is determined at pH conditions beyond the actual manufacturing range. If the inactivation step is operated outside of these, viral clearance may be compromised.

[0030] In some exemplary embodiments, the present application provides statistically designed experiments, e.g., experimental designs including controlled and uncontrolled factors related to the low-pH inactivation step, to determine the effects of protein type, pH conditions, temperature, acid titrant, NaCl content, spike timing, or post-spike filtration on viral inactivation at various time points. In some aspects, the present application provides experimental factors and conditions for defining operational conditions for reliable and effective viral clearance. In some aspects, X-MuLV is selected as a model endogenous enveloped virus. In some aspects, two types of monoclonal antibodies, such as IgG1 or IgG4, are subjected to testing various parameters, such as pH, temperature, acid titrant, ionic strength, spike timing, or post-spike filtration.

[0031] The present application's experimental design for multivariate analysis, e.g., Design of Experiments (DoE), involves characterizing viral inactivation at low pH retention. DoE is a methodology that allows for the systematic variation of multiple development factors within the context of a single experimental design. DoE results can be used to create mathematical models of the process being investigated. The true optimum of the investigated process can be identified by applying these mathematical models. Application of DoE results includes eliminating non-substantial development factors, identifying critical development factors for further study, and predicting the performance of the experimental process. DoE is performed in a systematic logical flow that includes stating objectives, selecting variables and models, creating an experimental design that supports the model, collecting data based on the design, performing analyses or validating the model at checkpoints, and reporting the results. The results of DoE and the resulting models can be used to confirm, reject, or modify existing understanding of the mechanism of low pH retention for viral clearance.

[0032] In some exemplary embodiments, the present application provides a method for purifying an antibody from a sample containing one or more impurities, including potentially infectious viral particles, the method comprising adjusting the ionic strength conditions of the sample, adjusting the pH conditions of the sample to an acidic pH, and then maintaining the sample at the ionic strength and pH conditions for at least about 15 minutes to inactivate a quantity of viral particles. In one aspect, the capacity of the method of the present application to inactivate the quantity of viral particles is at least about 3 LRF. In one aspect, the capacity of the method of the present application to inactivate the quantity of viral particles is at least about 4 LRF. In one aspect, the peptide or protein in the sample is an antibody produced in a host cell. In one aspect, the antibody is a monoclonal antibody or a bispecific antibody. In one aspect, the antibody in the method of the present application is an eluate from Protein A chromatography.

[0033] In one embodiment, the pH conditions of the sample in the method of the present application are about pH 3.90 or less. In one embodiment, the pH conditions of the sample are in the range of about pH 3.60 to about pH 3.90. In one embodiment, the pH conditions of the sample are in the range of about pH 3.65 to about pH 3.80. In some embodiments, the pH conditions in the statistically designed experiments of the present application are in the range of about pH 3.65 to about pH 3.80, such as about pH 3.65, about pH 3.73, or about pH 3.80, which is above the pH range of the manufacturing process. The pH range of pH 3.65 to 3.80 is larger than and outside the pH range suggested by ASTM E2888-12, e.g., pH 3.6 or less. It is known that lower pH conditions can cause faster viral inactivation (Brorson et al.). In some embodiments, the temperature in the statistically designed experiments of the present application is in the range of about 15°C to about 20°C, such as about 15°C or about 20°C, which is at or below the lower end of the temperature range for manufacturing processes. It is known that higher temperatures can cause inactivation more quickly (Brorson et al.). The temperature range of about 15°C to about 20°C is within the temperature range suggested by ASTM E2888-12, e.g., 15°C or higher. In some embodiments, the acid titrant in the statistically designed experiments of the present application is about 0.25M phosphoric acid or about 0.25M glycine HCl. In some embodiments, the NaCl content added to the starting material in the statistically designed experiments of the present application is in the concentration range of about 0 mM to about 100 mM.

[0034] In some embodiments, the methods of the present application further include optimizing the inactivation of a quantity of viral particles by performing a D-optimal DoE to evaluate various factors, including the pH conditions of the sample, the conductivity of the sample, the type of peptide or protein, the temperature of the sample, the acid titrant for adjusting the pH conditions of the sample, the spike timing for spiking the viral particles into the sample, or the presence of post-spike filtration. In some embodiments, the spike timing in the statistically designed experiments of the present application is a calibrated spike readjustment method or a spike adjustment method. The calibrated spike readjustment method can provide a constant desired pH range throughout the entire hold time. The spike adjustment method can represent reliable manufacturing conditions in which in-process intermediates containing endogenous retrovirus-like particles are acidified from the starting pH. In the calibrated spike readjustment method as shown in FIG. 1A, the sample is adjusted / titrated to a target pH and then spiked with a viral stock having a pH of approximately 7.2. The pH hold timing was initiated at the time of the spike. Upon observing a pH increase after spiking the virus stock, the sample pH is readjusted to the target pH, followed by holding at the desired temperature for the remainder of the pH hold period. In the spike adjustment method shown in Figure 1B, the sample is first spiked with the virus stock solution and then adjusted / titrated to the target pH. Once the target pH is reached, the timing of the pH hold begins, and the sample is incubated at the desired temperature. In some embodiments of the statistically designed experiments of the present application, the low pH hold can be performed with or without post-spike filtration using a filter material, such as an approximately 0.2 μm filter material. Post-spike filtration represents a reliable manufacturing operation that filters in-process intermediates containing endogenous retrovirus-like particles to maintain sterility. A filtration step that can remove viral aggregates can result in monodisperse viruses.

[0035] In some exemplary embodiments, the conductivity of the starting material has a strong effect on viral inactivation kinetics at various target pH conditions. In some exemplary embodiments, the present application provides a model for predicting viral clearance across a range of process conditions. In some aspects, in addition to the effect of pH on viral inactivation, increases in ionic strength, such as NaCl concentration, or conductivity can be important components affecting viral inactivation kinetics. In one aspect, pH conditions are an important factor when the load material has low ionic strength. For example, X-MuLV inactivation can depend on pH conditions at low ionic strength. In some aspects, the effect of pH conditions on viral inactivation decreases as ionic strength increases. For example, X-MuLV can be rapidly inactivated at all target pH conditions when ionic strength is increased. In some aspects, in the presence of about 50 mM or about 100 mM NaCl, complete and effective viral clearance, such as inactivation of X-MuLV, is observed at about 30 minutes under target pH conditions of about pH 3.65, about pH 3.73, and about pH 3.80. For pH conditions of about pH 3.65 and pH 3.73, complete inactivation of X-MuLV was observed after 15 minutes. In one embodiment, the ionic strength of the sample is adjusted using the addition of sodium chloride, with the concentration of sodium chloride ranging from about 1 mM to about 100 mM, from about 1 mM to about 500 mM, about 50 mM, or about 100 mM.

[0036] Low pH maintenance during the manufacturing of biopharmaceutical products can have a significant impact on the quality and stability of proteins such as monoclonal antibodies. Using the model of the present application, the conductivity of the low-pH starting material can be manipulated to predict effective clearance when viral inactivation is performed in the pH range of 3.60 to 3.90. In some embodiments, the present application provides that increasing the conductivity via the addition of NaCl to the starting material can achieve rapid and effective viral inactivation at various target pH conditions. In one embodiment, the sample in the method of the present application is maintained at the ionic strength and pH conditions of the sample for at least about 30 minutes to inactivate the amount of viral particles. In one embodiment, the sample is maintained at the ionic strength and pH conditions of the sample for about 15 to about 30 minutes to inactivate the amount of viral particles.

[0037] The statistical DoE test results for the low pH hold in this application are consistent with the ASTM standard for X-MuLV inactivation at 5.0 LRF below approximately pH 3.60. The results also demonstrate robust and effective inactivation at pHs above approximately 3.60. For ranges outside the ASTM comprehensive requirements, the results indicate that rapid and effective X-MuLV inactivation can be achieved by increasing the NaCl content. Typically, the pH of the hold depends on the protein's stability. The model in this application offers the advantage of predicting effective clearance when operating at pH 3.60 to 3.90 by manipulating the conductivity of the low-pH starting material.

[0038] The demand for improved product quality, efficacy, and safety of biopharmaceutical products has led to an increasing demand for effective and robust experimental designs to ensure viral inactivation. The present disclosure provides methods that meet the aforementioned demand. Exemplary embodiments disclosed herein meet the aforementioned demand by providing a method for purifying antibodies from samples containing one or more impurities, including viral particles. The present application fulfills a long-standing need by providing a model for predicting robust and effective inactivation of viruses via increasing the ionic strength of a low-pH starting material, and effective clearance at any pH manipulation by manipulating the ionic strength of the low-pH starting material.

[0039] The term "a" should be understood to mean "at least one," and the terms "about" and "approximately" should be understood to allow for standard variation as understood by one of ordinary skill in the art, and when ranges are provided, the endpoints are included. As used herein, the terms "include," "includes," and "including" are meant to be open-ended and are understood to mean "comprise," "comprises," and "comprising," respectively.

[0040] In some exemplary embodiments, the present application provides a method for purifying a peptide or protein, such as an antibody, from a sample containing one or more impurities, including viral particles. In some exemplary embodiments, the method of the present application includes adjusting the ionic strength conditions of the sample, adjusting the pH conditions of the sample to an acidic pH, and then maintaining the sample at the ionic strength and pH conditions for at least about 15 minutes to inactivate a quantity of viral particles. In one aspect, the peptide or protein in the sample is an antibody produced in a host cell. In one aspect, the peptide or protein is a monoclonal antibody or a bispecific antibody. In one aspect, the peptide or protein is an antibody, an antibody fragment, an Fab region of an antibody, an antibody-drug conjugate, a fusion protein, a protein pharmaceutical product, or a drug.

[0041] As used herein, the terms "protein" or "protein of interest" can include any amino acid polymer having covalently linked amide bonds. Proteins include one or more amino acid polymer chains, generally known in the art as "polypeptides." A "polypeptide" refers to a polymer composed of amino acid residues, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof linked via peptide bonds, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof. A "synthetic peptide or polypeptide" refers to a non-naturally occurring peptide or polypeptide. Synthetic peptides or polypeptides can be synthesized, for example, using an automated polypeptide synthesizer. Various solid-phase peptide synthesis methods are known to those skilled in the art. A protein can include one or more polypeptides to form a single functional biomolecule. In another exemplary embodiment, a protein can include an antibody fragment, a nanobody, a recombinant antibody chimera, a cytokine, a chemokine, a peptide hormone, or the like. The proteins may include any of biotherapeutic proteins, recombinant proteins used in research or therapy, trap proteins and other chimeric receptor Fc fusion proteins, chimeric proteins, antibodies, monoclonal antibodies, polyclonal antibodies, human antibodies, and bispecific antibodies. Proteins may be produced using recombinant cell-based production systems such as insect baculovirus systems, yeast systems (e.g., Pichia species), mammalian systems (e.g., CHO cells and CHO derivatives such as CHO-K1 cells), etc.For a recent review discussing biotherapeutic proteins and their production, see Darius Ghaderi et al., "Production platforms for biotherapeutic glycoproteins. Occurrence, impact, and challenges of non-human sialylation," 28 BIOTECHNOLOGY AND GENETIC ENGINEERING REVIEWS 147-176 (2012). In some exemplary embodiments, the proteins comprise modifications, adducts, and other covalently linked moieties. These modifications, adducts, and moieties include, for example, avidin, streptavidin, biotin, glycans (e.g., N-acetylgalactosamine, galactose, neuraminic acid, N-acetylglucosamine, fucose, mannose, and other monosaccharides), PEG, polyhistidine, FLAG tags, maltose-binding protein (MBP), chitin-binding protein (CBP), glutathione-S-transferase (GST) myc-epitopes, fluorescent labels, and other dyes. Proteins can be classified based on their composition and solubility and thus can include simple proteins, such as globular proteins and fibrous proteins; conjugated proteins, such as nucleoproteins, glycoproteins, mucoproteins, chromoproteins, phosphoproteins, metalloproteins, and lipoproteins; and derived proteins, such as primary and secondary derived proteins.

[0042] In some exemplary embodiments, the protein of interest may be a recombinant protein, an antibody, a bispecific antibody, a multispecific antibody, an antibody fragment, a monoclonal antibody, a fusion protein, an scFv, and combinations thereof.

[0043] As used herein, the term "recombinant protein" refers to a protein produced as a result of transcription and translation of a gene carried on a recombinant expression vector that has been introduced into a host cell. In certain exemplary embodiments, the recombinant protein may be a fusion protein. In certain exemplary embodiments, the recombinant protein may be an antibody, e.g., a chimeric antibody, a humanized antibody, or a fully human antibody. In certain exemplary embodiments, the recombinant protein may be an antibody of an isotype selected from the group consisting of IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgM, IgA1, IgA2, IgD, or IgE. In certain exemplary embodiments, the antibody molecule is a full-length antibody (e.g., an IgG1 or IgG4 immunoglobulin), or alternatively, the antibody may be a fragment (e.g., an Fc fragment or a Fab fragment).

[0044] The term "antibody," as used herein, includes immunoglobulin molecules comprising four polypeptide chains: two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, as well as multimers thereof (e.g., IgM). Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, CH1, CH2, and CH3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region comprises one domain (CL1). The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity-determining regions (CDRs), interspersed with more conserved regions, termed framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. In various embodiments of the present invention, the FRs of an anti-big-ET-1 antibody (or antigen-binding portion thereof) may be identical to human germline sequences or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on a parallel analysis of two or more CDRs. The term "antibody," as used herein, also includes antigen-binding fragments of an intact antibody molecule. The terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and the like, as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. Antigen-binding fragments of antibodies can be derived from intact antibody molecules using any suitable standard technique, such as proteolytic or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable domains and, optionally, constant domains. Such DNA is known and / or readily available, for example, from commercial sources, DNA libraries (including, for example, phage antibody libraries), or can be synthesized.The DNA can be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable and / or constant domains into a suitable configuration, or to introduce codons, create cysteine ​​residues, modify, add or delete amino acids, etc.

[0045] As used herein, "antibody fragment" includes a portion of an intact antibody, such as, for example, the antigen-binding or variable region of the antibody. Examples of antibody fragments include, but are not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, scFv fragments, Fv fragments, dsFv diabodies, dAb fragments, Fd' fragments, Fd fragments, and isolated complementarity-determining region (CDR) regions, as well as triabodies, tetrabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments. An Fv fragment is a combination of the variable regions of an immunoglobulin heavy and light chains, and an ScFv protein is a recombinant single-chain polypeptide molecule in which the variable regions of an immunoglobulin light and heavy chains are connected by a peptide linker. In some exemplary embodiments, an antibody fragment contains the full amino acid sequence of a parent antibody such that the fragment binds to the same antigen as the parent antibody; in some exemplary embodiments, the fragment binds to the antigen with an affinity comparable to that of the parent antibody and / or competes with the parent antibody for binding to the antigen. Antibody fragments can be produced by any means. For example, an antibody fragment may be enzymatically or chemically produced by fragmentation of an intact antibody and / or it may be recombinantly produced from a gene encoding a partial antibody sequence. Alternatively or additionally, an antibody fragment may be wholly or partially synthetically produced. An antibody fragment may optionally comprise a single-chain antibody fragment. Alternatively or additionally, an antibody fragment may comprise multiple chains linked together, for example, by disulfide linkages. An antibody fragment may optionally comprise a multimolecular complex. Functional antibody fragments typically comprise at least about 50 amino acids, more typically at least about 200 amino acids.

[0046] The phrase "bispecific antibody" includes antibodies that can selectively bind to two or more epitopes. Bispecific antibodies generally comprise two different heavy chains, each of which specifically binds to a different epitope, either on two different molecules (e.g., antigens) or on the same molecule (e.g., the same antigen). When a bispecific antibody can selectively bind to two different epitopes (a first epitope and a second epitope), the affinity of the first heavy chain for the first epitope is generally at least one to two, or three, or four orders of magnitude lower than the affinity of the first heavy chain for the second epitope, or vice versa. The epitopes recognized by a bispecific antibody can be on the same or different targets (e.g., on the same or different proteins). Bispecific antibodies can be generated, for example, by combining heavy chains that recognize different epitopes of the same antigen. For example, nucleic acid sequences encoding heavy chain variable sequences that recognize different epitopes of the same antigen can be fused to nucleic acid sequences encoding different heavy chain constant regions, and such sequences can be expressed in cells that express immunoglobulin light chains.

[0047] A typical bispecific antibody has two heavy chains, each with three heavy chain CDRs, followed by a CH1 domain, hinge, CH2 domain, and CH3 domain, and an immunoglobulin light chain that does not confer antigen-binding specificity but can either associate with each heavy chain, or with each heavy chain and associate with one or more of the epitopes bound by the heavy chain antigen-binding region, or with each heavy chain and enable binding to one or both epitopes, or one or both of the heavy chains. bsAbs can be divided into two major classes: those that retain the Fc region (IgG-like) and those that lack the Fc region, the latter usually being smaller than Fc-containing IgG and IgG-like bispecific molecules. IgG-like bsAbs can have various formats, including but not limited to triomabs, knob-into-hole IgG (kihIgG), cross-Mab, orth-Fab IgG, dual variable domain Ig (DVD-Ig), two-in-one or dual acting Fab (DAF), IgG-single chain Fv (IgG-scFv), or κλ bodies. Various non-IgG-like formats include tandem scFvs, diabody formats, single-chain diabodies, tandem diabodies (TandAbs), dual affinity retargeting molecules (DARTs), DART-Fc, nanobodies, or antibodies produced by the dock-and-lock (DNL) method (Gaowei Fan, Zujian Wang & Mingju Hao, Bispecific antibodies and their applications, 8 JOURNAL OF HEMATOLOGY & ONCOLOGY 130; Dafne Muller & Roland E. Kontermann, Bispecific Antibodies, HANDBOOK OF THERAPEUTIC ANTIBODIES 265-310 (2014)). Methods for producing bsAbs include, but are not limited to, quadroma technology based on somatic cell fusion of two different hybridoma cell lines, chemical conjugation using chemical crosslinkers, and genetic approaches using recombinant DNA technology. Examples of bsAbs include those disclosed in the following patent applications, which are incorporated herein by reference:U.S. Patent Application No. 12 / 823838 filed June 25, 2010, U.S. Patent Application No. 13 / 488628 filed June 5, 2012, U.S. Patent Application No. 14 / 031075 filed September 19, 2013, U.S. Patent Application No. 14 / 808171 filed July 24, 2015, U.S. Patent Application No. 15 / 713574 filed September 22, 2017, U.S. Patent Application No. 15 / 713574 filed September 22, 2018, U.S. Patent Application No. 15 / 713574 filed September 22, 2019, U.S. Patent Application No. 15 / 713574 filed September 22, 20 ... No. 15 / 713,569, filed September 22, 2017; U.S. Patent Application No. 15 / 386,453, filed December 21, 2016; U.S. Patent Application No. 15 / 386,443, filed December 21, 2016; U.S. Patent Application No. 15 / 22,343, filed July 29, 2016; and U.S. Patent Application No. 15,814,095, filed November 15, 2017. Low levels of homodimeric impurities may be present at several steps during the manufacture of bispecific antibodies. Detection of such homodimeric impurities can be difficult when performed using intact mass spectrometry due to the low abundance of homodimeric impurities when performed using conventional liquid chromatography methods and the co-elution of these impurities with the main species.

[0048] As used herein, a "multispecific antibody" or "Mab" refers to an antibody that has binding specificities for at least two different antigens. Such molecules typically bind only two antigens (i.e., bispecific antibodies, BsAbs), although antibodies with additional specificities, such as trispecific antibodies and KIH trispecifics, can also be addressed by the systems and methods disclosed herein.

[0049] The term "monoclonal antibody," as used herein, is not limited to antibodies made through hybridoma technology. Monoclonal antibodies may be derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, by any means available or known in the art. Monoclonal antibodies useful in the present disclosure may be prepared using a wide variety of techniques known in the art, including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof.

[0050] In some exemplary embodiments, the protein of interest may be purified from mammalian cells. Mammalian cells may be of human or non-human origin, including primary epithelial cells (e.g., keratinocytes, cervical epithelial cells, bronchial epithelial cells, tracheal epithelial cells, renal epithelial cells, and retinal epithelial cells), established cell lines and their derivatives (e.g., 293 embryonic kidney cells, BHK cells, HeLa cervical epithelial cells and PER-C6 retinal cells, MDBK (NBL-1) cells, 911 cells, CRFK cells, MDCK cells, CHO cells, BeWo cells, Chang cells, Detroit562 cells, HeLa229 cells, HeLaS3 cells, Hep-2 cells, KB cells, LSI80 cells, LS174T cells, NCI-H-548 cells, RPMI2650 cells, SW-13 cells, T24 cells, WI-28 cells, and the like).VA13, 2RA cells, WISH cells, BS-CI cells, LLC-MK2 cells, clone M-3 cells, 1-10 cells, RAG cells, TCMK-1 cells, Yl cells, LLC-PKi cells, PK(15) cells, GHi cells, GH3 cells, L2 cells, LLC-RC256 cells, MHiCi cells, XC cells, MDOK cells, VSW cells, and TH-I, B1 cells, BSC-1 cells, RAf cells, RK cells, PK-15 cells or their derivatives), fibroblasts derived from any tissue or organ. Cells (heart, liver, kidney, colon, intestine, esophagus, stomach, nervous tissue (brain, spinal cord), lung, vascular tissue (arteries, veins, capillaries), lymphatic tissue (lymph glands, adenoids, tonsils, bone marrow, and blood), spleen, and fibroblast and fibroblast-like cell lines (e.g., CHO cells, TRG-2 cells, IMR-33 cells, Don cells, GHK-21 cells, citrullinemia cells, Dempsey cells, Detroit551 cells, Detroit510 cells, Detroit525 cells, Detroit5 29 cells, Detroit532 cells, Detroit539 cells, Detroit548 cells, Detroit573 cells, HEL299 cells, IMR-90 cells, MRC-5 cells, WI-38 cells, WI-26 cells, Midi cells , CHO cells, CV-1 cells, COS-1 cells, COS-3 cells, COS-7 cells, Vero cells, DBS-FrhL-2 cells, BALB / 3T3 cells, F9 cells, SV-T2 cells, M-MSV-BALB / 3T3 cells, K-BALB cells. The present invention may include, but is not limited to, mouse L cells, BLO-11 cells, NOR-10 cells, C3H / IOTI / 2 cells, HSDMiC3 cells, KLN205 cells, McCoy cells, mouse L cells, line 2071 (mouse L) cells, LM line (mouse L) cells, L-MTK' (mouse L) cells, NCTC clones 2472 and 2555, SCC-PSA1 cells, Swiss / 3T3 cells, Indian muntjac cells, SIRC cells, Cn cells, and Jensen cells, Sp2 / 0, NS0, NS1 cells or derivatives thereof).

[0051] In some exemplary embodiments, the protein of interest may be a VEGF antagonist. As used herein, a "VEGF antagonist" is any agent that binds to or interacts with VEGF, inhibits VEGF binding to its receptors (VEGFR1 and VEGFR2), and / or inhibits the biological signaling and activity of VEGF. VEGF antagonists include molecules that interfere with the interaction between VEGF and a native VEGF receptor, such as molecules that bind to VEGF or a VEGF receptor and prevent or otherwise disrupt the interaction between VEGF and a VEGF receptor. Specific exemplary VEGF antagonists include anti-VEGF antibodies (e.g., ranibizumab [LUCENTIS®]), anti-VEGF receptor antibodies (e.g., anti-VEGFR1 antibodies, anti-VEGFR2 antibodies, etc.), and VEGF receptor-based chimeric molecules or VEGF inhibitory fusion proteins (also referred to herein as "VEGF-traps" or "VEGF mini-traps"), such as aflibercept, ziv-aflibercept, and proteins having the amino acid sequence of SEQ ID NO: 42. Other examples of VEGF-traps are ALT-L9, M710, FYB203, and CHS-2020. Additional examples of VEGF-traps can be found in U.S. Patent Nos. 7,070,959, 7,306,799, 7,374,757, 7,374,758, 7,531,173, 7,608,261, 5,952,199, 6,100,071, 6,383,486, 6,897,294, and 7,771,721, which are specifically incorporated by reference herein in their entireties.

[0052] VEGF receptor-based chimeric molecules include chimeric polypeptides containing two or more immunoglobulin (Ig)-like domains of a VEGF receptor, such as VEGFR1 (also known as Flt1) and / or VEGFR2 (also known as Flk1 or KDR), and may also contain a multimerization domain (e.g., an Fc domain that promotes multimerization, such as dimerization, of two or more chimeric polypeptides). An exemplary VEGF receptor-based chimeric molecule is a molecule designated VEGFR1R2-FcΔC1(a) (also known as aflibercept, commercially available under the product name EYLEA®).

[0053] As used herein, the term "protein pharmaceutical product" includes an active ingredient that may be completely or partially biological in nature. In some exemplary embodiments, the protein pharmaceutical product may include a peptide, a protein, a fusion protein, an antibody, an antigen, a vaccine, a peptide-drug conjugate, an antibody-drug conjugate, a protein-drug conjugate, a cell, a tissue, or a combination thereof. In some other exemplary embodiments, the protein pharmaceutical product may include a recombinant, engineered, modified, mutated, or truncated version of a peptide, a protein, a fusion protein, an antibody, an antigen, a vaccine, a peptide-drug conjugate, an antibody-drug conjugate, a protein-drug conjugate, a cell, a tissue, or a combination thereof.

[0054] Illustrative Embodiments Embodiments disclosed herein provide methods for purifying peptides or proteins, such as antibodies, from samples containing one or more impurities, including viral particles. Embodiments disclosed herein also provide methods for viral clearance using a low pH hold based on statistical experimental design.

[0055] In some exemplary embodiments, the present application provides a method for purifying a peptide or protein, such as an antibody, from a sample containing one or more impurities, including viral particles. In some exemplary embodiments, the method of the present application includes adjusting the ionic strength conditions of the sample and adjusting the pH conditions of the sample to an acidic pH, and then maintaining the sample at the ionic strength and pH conditions for at least about 15 minutes to inactivate a quantity of viral particles.

[0056] In one embodiment, the pH condition of the sample in the method of the present application is an acidic pH, about pH 7 or less, about pH 6 or less, about pH 5 or less, about pH 4 or less, about pH 3.90 or less, about pH 3.80 or less, about pH 3.70 or less, about pH 3.60 to about pH 3.90, or about pH 3.65 to about pH 3.80.

[0057] In one embodiment, the sample in the method of the present application is maintained at the ionic strength and pH conditions of the sample for at least about 30 minutes, at least about 15 minutes, about 15 minutes to about 30 minutes, at least about 10 minutes, at least about 20 minutes, at least about 25 minutes, at least about 35 minutes, about 10 minutes to about 30 minutes, about 15 minutes to about 35 minutes, about 20 minutes to about 30 minutes, about 20 minutes to about 35 minutes, or about 25 minutes to about 40 minutes to inactivate the amount of virus particles.

[0058] In one aspect, the ionic strength of the sample is adjusted using the addition of sodium chloride, wherein the concentration of sodium chloride is in the range of about 1 mM to about 200 mM, about 1 mM to about 500 mM, about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 72 mM, about 80 mM, about 82 mM, about 90 mM, about 100 mM, about 150 mM, about 200 mM, about 250 mM, about 300 mM, about 350 mM, about 400 mM, about 450 mM, or about 500 mM.

[0059] It is understood that the present methods are not limited to any of the aforementioned peptides, proteins, antibodies, D-optimal designs, viruses, retroviruses, viral inactivation, viral clearance, ionic strength, or pH conditions.

[0060] The sequential labeling of method steps provided herein with numbers and / or letters is not intended to limit the method or any embodiment thereof to the particular order indicated. Various publications, including patents, patent applications, published patent applications, accession numbers, technical papers, and academic papers, are cited throughout this specification. Each of these cited references is incorporated herein by reference in its entirety and for all purposes. Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The present disclosure will be more fully understood by reference to the following examples, which are provided to more fully illustrate this disclosure. They are intended to be illustrative and should not be construed as limiting the scope of the disclosure. [Example]

[0061] Materials and Reagents 1. Model Protein and Buffer Different isotypes of monoclonal antibodies expressed in CHO cells were used as model proteins, mAb1, which represents an IgG4 isotype, and mAb2, which represents an IgG1 isotype. The isoelectric points of the model proteins were estimated to be 8.8 for mAb1 and pH 9.3 for mAb2. The monoclonal antibodies were purified using standard protein A chromatography and eluted with 40 mM acetic acid, yielding a pool at approximately pH 4.2. The concentration of the monoclonal antibodies was approximately 15 g / L.

[0062] 2. Spiking and Virus Stock for Infectivity Assays X-MuLV was used to evaluate virus inactivation. X-MuLV virus stock was prepared by WuXi AppTec (Philadelphia, PA). One lot of X-MuLV virus stock solution contains approximately 10 7 The titers were in PFU / mL. For all runs, the virus stock solution was sonicated, filtered, and then spiked into the load material. A 0.5 mL assay volume of infectious samples was removed and analyzed at 15 minutes. A larger 7 mL assay volume of infectious samples was removed and tested at 30 minutes. All samples were quantified for X-MuLV infectivity using PG4 indicator cells. Prior to seeding, samples were neutralized to pH 6.5-7.5. The viral LRF was calculated by comparing the amount of spiked virus present in the load with the amount of virus present in the pool, as described in Equation 1. Here, C(virus, load) denotes the virus concentration in the load, C(virus, pool) denotes the virus concentration in the pool, V(load) denotes the load volume, and V(pool) denotes the pool volume. Formula 1. Virus log 10 Calculating the LRF TIFF2026041901000002.tif12128 samples were evaluated for the cytotoxic effect of the sample matrix on the indicator cell line in the infectivity assay prior to performing the study. Additional controls were performed during the spiking study to ensure that viral inactivation was caused by the presence of a low pH hold and not associated with the protein matrix itself.

[0063] Methods and devices 1. Method for pH titration and spiking: Samples were titrated to the desired pH using either 0.25 M phosphoric acid or 0.25 M glycine HCl. Acidic samples were neutralized using 2 M Tris base. Each inactivation experiment was performed using one of two spiking methods, guided by the experimental design. In spike method 1, e.g., the adjusted spike / readjust method, the sample was adjusted / titrated to the target pH and then spiked with a virus stock solution at a pH of approximately 7.2. The pH hold timing was initiated at the time of the spike. Upon observing a pH increase after spiking the virus stock, the sample's pH was readjusted to the target pH, after which it was held at the desired temperature for the remainder of the pH hold. In spike method 2, e.g., the adjusted spike method, the sample was first spiked with the virus stock solution and then adjusted / titrated to the target pH. Acid addition was performed using a syringe pump with appropriate mixing times and speeds representative of large-scale manufacturing. Once the target pH was reached, the pH hold timing was initiated, and the sample was incubated at the desired temperature.

[0064] For both spike methods 1 and 2, the starting load material was spiked with 2% X-MuLV virus stock (v / v). Where post-spiking filtration was indicated as a factor in the experimental design, 0.2 μm PES filter media (polyethersulfone filter media) was used. The bulk material was incubated in a water bath during the pH hold period. The temperature of the water bath was a factor in the experimental design and was monitored using a calibrated thermometer. All pH measurements were performed offline by removing 2 mL samples from the bulk material in a biosafety cabinet.

[0065] 2. pH measurement device pH measurements were performed using an In Lab Expert Pro pH probe and a SevenCompact S220 pH meter (Mettler Toledo, Columbus, OH). The meter was set to linear calibration mode, exact endpoint format, and automatic temperature compensation. The pH meter was set to display results to two decimal points. The probe was equilibrated in the electrode storage solution for 30–60 minutes before use. The probe (VWR, Radnor, PA) was calibrated using buffer solutions with pH 1.68, pH 4.01, or pH 7.00. The three-point calibration slope criteria were within the range of 97.0–103.0%. After successful calibration, independent buffer standards at pH 3.00 and pH 6.00 were measured to confirm accuracy within ±0.03 pH units of the buffer pH. The independent standards were remeasured daily after completion of the final sample to confirm that the pH was maintained at the target pH. A new pH probe was calibrated against the same standard and used on each day of testing to minimize probe variability over time.

[0066] 3. Statistical Design of Experiments (DoE) for Low pH Retention A statistical design of experiments (DoE) was used to evaluate and characterize the effectiveness of the low-pH hold step for retroviral inactivation. The effects of several factors associated with the low-pH hold step were evaluated, including protein type, pH, temperature, acid titrant, NaCl content, spike timing, and post-spike filtration. To investigate the influence of these factors, statistical DoE was designed using software. D-optimal model designs, such as 15 runs or 30 runs, were generated using JMP software v.13 (SAS, Cary, NC). A scatterplot matrix and multivariate correlations for the D-optimal design are shown in Figure 2A. Seven factors, listed in Table 1, were incorporated into the study design, including: The seven factors included protein type, such as two monoclonal antibodies (mAbs) of different isotypes (e.g., IgG1 or IgG4); pH, such as pH 3.65, pH 3.73, or pH 3.80; temperature, such as 15°C or 20°C; acid titrant, such as 0.25 M phosphate or 0.25 M glycine HCl; NaCl content in the starting solution, such as 0 mM, 50 mM, or 100 mM; spike timing, such as spike adjustment method or spike adjustment method; and post-spike filtration, with or without post-spike filtration. The spike solution contained X-MuLV purified stock for 2% v / v spiking. Table 1 illustrates the implementation of these seven factors into a design matrix, including the specific level or type of each factor.

[0067] Table 1: Factors used in experimental design TIFF2026041901000003.tif67167

[0068] The design evaluated all main effects and several interactions. Fifteen conditions with two replicates, a total of 30 runs, were performed by the same analyst over four days. Additional controls were included for each monoclonal antibody at three levels of NaCl content: 0 mM, 50 mM, or 100 mM NaCl. Samples were analyzed at two time points (T), such as 15 or 30 minutes, using an infectivity assay. The response was X-MuLV LRF at 15 and 30 minutes. Least-squares modeling was performed using JMP® (statistical software from SAS). Analysis of variance (ANOVA) was used to identify statistically significant (P<0.05) factors and interactions. All non-significant factors (P>0.05), collinear factors (VIF>5), and outlier runs (determined by jackknife distance) were removed.

[0069] Neutral controls, shown in Figure 2B, were performed for all monoclonal antibody conditions. These controls were performed to ensure that the measured viral activity was the result of chemical inactivation at low pH. The pH of the load material was adjusted to approximately pH 6.5-7.5. No clearance was expected to be observed to demonstrate that viral inactivation was not due to the protein matrix alone. Samples were taken from pre- and post-filtration steps to monitor potential viral loss to the filter media. Each monoclonal antibody condition had a representative neutral control to serve as the load material for data analysis. Environmental temperature can affect pH measurements. All virus processing, adjustments, and pH measurements were performed at room temperature. Bulk test articles were incubated in a water bath at two different temperatures. Measurements were performed using a Mettler Toledo Expert Pro.

[0070] Example 1. Investigating factors affecting low pH retention As shown in Table 1, the effects of protein type, pH, temperature, acid titrant, NaCl content, spike timing, and post-spike filtration on X-MuLV inactivation for low pH hold were investigated via D-optimal experimental design. Fifteen duplicate runs were performed on four consecutive days using the same virus stock, personnel, buffer, and equipment. A neutral control was performed for each monoclonal antibody (mAb) condition (mAb type and NaCl content, N = 6) and served as the challenge sample for data analysis. The LRF values ​​obtained for each run condition and both the 15 and 30 min time points are listed in Table 2. A summary of the data for the experimental design is shown in Table 2. No virus was detected in 22 of the 30 runs after 30 min. The LRFs for each of the replicate runs after 30 min were within 0.5 LRF of each other, except for two runs that were removed as outliers during data analysis. There was no significant day-to-day or run-to-run variability in the data set based on pH measurements, acid addition, or challenge material.

[0071] In Table 2, for target pH, samples were adjusted to the target pH within ±0.02 pH units. For NaCl content, NaCl was added to the bulk material before titration to a lower pH with acid. The ">" symbol indicates that the virus was reduced below the detection limit of the assay. P indicates phosphate. G indicates glycine HCl. Some specific runs were determined to be outliers by jackknife distance and removed before further data analysis. Prior to the infectivity assay, the sample matrix was evaluated for interference with virus growth in the indicator cell line so that non-interfering dilutions could be identified. Based on observed interference, runs were reported at 3x dilutions. One replicate run unexpectedly interfered with the infectivity assay at 3x dilutions; the reported value was a 10x dilution that prevented interference. This run was determined to be an outlier by jackknife distance and removed from data analysis.

[0072] Table 2: Data summary for experimental design TIFF2026041901000004.tif175170

[0073] Example 2. Analysis of X-MuLV inactivation kinetics at various target pHs Statistical experimental design (DoE) was used to evaluate and characterize the effect of the low pH hold step on virus inactivation, including evaluation of several factors, such as protein type, pH conditions, temperature, acid titrant, ionic strength of the starting solution, spike timing, and post-spike filtration. Statistical DoE was used to evaluate and characterize the effect of the low pH hold step on virus inactivation. The inactivation kinetics of X-MuLV was analyzed considering various target pHs. Samples at 15 and 30 minutes were tested for X-MuLV infectivity, and the initial inactivation (e.g., 15 minutes) and end-of-low pH hold inactivation (e.g., 30 minutes) were evaluated. Figures 3A-3C show the inactivation kinetics of X-MuLV at target pHs of 3.65, 3.73, or 3.80. For each pH value, an LRF curve was obtained by plotting the LRF value against the time point. As shown in Figures 3A-3C, open circles indicate that no virus was detected in the sample, and crosses indicate that virus was detected in the sample. The red line corresponds to runs performed at pH 3.65 ± 0.02, the green line corresponds to runs performed at pH 3.73 ± 0.02, and the blue line corresponds to runs performed at pH 3.80 ± 0.02. As shown in Figure 3A, for the pH 3.65 runs, no virus was detected in any of the samples at 15 minutes, indicating rapid inactivation of the virus (detection limit: approximately 2.5 PFU / mL). For the pH 3.65 runs, two runs showed virus detection at 30 minutes. However, the results for these two runs approached the detection limit of the assay (approximately 1.0 PFU / mL). At pH 3.65 ± 0.02, all LRFs were above 5.0 at 30 minutes. For the pH 3.73 and pH 3.80 runs, there was some discrepancy between runs, as shown in Figures 3B and 3C. Some runs at pH 3.73 or pH 3.80 showed rapid inactivation similar to the runs at pH 3.65. One or two runs at pH 3.73 or pH 3.80 showed incomplete inactivation at 30 minutes. The variability of these runs suggested that another factor may have a stronger effect than pH conditions.

[0074] Example 3. Analysis of X-MuLV inactivation kinetics at different NaCl contents Further analysis was performed on the runs in Table 2 that showed retrovirus detection at 30 min. Common factors, such as NaCl content, were analyzed in these runs that detected virus after 30 min. Runs were performed without adding NaCl to the starting load material. LRF curves were obtained by plotting LRF values ​​against time points for each level of added NaCl. Figures 4A-4C show the inactivation kinetics of X-MuLV at NaCl contents of 0 mM (Figure 4A), 50 mM (Figure 4B), or 100 mM (Figure 4C). As shown in Figures 4A-4C, open circles indicate that no virus was detected in the sample, and crosses indicate that virus was detected in the sample. The red line corresponds to runs performed at approximately pH 3.65 ± 0.02, the green line corresponds to runs performed at approximately pH 3.73 ± 0.02, and the blue line corresponds to runs performed at pH 3.80 ± 0.02.

[0075] The results showed that the conductivity of the starting solution strongly influenced the inactivation kinetics of X-MuLV at various target pH conditions. NaCl content was determined to have the greatest effect on retroviral inactivation. As shown in Figure 4A, when the loading material had low ionic strength or when no additional NaCl was added to the starting material, pH conditions were an important factor. The data suggested that X-MuLV inactivation was pH-dependent at low ionic strength. However, when the ionic strength, e.g., NaCl concentration, was increased, X-MuLV was rapidly inactivated at all three target pH conditions, so no effect of the various target pH conditions was observed. In other words, when the NaCl concentration was increased, the effect of pH conditions on X-MuLV inactivation decreased. When 50 mM or 100 mM NaCl was added, complete and effective clearance of X-MuLV was observed for all runs at the 30-minute time point for the target pH conditions of pH 3.65, pH 3.73, and pH 3.80, as shown in Figure 4B (50 mM NaCl) or Figure 4C (100 mM NaCl), respectively. At low conductivity (e.g., low NaCl content), pH has a strong effect on X-MuLV inactivation. At higher conductivity (e.g., higher NaCl content), pH has no effect on X-MuLV inactivation. 0.25 M glycine HCl showed increased X-MuLV inactivation compared to 0.25 M phosphate.

[0076] Example 4. Generating a multivariate linear regression model To investigate the influence of various factors, multivariate linear regression models were generated to explore the main, secondary, and interaction effects of the evaluated factors. Furthermore, the effect of ionic strength on X-MuLV inactivation at both the 15 and 30 minute time points was quantified. The generated models had an adjusted R of approximately 99% with an error estimate of less than 0.15 LRF, as shown in Table 3. 2 values, while the reported variability of the infectivity assay is 0.5 LRF (ICH Q5A(R1)). In Table 3, R 2 indicates the correlation coefficient, adjusted R 2indicates the amount of variation explained by the model, and RMSE indicates the root mean square error for estimating the error in the method. Table 4 lists significant (P<0.05) factors for each model. Actual vs. predicted plots are shown in Figures 5A and 5B. Plots of actual vs. predicted values ​​of the multivariate linear regression model of LRF for two time points are shown in Figures 5A and 5B, e.g., 15 minutes (Figure 5A) or 30 minutes (Figure 5B). In Figures 5A and 5B, the red line indicates the fitted line, the blue line indicates the mean line, and the red shaded lines indicate the 95% confidence interval.

[0077] Table 3. Fitted significant multivariate linear regression models for each time point. TIFF2026041901000005.tif23136

[0078] Table 4. Significant factors determined from the LRF multivariate linear regression model generated for each time point. TIFF2026041901000006.tif107158

[0079] The significant parameters of the LRF models generated for 15 and 30 min were the same, showing similar trends in effect size. However, the interaction between mAb and pH was an exception, as it was only significant for the 30-min LRF model. pH conditions were known to be important for viral inactivation. However, the model showed that NaCl had the strongest effect on X-MuLV inactivation at both the 15 and 30 min time points. As NaCl concentration increased, the LRF of X-MuLV increased accordingly until ionic strength no longer affected inactivation. Other factors determined to be important were the secondary nature of NaCl content, the interaction between pH and NaCl content, and pH conditions. The effect of NaCl concentration on inactivation kinetics can be explained according to Figure 6. As NaCl concentration increased, the effect of pH on X-MuLV inactivation decreased.

[0080] The main effects of temperature, mAb, post-spike filtration, acid titrant, spike method, and the interaction between pH and mAb were all significant within the linear regression model. However, the effect sizes of these factors were smaller than the reported variability of the infectivity assay (e.g., 0.5 LRF). Although these factors were significant within the generated model (P < 0.05), they were not practically significant because their effect sizes were smaller than the variability of the infectivity assay. Temperature had a known effect on virus inactivation, as decreasing temperature correlates with decreased inactivation kinetics. However, this dataset supported minimal differences in inactivation between 15°C and 20°C.

[0081] A predictive profiler was created to optimize the model to achieve greater than 4 LRF after both the 15 and 30 minute time points, as shown in Figure 6. Figure 6 shows the predictive profiler representing X-MuLV inactivation as a function of significant factors evaluated in the D-optimal DoE. The red line indicates the fit, the blue line indicates the mean, and the red shaded lines indicate the 95% confidence intervals. P indicates phosphate, and G indicates glycine HCl. When all other factors remain constant, increasing conductivity via the addition of NaCl in viral inactivation procedures in the pH range of approximately 3.60-3.90 can result in greater retroviral LRF.

[0082] The statistical DoE test results for the low pH hold in this application were consistent with the ASTM standard for X-MuLV inactivation in 5.0 LRF at pH below 3.60. The results also demonstrated robust and effective inactivation at pHs above 3.60. For ranges outside the ASTM comprehensive requirements, the results indicated that rapid and effective X-MuLV inactivation could be achieved by increasing the NaCl content. Typically, the pH of the hold depends on protein stability. The model in this application can be used to predict effective viral clearance when operating in the pH range of 3.60 to 3.90 by manipulating the conductivity of the low-pH starting material.

[0083] High protein concentrations, such as above 25 g / L, have been reported to negatively impact X-MuLV inactivation (ASTM). However, previous Regeneron research demonstrated that higher protein concentrations could potentially improve X-MuLV inactivation kinetics under conditions where inactivation may not be complete. Conditions with increased ionic strength, such as higher buffer concentrations, weak acid titration, or higher protein concentrations, correlated with higher LRFs at higher pH (Chinniah et al.). While the data set in this application used two monoclonal antibodies with similar concentrations, the data conclusions were consistent in that increasing protein concentration increases inactivation. Due to the increase in acid titrant required to achieve the desired pH, more ions were added during titration. This increased ionic strength of the solution would suggest greater inactivation kinetics.

[0084] Similar to the effect of protein concentration, significant differences were observed for the acid titrant, with glycine HCl (a weaker acid) correlated with higher LRF values ​​than phosphate (a stronger acid). This conclusion was not practically significant because the effect size was less than 0.5 LRF. The results supported increased inactivation with increasing ion concentration in solution. Previous studies have shown that lower temperatures result in decreased clearance due to the thermodynamics of viral inactivation. Temperature was statistically significant in the generated linear regression model, but with a minimal effect size within the study range (15–20°C). Previous studies have concluded that there is no statistically significant difference in viral inactivation between 15°C and 16+°C (Mattila et al., Retrospective evaluation of low-pH virus inactivation and viral filtration data from a multiple company collaboration, PDA Journal of Pharmaceutical Science and Technology 70.3 (2016): 293–299). The experiments in this application supported the ASTM comprehensive viral clearance requirements and the identification of solutions to achieve effective retrovirus inactivation above pH 3.60. At higher pH, increasing the ionic strength of the solution can promote viral dispersion. In low-conductivity solutions, retroviral glycoproteins can potentially aggregate at low pH, protecting themselves from chemical damage. With the addition of 50 mM and 100 mM NaCl, all runs at all pH set points demonstrated complete and effective clearance after 30 minutes. In summary, an operating space can be defined for effective X-MuLV inactivation when experiments are operated at pHs outside the ASTM module requirements.

[0085] Example 5. Evaluating Existing and Redesigned Operating Conditions A statistical design of experiments (DoE) was used to evaluate and characterize the effect of a low pH hold step for virus inactivation, including evaluation of several factors such as protein type, pH conditions, temperature, acid titrant, NaCl content, spike timing, and post-spike filtration. Using the DoE for the low pH hold step, several existing operating conditions at pH 3.70-3.75 were evaluated for low pH hold. A predicted profiler with parameter estimates was generated, as shown in Figure 7.

[0086] Using the DoE for the low pH hold step, several existing operating conditions at pH 3.65-3.70 were also evaluated for the low pH hold. A predicted profiler with parameter estimates was generated, as shown in Figure 8. The redesigned operating conditions at approximately pH 3.65-3.70 failed to meet the clearance of X-MuLV in 4LRF at the 30-minute time point with a failure rate of 1.5%.

[0087] Example 6. Evaluating the Factor of Protein Type Statistical DoE was used to evaluate and characterize the effect of a low-pH hold step on virus (X-MuLV) inactivation, including evaluation of several factors, such as protein type, pH conditions, temperature, acid titrant, NaCl content, spike timing, and post-spike filtration. As shown in Figure 9, the DoE demonstrated statistical significance in predicting multivariate models for protein types, such as monoclonal antibody isotypes, but differences between protein types, such as IgG1 and IgG4, were not significant within the range studied. Figure 9 shows the scaled estimated LRFs for the evaluated factors, including NaCl, pH, acid titrant, temperature, protein type (mAb, monoclonal antibody), spike timing, and their combinations. Retrospective data for existing operating conditions showed significant differences between monoclonal antibody isotypes, such as IgG1 and IgG4. However, these differences may be due to differences in operating pH ranges. Figure 9 also shows the X-MuLV LRF at 30 minutes for protein types, including IgG1 and IgG2, based on retrospective data.

[0088] Example 7. Evaluating the Factor of Temperature A statistical DoE was used to evaluate and characterize the effect of a low-pH hold step on virus (X-MuLV) inactivation, including evaluation of several factors, such as protein type, pH conditions, temperature, acid titrant, NaCl content, spike timing, and post-spike filtration. As shown in Figure 10, the DoE demonstrated statistical significance in the multivariate model predictions for temperature, but differences between various temperature conditions had minimal effect on X-MuLV clearance in the study range. Figure 10 shows the scaled estimated LRFs for the evaluated factors, including NaCl, pH, acid titrant, temperature, protein type (mAb, monoclonal antibody), spike timing, and their combinations. Retrospective data from industrial operating conditions in the range of 15°C to 20°C (Mattila et al.) showed no statistically significant differences between 15±1°C and 16+°C. Figure 10 also shows the retrovirus LRFs for various temperature conditions based on retrospective industrial data.

[0089] Example 8. Evaluating spike timing factors A statistical DoE was used to evaluate and characterize the effect of a low pH hold step on virus (X-MuLV) inactivation, including evaluation of several factors, such as protein type, pH conditions, temperature, acid titrant, NaCl content, spike timing, and post-spike filtration. The spike timing in this statistical DoE is the adjusted spike readjustment method or spike adjustment method. In the adjusted spike readjustment method, the sample is adjusted / titrated to a target pH and then spiked with a virus stock having a pH of 7.2. The pH hold timing was initiated at the time of the spike. Upon observing a pH increase after spiking the virus stock, the pH of the sample is readjusted to the target pH, after which it is held at the desired temperature for the remainder of the pH hold. In the spike adjustment method, the sample is first spiked with the virus stock solution and then adjusted / titrated to the target pH. Once the target pH is reached, the pH hold timing is initiated and the sample is incubated at the desired temperature.

[0090] As shown in Figure 11, the difference in spike timing for the two different methods does not significantly affect X-MuLV clearance in the range studied. Figure 11 shows the scaled estimated LRF for the factors evaluated, including NaCl, pH, acid titrant, temperature, protein type (mAb, monoclonal antibody), spike timing, and their combinations. Figure 11 also shows the spike adjustment times for the two methods.

Claims

1. 1. A method for purifying a peptide or protein from a sample, comprising: The method comprises: subjecting the sample to increasing ionic strength by addition of salt; subjecting the sample to an acidic pH; The sample is then maintained at the ionic strength and pH conditions for at least about 15 minutes to inactivate a quantity of viral particles. Including, The method, wherein the sample contains one or more impurities, including the virus particles.

2. 10. The method of claim 1, wherein the amount of viral particle inactivation is at least about 3 LRF (log reduction).

3. 10. The method of claim 1, wherein the amount of viral particle inactivation is at least about 4 LRF.

4. 10. The method of claim 1, wherein the pH condition of the sample is about pH 3.90 or less.

5. 10. The method of claim 1, wherein the pH condition of the sample ranges from about pH 3.60 to about pH 3.

90.

6. 10. The method of claim 1, wherein the pH condition of the sample ranges from about pH 3.65 to about pH 3.

80.

7. The method of claim 1 , wherein the peptide or protein is an antibody produced in a host cell.

8. 10. The method of claim 1, wherein the sample is maintained at said ionic strength and pH conditions for at least about 30 minutes to inactivate said quantity of infectious viral particles.

9. 10. The method of claim 1, wherein the sample is maintained at the ionic strength and pH conditions for about 15 minutes to about 30 minutes to inactivate the quantity of infectious viral particles.

10. D - Optimizing the ionic strength and pH conditions of the sample for inactivation of the amount of infectious viral particles by performing an optimal experimental design. The method of claim 1 further comprising:

11. 11. The method of claim 10, wherein the D-optimal experimental design evaluates the pH conditions of the sample and the ionic strength of the sample and adjusts the pH conditions of the sample and the ionic strength of the sample to inactivate a quantity of infectious viral particles.

12. The D-optimal experimental design the conductivity of the sample; the type of the peptide or protein; the temperature of the sample; an acid titrant for adjusting the pH condition of the sample; a method for spiking said viral particles into said sample; or Presence of post-spike filtration The method of claim 11 further comprising evaluating and adjusting one or more of:

13. 2. The method of claim 1, wherein the sample is an eluate from Protein A chromatography.

14. the ionic strength of the sample is adjusted using the addition of sodium chloride; the concentration of the sodium chloride is in the range of about 1 mM to about 100 mM; The method of claim 1.

15. 10. The method of claim 1, wherein the concentration of the sodium chloride ranges from about 1 mM to about 500 mM.

16. 2. The method of claim 1, wherein the concentration of sodium chloride is about 25 mM, about 50 mM, or about 100 mM.

17. 10. The method of claim 1, wherein the pH condition of the sample is adjusted using phosphoric acid or glycine HCl.

18. The method of claim 1, wherein the peptide or protein is an antibody having an IgG1 isotype or an IgG4 isotype.

19. The method of claim 1 , wherein the peptide or protein is a monoclonal antibody or a bispecific antibody.

20. The method of claim 1, wherein the peptide or protein is an antibody, an antibody fragment, a Fab region of an antibody, an antibody-drug conjugate, a fusion protein, a protein pharmaceutical product, or a drug.

21. 1. A method for producing a preparation containing a protein of interest and a reduced amount of viral particles from a sample having said protein of interest and infectious viral particles, comprising: subjecting the sample to a pH greater than about 3.6; subjecting the sample to conditions of increased ionic strength by the addition of salt to the starting solution; maintaining the sample at the pH and ionic strength conditions for an appropriate period of time to produce the preparation containing the protein of interest and the reduced amount of infectious viral particles; A method comprising:

22. 22. The method of claim 21, wherein the concentration of the protein of interest in the sample is greater than about 25 g / L.

22. 22. The method of claim 21, wherein the suitable period of time is about 15 minutes, about 20 minutes, about 25 minutes, or about 30 minutes.

23. 22. The method of claim 21, wherein the amount of infectious viral particles from a sample is reduced by about 3 LRF (log reduction).

24. 22. The method of claim 21, wherein the amount of infectious viral particles from a sample is reduced by about 4 LRF (log reduction factor).

25. 22. The method of claim 21, wherein the pH condition of the sample is greater than about pH 3.70, about 3.80, about pH 3.90, or about pH 4.

0.

26. 22. The method of claim 21, wherein the pH condition of the sample ranges from about pH 3.60 to about pH 4.

0.

27. 22. The method of claim 21, wherein the sample is an eluate from Protein A chromatography.

28. the ionic strength of the sample is adjusted using the addition of sodium chloride; the concentration of the sodium chloride is in the range of about 1 mM to about 200 mM; 22. The method of claim 21.

29. 22. The method of claim 21, wherein the concentration of the salt is greater than about 50 mM or about 100 mM.

30. 22. The method of claim 21, wherein the pH condition of the sample is adjusted using phosphoric acid or glycine HCl.

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