Methods for virus inactivation

Abstract

Embodiments of the present disclosure relate to systems and methods for adjusting the pH in a mixture containing a polypeptide. The pH can be adjusted for a suitable purpose, such as to inactivate viruses in the mixture. The method can include eluting the mixture (e.g., eluate) from a chromatography column, the mixture having a pH greater than 3.9, e.g., less than 8.5. The method can further include one or more of measuring the protein concentration of the mixture and measuring the pH of the mixture. The amount of acid required to lower the pH of the mixture to a target pH can then be calculated based on the protein concentration of the mixture, the pH of the mixture, or both. After calculating the amount of acid to be added, a portion of the acid can be added to the mixture, where the portion of the acid is sufficient to achieve the target pH.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims priority to U.S. Provisional Patent Application No. 62 / 881,692, filed on August 1, 2019, the entire disclosure of which is incorporated herein by reference.

[0002] Technical Field The present disclosure generally relates to methods for achieving a target pH in a mixture containing a polypeptide. More specifically, the present disclosure relates to methods for achieving a target pH in a mixture containing a polypeptide to help ensure that enveloped viruses or virus - like particles are inactivated.

Background Art

[0003] In the production of polypeptides, it may be necessary to separate a target molecule (e.g., the target polypeptide component of a drug product) from a culture medium. For example, a separation process such as affinity chromatography can be carried out as part of the target molecule preparation process. After such a separation process, the resulting mixture containing the polypeptide can potentially contain unwanted viruses or other contaminants that are undesirable to be present in a drug product. Therefore, methods for removing or inactivating such contaminants are desirable.

[0004] Process Analytical Technology (PAT) may be employed in some commercial-scale target molecule synthesis processes. PAT includes systems and methods involved in the design, analysis, and control of the target molecule manufacturing process. PAT includes identifying process parameters that affect product quality and regularly monitoring these parameters to ensure product quality is maintained. PAT is encouraged by regulatory bodies to reduce overall risks associated with target molecules and pharmaceutical products. PAT can provide statistical verification or confirmation that one or more process conditions are met that can improve or maintain the quality of the target molecule and / or product. [Overview of the project]

[0005] The methods and systems disclosed herein may improve the efficiency and / or productivity of polypeptide preparation methods, including virus inactivation. The methods and systems disclosed herein may also improve the efficiency and / or productivity of pharmaceutical product preparation methods and may address one or more of the problems identified above.

[0006] overview Embodiments of the present disclosure may relate to a method for inactivating a virus in a mixture, for example, an eluate. The method may include eluting the mixture from a chromatography column at a pH greater than 3.9 and less than 8.5. The method may further include measuring the protein concentration of the mixture and measuring the pH of the mixture. Then, based on the protein concentration of the mixture, the amount of acid required to lower the pH of the mixture to an inactivation pH can be calculated. After calculating the amount of acid to add, a first portion of the acid, which is 68% to 99% of the amount of acid to add, may be added to the mixture. The method may further include adding an additional portion of the acid to the mixture so that the pH of the mixture is below the inactivation pH. In the method of the present disclosure, the mixture may be configured to be held at the inactivation pH for an inactivation interval, thereby inactivating the virus in the mixture.

[0007] In some embodiments of this disclosure, a method for inactivating a virus in a mixture may include loading a mixture containing the target molecule into a chromatographic column, the loading of which may be performed at a pH between about 5.0 and about 8.5. This method may further include eluting an eluted mixture containing the target molecule from the chromatographic column at a pH between about 3.9 and about 5.0. This method may also include adding an acid to the mixture to form a mixture-acid combination, the combination configured to exhibit effective virus inactivation, and the combination having a pH between about 3.8 and about 3.0. The expected pH of the combination can be predetermined using a pH confirmation model. Furthermore, the pH of the combination may be measured and / or recorded. The difference between the expected pH and the recorded pH may be calculated, and corrective measures may be taken based on the calculated difference between the expected pH and the recorded pH.

[0008] Further embodiments of this disclosure may include a method for developing an acidic inactivation protocol. This method may include preparing a pool of eluents, each eluent in the pool containing a target molecule purified in a protein affinity capture process. This method may include measuring the pH and / or protein concentration of each eluent in the pool. Furthermore, each eluent in the pool can be titrated to determine the amount of acid required to bring the eluent to an inactivation pH. The relationship between the amount of acid added, the eluent protein concentration, the eluent pH, and the inactivation pH can then be regression-dependent.

[0009] In some embodiments of this disclosure, a method for inactivating a virus in a mixture may include measuring the protein concentration of the mixture. Then, based on the protein concentration of the mixture, the amount of acid required to lower the pH of the mixture to an inactivation pH can be calculated. After calculating the amount of acid to be added, the amount of acid required to lower the pH of the mixture may be added to the mixture.

[0010] Brief explanation of the drawing The accompanying drawings incorporated herein and constituting part of this specification illustrate various exemplary embodiments and, together with the descriptions, serve to illustrate the principles of the disclosed embodiments. Any feature of the embodiments or examples described herein (e.g., compositions, formulations, methods, etc.) can be combined with any other embodiments or examples, and all such combinations are encompassed by this disclosure. Furthermore, the systems and methods described herein are not limited to any single aspect or embodiment thereof, nor to any combination or permutation of such aspects and embodiments. For the sake of brevity, certain substitutions and combinations are not individually described and / or illustrated herein. [Brief explanation of the drawing]

[0011] [Figure 1] Figure 1 shows an exemplary process for inactivating the virus in the eluate according to this disclosure, in flowchart format.

[0012] [Figure 2] Figure 2 shows an exemplary process for inactivating the virus in the eluate according to this disclosure, in flowchart format.

[0013] [Figure 3] Figure 3 shows an exemplary process for developing an acidic inactivation protocol according to this disclosure, in flowchart format. [Modes for carrying out the invention]

[0014] As used herein, the terms “comprises,” “comprising,” or other variations thereof are intended to cover non-exclusive inclusion, and a process, method, article, or apparatus containing a list of elements may not contain only those elements, but may also contain other elements not expressly listed or inherent in such process, method, article, or apparatus. The term “exemplary” is used in the sense of “example,” not “ideal.” The terms “for example” and “such as” and their grammatical equivalents are understood to be followed by the phrase “and without limitation,” unless otherwise explicitly stated.

[0015] As used herein, the term "about" means to account for variations due to experimental error. When applied to numerical values, the terms "about" and "approximately" may indicate a variation of + / - 5% from the disclosed value unless a different variation is specified. When applied to pH values, the terms "about" and "approximately" may indicate a variation of + / - 0.05. As used herein, the singular forms "a," "an," and "the" include multiple criteria unless the context clearly indicates otherwise.

[0016] It should be noted that all numerical values ​​disclosed herein (including all disclosed values, limits, and ranges) may have a variation of + / - 5% from the disclosed value unless different variations are specified. pH values ​​disclosed herein may have a variation of + / - 0.05. Furthermore, all ranges are understood to include the endpoint; for example, 1 cm to 5 cm includes lengths of 1 cm and 5 cm, and all distances between 1 cm and 5 cm.

[0017] Detailed explanation This disclosure is not limited to any specific composition, formulation, material manufacturer, drug product, device, system, experimental conditions, or method disclosed herein, and many modifications are possible within the scope of those skilled in the art. The terms used herein are for the purpose of describing only specific embodiments and are not intended to limit them.

[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this disclosure belongs. Any suitable methods and materials (e.g., similar or equivalent to those described herein) may be used in the implementation or testing of this disclosure, but specific methods are described herein. All publications described herein are incorporated herein by reference.

[0019] As used herein, the term "polypeptide" refers to any amino acid polymer having more than about 20 amino acids covalently linked via amide bonds. Proteins contain one or more amino acid polymer chains (e.g., polypeptides). Thus, polypeptides can be proteins, and proteins can contain multiple polypeptides to form a single functional biomolecule.

[0020] Post-translational modifications can alter or change the structure of polypeptides. For example, disulfide crosslinks (e.g., disulfide bonds between cysteine ​​residues) can be formed post-translation in some proteins. Some disulfide crosslinks are essential for the proper structure, function, and interactions of polypeptides, immunoglobulins, proteins, cofactors, and substrates. In addition to disulfide bond formation, proteins can undergo other post-translational modifications such as lipidation (e.g., myristoylation, palmitoylation, farnesoylation, geranylgeranylation, and glycosylphosphatidylinositol (GPI) anchoring), alkylation (e.g., methylation), acylation, amidation, glycosylation (e.g., addition of glycosyl groups to arginine, asparagine, cysteine, hydroxylysine, serine, threonine, tyrosine, and / or tryptophan), and phosphorylation (i.e., addition of phosphate groups to serine, threonine, tyrosine, and / or histidine). Post-translational modifications can affect hydrophobicity, electrostatic surface properties, or other properties that determine the surface interactions involving polypeptides.

[0021] As used herein, the term "protein" includes biotherapeutic proteins, recombinant proteins used in research or therapy, trap proteins and other Fc fusion proteins, chimeric proteins, antibodies, monoclonal antibodies, human antibodies, bispecific antibodies, antibody fragments, antibody-like molecules, nanobodies, recombinant antibody chimeras, cytokines, chemokines, peptide hormones, and the like. A protein-of-interest (POI) can include any polypeptide or protein that is desired to be isolated, purified, or otherwise prepared. The POI can include a target polypeptide or other polypeptide produced by a cell, including an antibody.

[0022] As used herein, the term "antibody" includes immunoglobulins consisting of four polypeptide chains: two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. Typically, an antibody has a molecular weight of more than 100 kDa, for example, 130 kDa to 200 kDa, such as about 140 kDa, 145 kDa, 150 kDa, 155 kDa, or 160 kDa. Each heavy chain includes a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region consists of three domains, CH1, CH2, and CH3. Each light chain includes a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region includes one domain, CL. The VH and VL regions can be further divided into hypervariable regions called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, which are arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (heavy chain CDRs can be abbreviated as HCDR1, HCDR2, and HCDR3; light chain CDRs can be abbreviated as LCDR1, LCDR2, and LCDR3).

[0023] For example, a type of immunoglobulin called immunoglobulin G (IgG) is commonly found in human serum and contains four polypeptide chains, two light chains and two heavy chains. Each light chain is bound to one heavy chain via a cystine disulfide bond, and the two heavy chains are bound to each other via two cystine disulfide bonds. Other classes of human immunoglobulins include IgA, IgM, IgD, and IgE. In the case of IgG, there are four subclasses: IgG1, IgG2, IgG3, and IgG4. Each subclass may have different effector functions because their constant regions are different. In some embodiments described herein, the POI may include a target polypeptide that includes IgG. In at least one embodiment, the target polypeptide includes IgG4.

[0024] As used herein, the term “antibody” also includes antigen-binding fragments of a complete antibody molecule. Terms such as “antigen-binding portion of an antibody,” “antigen-binding fragment of an antibody,” as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Antigen-binding fragments of an antibody can be derived, for example, from a complete antibody molecule using any suitable standard techniques such as proteolytic digestion or recombinant genetic engineering techniques that involve manipulation and expression of DNA encoding the antibody variable domain and optionally the constant domain. Such DNA is readily available, for example, from commercial sources, DNA libraries (including phage-antibody libraries), and / or can be synthesized. The DNA can be sequenced and manipulated, for example, using chemical or molecular biology techniques, to place one or more variable domains and / or constant domains in a suitable configuration, introduce codons, generate cysteine residues, modify amino acids, add, or delete amino acids.

[0025] The target molecule (e.g., target polypeptide) can be produced using recombinant cell-based production systems such as insect baculovirus systems, yeast systems (e.g., Pichia sp.), or mammalian systems (e.g., CHO cells and CHO derivatives such as CHO-K1 cells). The term “cell” includes cells suitable for expressing recombinant nucleic acid sequences. Cells include prokaryotes and eukaryotes (unicellular or multicellular), bacterial cells (e.g., strains such as E. coli, Bacillus spp., and Streptomyces spp.), mycobacteria cells, fungal cells, yeast cells (e.g., S. cerevisiae, S. pombe, P. pastoris, P. methanolica, etc.), plant cells, insect cells (e.g., SF-9, SF-21, baculovirus-infected insect cells, Trichoplusiani, etc.), non-human animal cells, human cells, or cell fusions such as hybridomas or quadromas. In some embodiments, cells may be human, monkey, ape, hamster, rat, or mouse cells. In some embodiments, the cells may be eukaryotes and may be selected from the following cells: CHO (e.g., CHO K1, DXB-11 CHO, Veggie-CHO), COS (e.g., COS-7), retinal cells, Vero, CV1, kidney (e.g., HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK), HeLa, HepG2, WI38, MRC 5, Colo205, HB 8065, HL-60, (e.g., BHK21), Jurkat, Daudi, A 431 (epidermal), CV-1, U937, 3T3, L cells, C127 cells, SP2 / 0, NS-0, MMT 060562, Sertoli cells, BRL 3A cells, HT1080 cells, myeloma cells, tumor cells, and cell lines derived from the above cells. In some embodiments, the cells may include one or more viral genes, for example, retinal cells expressing the viral gene (e.g., PER.C6(TM) cells).

[0026] The term “target molecule” may be used herein to refer to a target polypeptide (e.g., an antibody, antibody fragment, or other protein or protein fragment), or other molecules intended to be manufactured, isolated, purified, and / or included in a pharmaceutical product (e.g., adeno-associated virus (AAV) or other molecules for therapeutic purposes). While the methods according to this disclosure may refer to target polypeptides, they may also be applied to other target molecules. For example, AAV may be prepared by a suitable method (e.g., depth filtration, affinity chromatography, etc.), and a mixture containing AAV (e.g., an eluate containing AAV) may be subjected to a method according to this disclosure. Before or after following one or more methods according to this disclosure, the mixture containing AAV may be subjected to additional steps (e.g., removal of an “empty cassette” or AAV that does not contain the target sequence).

[0027] The term "viral content" refers to a qualitative description of a mixture. For example, if a mixture contains viruses or virus-like particles, then the mixture has a viral content. In some embodiments, viral content can be quantified by the number of viral particles or infectious units (i.e., concentration) per unit volume of the mixture. The term "viral concentration" may mean the concentration of viral particles (e.g., active and inactive viral particles) or the concentration of infectious units.

[0028] Exemplary methods for virus inactivation may include adding an acid to a mixture to achieve a pH known to inactivate certain viruses and virus-like particles, and holding the mixture at the achieved pH for a predetermined time. For example, in some embodiments, the methods herein may inactivate retroviruses and retrovirus-like particles. In some embodiments, a method for preparing a target molecule from a mixture containing the target molecule may include contacting the mixture with a chromatographic apparatus. Such a chromatographic apparatus may include pre-fabricated apparatus (e.g., Cadence™ BioSMB (Pall Biosciences), BioSC® (novasep), Varicol® (novasep), Octave (Semba® Biosciences)), custom-fabricated apparatus, manually assembled apparatus, or simply two or more standard batch chromatographic apparatuses used in tandem.

[0029] In some embodiments, the target molecule can be eluted from the chromatograph by contacting the chromatograph (e.g., a chromatographic column) with a stripping buffer and / or by contacting the chromatograph with an equilibration buffer. In some embodiments, the stripping buffer may include water, an alkaline solution, or a solution containing alcohol. For example, water such as deionized water may have less than 5% by volume (vol.%) of dissolved ions, less than 1% by volume, less than 0.1% by volume, or less than 0.01% by volume. According to some embodiments, the alkaline solution may include one or more alkali ion compounds such as LiOH, NaOH, KOH, Ca(OH)2, NH4OH, or other alkali compounds. The concentration of the alkaline compound in the stripping buffer may be in the range of, for example, approximately 0.1N to 1.5N, approximately 0.1N to 1N, approximately 0.1N to 1.5N, approximately 0.5N to 1.5N, approximately 0.1N to 0.8N, approximately 0.1N to 0.6N, approximately 0.1N to 0.5N, approximately 0.1N to 0.4N, or approximately 0.1N to 0.3N. For example, the concentration of the alkaline compound in the stripping buffer can be approximately 0.1N, approximately 0.2N, approximately 0.3N, approximately 0.4N, approximately 0.5N, approximately 0.6N, approximately 0.7N, approximately 0.8N, approximately 0.9N, approximately 1N, approximately 1.1N, approximately 1.2N, approximately 1.3N, approximately 1.4N, or approximately 1.5N. The alcohol-containing stripping buffer may include methanol, ethanol, propanol, benzyl alcohol, or other alcohols. The alcohol concentration in the stripping buffer is approximately 0.1 vol.% to approximately 30 vol.%, for example, 0.5 vol.% to approximately 30 vol.%, approximately 0.5 vol.% to approximately 25 vol.%, or approximately 0.5 vol.% to approximately 25 vol.% based on the total weight of the stripping buffer.The alcohol concentration in the stripping buffer can be approximately 0.5% to 25% by volume, approximately 1% to 20% by volume, approximately 1% to 15% by volume, approximately 1% to 10% by volume, approximately 10% to 50% by volume, approximately 10% to 40% by volume, approximately 10% to 30% by volume, approximately 10% to 25% by volume, approximately 15% to 25% by volume, or approximately 20% to 25% by volume. For example, the alcohol concentration in the stripping buffer can be approximately 0.1% by volume, approximately 0.5% by volume, approximately 1% by volume, approximately 2% by volume, approximately 3% by volume, approximately 5% by volume, approximately 10% by volume, approximately 15% by volume, approximately 20% by volume, or approximately 25% by volume.

[0030] In some embodiments, the equilibration buffer may have a composition similar to or identical to that of the stripping buffer. In other embodiments, the equilibration buffer may have a different composition compared to the stripping buffer. In some embodiments, the equilibration buffer may contain one or more salts, such as sodium, potassium, magnesium, calcium, citrate, acetate, phosphate, sulfate, Tris, or other salts.

[0031] In one or more embodiments, a method for virus inactivation may be used after a mixture containing the target molecule has been eluted from a chromatographic apparatus (e.g., a packed bed affinity chromatography column, a hydrophobic interaction chromatography column, an ion exchange chromatography column, and / or a size exclusion chromatography column). The loading of the target molecule into the chromatographic apparatus may vary depending on the upstream process, but the mixture containing the target molecule can be eluted from the chromatographic apparatus in a constant volume. In some embodiments, the mixture containing the target molecule is loaded into the chromatographic column at a pH of about 5.0 to about 8.5, for example, about 5.5 to about 8.5, about 6.0 to about 8.5, or about 5.0 to about 6.5. As a result of variations in loading, the protein concentration and / or pH of the eluate (e.g., the eluate eluted from the chromatographic apparatus) may vary. This variation also causes variations in the amount of acid required for virus inactivation.

[0032] In conventional manufacturing processes, low-pH virus inactivation is achieved through trial and error, where a predetermined amount of acid is added to the eluate, the pH of the eluate and acid mixture is measured, and the addition and measurement steps are repeated until an inactivation pH is reached. Due to the potential costs and losses resulting from the addition of excessive acid, such processes are conservative, involving small amounts of acid and often long inactivation process times on a time scale.

[0033] Aspects of this disclosure may offer various advantages to processes for preparing target polypeptides or other target molecules. For example, one or more methods and / or mathematical models described herein can be used to determine the amount of acid to be added, e.g., the amount of acid required to bring a mixture (containing the target molecule and a potentially undesirable virus or virus-like particle) to an inactivating pH. An amount of acid approximately equal to the amount to be added may be added to the mixture. As will be described in more detail below, an amount of acid equivalent to the amount to be added may be added as a single bolus or in two or more doses of acid. Dosing acid for virus inactivation in this manner is more efficient and less susceptible to error than conventional trial-and-error methods.

[0034] In some aspects of this disclosure, as part of the process, the viral content or infectious unit content in the mixture may be known or expected to be minimal or absent. In some such aspects of this disclosure, the systems and methods disclosed herein can be advantageously incorporated into a manufacturing process as part of a PAT to, for example, reduce potential variability within the process, provide real-time verification of compliance with process standards, and / or enhance the reliability of process integrity.

[0035] Further advantages and merits of the embodiments of this disclosure will be apparent to those skilled in the art.

[0036] As suggested above, after preparing the target molecule using one or more chromatographic and / or separation processes, a mixture (e.g., eluate) can be obtained. In some embodiments, one or more measurements of the mixture may be performed, including, for example, protein concentration, target molecule concentration, pH, or a combination thereof. Protein concentration can be measured by any suitable method, including, for example, ultraviolet / visible light spectroscopy. In some embodiments, protein concentration is measured using wavelengths that are characteristically absorbed by the target molecule, which may be a polypeptide. In such embodiments, the overall protein concentration may be approximately equivalent to the concentration of the target molecule (e.g., target polypeptide). In some embodiments, the mixture containing the target molecule may have protein concentrations of about 7.0 g / L to about 35.0 g / L, about 7.0 g / L to about 20.0 g / L, about 8.5 g / L to about 18.5 g / L, or about 10.0 g / L to about 17.0 g / L per liter (g / L) of eluate.

[0037] The pH of a mixture can be measured by any suitable method. Accurate and consistent pH measurement is crucial for successful inactivation of viral proteins by low pH. pH measurement can be affected by temperature, the type of pH probe used, individual differences between pH probes of the same type, and / or physical interactions between the medium being measured and the pH probe. Even with standardized pH measurement processes, variations of ±0.05 pH are common. In the fields of polypeptide production or viral inactivation, variations on the scale of ±0.05 pH represent approximately 20% of the working pH range and can have detrimental effects on viral inactivation and / or process validation. Such variations can become compound over time, even leading to instrument drift and more extreme variations in pH measurements. Therefore, in some embodiments, variations in pH measurement can be taken into account when measuring pH. In some embodiments, as suggested, the pH of the eluate can be measured by a standardized method with the aim of reducing or eliminating variability in pH measurement. Standardization of the pH measurement method may include using a single manufacturer of pH probes, using a single lot of pH probes, measuring pH at a predetermined temperature, and standardizing the measurement sample matrix. In some embodiments, the pH of the eluate can be measured by a pH meter, such as a potentiometer pH meter. In some embodiments, the eluate containing the target molecule may have a pH between about 3.9 and about 8.5, for example, about 3.9 to about 6.5, about 3.9 to about 5.5, about 4.5 to about 6.5, about 4.0 to about 4.4, about 3.9 to about 4.4, or about 4.0 to about 4.3. In this specification, pH values ​​or ranges of pH values ​​may have variations of ±0.05 pH units.

[0038] The presence of certain viruses and virus-like particles (e.g., enveloped viruses, retroviruses, retrovirus-like particles, pseudorabies, herpesviruses, etc.) in mixtures (e.g., eluates), formulations, and / or pharmaceutical products may affect the components, properties, or usability of such mixtures, formulations, and / or pharmaceutical products. For example, the presence of unwanted viruses or virus-like particles in a pharmaceutical product may affect the stability of the product, shorten its shelf life, or cause the product to fail to comply with internal standards, pharmacopoeias, or regulations (e.g., the U.S. Food and Drug Administration). Some viruses and virus-like particles can cause clinical effects, such as immunogenic reactions, upon administration of a formulation containing the virus. Embodiments of the Disclosure may be useful in inactivating viruses or virus-like particles to reduce or eliminate any or all of such undesirable effects. For example, embodiments of the Disclosure may be applicable to a mixture (e.g., eluate) containing a virus after one or more polypeptide purification processes (e.g., a separation process including a protein A affinity column).

[0039] In some embodiments, the conductivity of a mixture can be measured before virus inactivation. In some embodiments, one or more salts can be added to the mixture before virus inactivation to adjust its conductivity (e.g., increase its conductivity). Such one or more salts may include alkali metal salts, alkaline earth metal salts, halides, and / or one or more other ionic active compounds. Without being limited by theory, the addition of one or more salts to adjust conductivity may reduce the aggregation of target molecules, viruses, or retrovirus-like particles. Aggregation of target molecules, viruses, or retrovirus-like particles may affect how the surfaces of these species interact with acids. Adding salts to a mixture may increase the ionic activity of the mixture, increase its conductivity, and reduce the aggregation of target molecules or viruses. Thus, in some embodiments, the conductivity of the mixture may be related to the degree of aggregation of target molecules or viruses.

[0040] In some cases, embodiments of the present disclosure may be applicable to mixtures with extremely low viral content (e.g., less than or equal to about 0.0001 viral particles or infectious units per mL) or no viral content at all. Chromatography and other separation processes, alone or in combination, can adequately purify and / or separate target molecules and remove unwanted viruses or virus-like particles from the mixture. In such cases, the methods according to the present disclosure may be useful to additionally ensure that viruses or virus-like particles are inactivated and to guarantee product stability, product safety, product efficacy, and compliance with internal or regulatory specifications. Thus, embodiments of the present disclosure may also be applicable to mixtures with no known viral content, for example, to ensure that regulatory guidelines are met and / or to provide redundant quality control.

[0041] The virus inactivation protocols and methods described herein can be carried out without adversely affecting certain types of viruses, such as AAV (e.g., AAV containing a target sequence). For example, advantageously, the protocols and methods described herein can be carried out without degrading AAV. Therefore, the methods described herein may be suitable for use in mixtures containing AAV as a target molecule.

[0042] As suggested above, viruses in a mixture can be inactivated by holding the mixture at an inactivation pH for an inactivation time. The inactivation pH may be between 3.8 and 3.0, for example, 3.35-3.8, 3.75-3.0, 3.7-3.0, 3.65-3.0, 3.6-3.0, 3.55-3.0, 3.5-3.0, 3.45-3.0, 3.40-3.0, 3.35-3.75, 3.5-3.8, 3.5-3.75, 3.5-3.7, 3.5-3.6, or 3.5-3.65. As used herein, pH values ​​or ranges of pH values ​​may have variations of ±0.05 pH units. If the inactivation pH is too high, variability may occur within the inactivation process, potentially resulting in insufficient virus inactivation. Lowering the inactivation pH too low carries the risk of denaturing target molecules or other proteins, or altering the mixture in undesirable ways.

[0043] The inactivation time refers to the interval of time during which the mixture is held at an inactivating pH. The inactivation time can range from approximately 20 to 90 minutes, for example, approximately 30 minutes, 45 minutes, 60 minutes, approximately 30 to 45 minutes, approximately 30 to 60 minutes, approximately 30 to 75 minutes, approximately 30 to 90 minutes, approximately 45 to 60 minutes, approximately 45 to 75 minutes, approximately 45 to 90 minutes, approximately 60 to 75 minutes, or approximately 60 to 95 minutes. By holding the mixture at an inactivating pH for the inactivation time, the viral activity in the mixture can be reduced, removed, or reliably inactivated. A low pH environment can denature viral proteins, such as viral envelope proteins. Denatured viral proteins can inactivate retroviruses and retrovirus-like particles, thereby reducing the undesirable viral activity of the mixture.

[0044] The reduction in viral activity in a mixture can be quantified by the reduction factor. The reduction factor can be calculated according to Equation 1 shown below.

number

[0045] According to one or more embodiments, the amount of acid added can be calculated based on the protein concentration and inactivation pH of the mixture. For example, the amount of acid added can be calculated according to Equation 2 below. w = Ax + By + C Equation (2) In the formula, x is the protein concentration of the mixture expressed in grams per liter (g / L), y is the inactivation pH, w is the amount of acid added expressed in moles of acid per kilogram of the mixture (mol / kg), and A, B, and C are constants. The constant A in formula 2 has the unit of liters-moles of acid per gram-kilogram of the mixture (L·mol / g·kg). The constant A may be between 0.0003 L·mol / g·kg and 0.0006 L·mol / g·kg, for example, approximately 0.0003 L·mol / g·kg to approximately 0.0005 L·mol / g·kg, approximately 0.0004 L·mol / g·kg to approximately 0.0006 L·mol / g·kg, approximately 0.00035 L·mol / g·kg to approximately 0.0005 L·mol / g·kg, approximately 0.00035 L·mol / g·kg to approximately 0.0006 L·mol / g·kg, or approximately 0.0004 L·mol / g·kg to approximately 0.00055 L·mol / g·kg. The constants B and C in Equation 2 have units (mol / kg) representing the number of moles of acid per kilogram of the mixture. The constant B may be between -0.1 mol / kg and 0 mol / kg, for example, approximately -0.1 mol / kg to approximately 0 mol / kg, approximately -0.1 mol / kg to approximately -0.05 mol / kg, approximately -0.05 mol / kg to approximately 0 mol / kg, or approximately -0.08 mol / kg to approximately -0.01 mol / kg. The constant C may be between 0.02 mol / kg and 0.1 mol / kg, for example, approximately 0.02 mol / kg to approximately 0.1 mol / kg, approximately 0.02 mol / kg to approximately 0.05 mol / kg, approximately 0.05 mol / kg to approximately 0.1 mol / kg, or approximately 0.04 mol / kg to approximately 0.08 mol / kg.

[0046] In some embodiments, the amount of acid added can be calculated based on the protein concentration of the mixture, the inactivation pH, and the pH of the mixture. For example, the amount of acid added can be calculated according to Equation 3 shown below. w=Ex+Fy+Gz+H Formula (3) In the formula, x is the protein concentration of the mixture expressed in grams per liter (g / L), y is the inactivation pH, z is the pH of the mixture, w is the amount of acid added expressed in moles of acid per kilogram of the mixture (mol / kg), and E, F, G, and H are constants. The constant E in formula 3 has units of liters-moles of acid per gram-kilogram of the mixture (L·mol / g·kg), and the constants F, G, and H have units of moles of acid per kilogram of the mixture (mol / kg). The constant E can be greater than or equal to 0.00005 L·mol / g·kg and less than or equal to 0.0005 L·mol / g·kg, for example, approximately 0.00005 L·mol / g·kg to approximately 0.0005 L·mol / g·kg, approximately 0.0001 L·mol / g·kg to approximately 0.0005 L·mol / g·kg, approximately 0.00005 L·mol / g·kg to approximately 0.00045 L·mol / g·kg, approximately 0.0001 L·mol / g·kg to approximately 0.00035 L·mol / g·kg, or approximately 0.00035 L·mol / g·kg to approximately 0.0005 L·mol / g·kg. The constant F may be between -0.2 mol / kg and 0 mol / kg, for example, approximately -0.1 mol / kg to approximately 0 mol / kg, approximately -0.1 mol / kg to approximately -0.05 mol / kg, approximately -0.05 mol / kg to approximately 0 mol / kg, or approximately -0.08 mol / kg to approximately -0.01 mol / kg. The constant G may be between 0 mol / kg and 0.03 mol / kg, for example, approximately 0 mol / kg to approximately 0.03 mol / kg, approximately 0.001 mol / kg to approximately 0.03 mol / kg, approximately 0.005 mol / kg to approximately 0.3 mol / kg, or approximately 0.005 mol / kg to approximately 0.025 mol / kg. The constant H can be between -0.1 mol / kg and 0.1 mol / kg, for example, approximately -0.1 mol / kg to approximately 0.1 mol / kg, approximately -0.08 mol / kg to approximately 0.08 mol-10 / kg, approximately -0.05 mol / kg to approximately 0.1 mol / kg, or approximately -0.1 mol / kg to approximately 0.05 mol / kg.

[0047] The specific formula relating the mixture protein concentration to the amount of acid added and the inactivation pH (which may depend on the mixture pH as needed) may vary depending on the target molecule and / or acid system used. The values ​​of the constants defined above, applicable to a given target molecule and acid system, can be determined by regression according to the general formula defined above. As described in the Examples section below, it is unexpectedly found that the amount of acid added has a strong correlation with the mixture protein concentration according to the defined formula above. This unexpectedly strong correlation makes it possible to incorporate the general formulas and their derivatives described herein into PAT.

[0048] In one or more embodiments, the amount of acid added is 0.002 moles (mol / kg) to about 0.025 mol / kg per kg of the mixture, for example, about 0.002 mol / kg to about 0.025 mol / kg, about 0.01 mol / kg to about 0.025 mol / kg, about 0.002 mol / kg to about 0.020 mol / kg, or about 0.005 mol / kg to about 0.020 mol / kg.

[0049] In some embodiments, after the amount of acid to be added has been calculated, the acid can be added to the mixture to bring the mixture to an inactivating pH. For example, in at least one embodiment, a bolus of acid equivalent to the amount of acid to be added can be added to the mixture to bring the pH of the mixture below the inactivating pH. In other embodiments, a first portion of the acid may be added to the mixture, and then one or more additional portions of the acid may be added to the mixture so that the pH of the mixture is below the inactivating pH. In such embodiments, the first portion of the acid is 68% to 99% of the amount of acid added, for example, about 75% to about 99%, about 80% to about 99%, about 85% to about 99%, about 90% to about 99%, about 85% to about 95%, or about 90% to about 99%, etc.

[0050] The first portion of the acid may be allocated such that the target molecule in the mixture has the lowest possible pH is not denatured by the addition of the first portion. Additional portions of the acid may include one or more additions of the acid made after the addition of the first portion, such as three additions, four additions, or five additions. Each of the one or more additions of the acid may be in amounts ranging from 0.1% to 32% of the total amount of acid added, for example, about 0.1% to 30%, about 0.1% to 25%, about 0.1% to 20%, about 1% to 25%, about 0.1% to 15%, about 0.1% to 10%, about 1% to 15%, about 1% to 10%, or about 0.1% to 5%. Each of the one or more additions of acid may be in the same amount as the other one or more additions of acid. In other embodiments, each addition of acid is in the same amount as the other additions of acid.

[0051] In some embodiments, the pH can be measured after the first portion of the acid has been added to the mixture, but before the addition of any additional portions of the acid. In some such embodiments, the pH of the mixture measured after the addition of the first portion of the acid is between 3.5 and 3.75, for example, about 3.5 to about 3.75, about 3.6 to about 3.75, about 3.5 to about 3.65, about 3.6 to about 3.75, or about 3.5 to about 3.65. In this specification, pH values ​​or ranges of pH values ​​may have variations of ±0.02 pH units.

[0052] Acids can be added in the form of one or more acid solutions. The acid solutions may include any suitable acid, such as HCl, HBr, H3PO4, HO2C2O2H, C6H8O7, H2SO3, H3PO4, HNO2, C6H5CO2H, CH3CO2H, HClO, HCN, H3BO3, or combinations thereof. In addition, or alternatively, the acid solutions may include one or more salts, such as glycine, arginine, sodium acetate, and / or sodium chloride.

[0053] After adding an amount of acid to a mixture containing the target molecule, the resulting mixture has a pH below the inactivation pH. In some embodiments, the pH of the formed mixture can be measured, for example, to confirm that it is within a desired range. As previously mentioned, the mixture may be held at the inactivation pH for an inactivation time. After holding the mixture at the inactivation pH for an inactivation time, a decrease in viral activity may occur, equivalent to a reduction factor of, for example, 2.5 or higher, 3 or higher, 3.5 or higher, or 4 or higher. After holding the mixture at the inactivation pH for an inactivation time, the mixture can be titrated by adding an alkaline solution so that the pH of the mixture is between 4.5 and 8.5, for example, 4.5 or higher, 5.0 or higher, 5.8 or higher, 5.9 or higher, 6.0 or higher, 6.1 or higher, 6.2 or higher, 6.3 or higher, 6.4 or higher, etc. In this specification, pH values ​​or ranges of pH values ​​may have variations of ±0.02 pH units. The alkaline solution may contain one or more bases, such as NaOH, KOH, LiOH, Ca(OH)2, NH4OH, NaCH3CO2 and / or (HOCH2)3CNH2.

[0054] In some embodiments, the mixture is titrated for less than one hour after the first portion of the acid is added, for example, less than about 50 minutes, less than about 45 minutes, less than about 40 minutes, less than about 35 minutes, or less than about 30 minutes.

[0055] Aspects of this disclosure may also include a method for determining a function for predicting the pH of a combination (i.e., a combination of a mixture and an acid). For example, a function for predicting the pH of a combination (e.g., inactivation pH) can be determined based on the measured protein concentration and / or pH of the mixture and the amount of acid added to the mixture. Such a function can be used to detect processing errors (e.g., insufficient mixing, insufficient sampling, etc.) or instrument malfunctions (e.g., instrument error, instrument drift, pH probe anomaly, etc.).

[0056] In some embodiments, the expected pH of the acidified mixture can be predetermined according to a confirmation model. The confirmation model may be in the form of Equation 4 shown below. Equation (4) y = Kx + Lw + Mz + N In the formula, y is the inactivation pH, x is the protein concentration of the mixture expressed in grams per liter (g / L), z is the pH of the mixture, w is the amount of acid added expressed in moles of acid per kilogram of the mixture (mol / kg), and K, L, M, and N are constants. In formula 4, the constant K has units of liters per gram, the constant L has units of kilograms of the mixture per mole of acid, and the constants M and N are unitless. The constant K can be between 0 L / g and 0.03 L / g, for example, about 0 L / g to about 0.03 L / g, about 0.001 L / g to about 0.03 L / g, about 0 L / g to about 0.025 g / L, or about 0.001 g / L to about 0.025 L / g. The constant L may be between -80 kg / mol and -60 kg / mol, for example, approximately -75 kg / mol to approximately -60 kg / mol, approximately -80 kg / mol to approximately -65 kg / mol, or approximately -75 kg / mol to approximately -65 kg / mol. The constant M may be between 0 and 2.0, for example, approximately 0 to approximately 2.0, approximately 0.3 to approximately 2.0, approximately 0 to approximately 1.7, or approximately 0.3 to approximately 1.7. The constant N may be a number between -1.0 and 0, for example, approximately -1.0 to approximately 0, approximately -1.0 to approximately -0.1, or approximately -0.9 to approximately -0.1.

[0057] In some embodiments, the pH and protein concentration of the mixture before acid addition can be measured and / or recorded. Based on the mixture pH, the mixture protein concentration, and the amount of acid added, the predicted pH of the acidified mixture can be predetermined using a verification model. The actual pH of the acidified mixture can be measured, recorded, and / or compared to the predicted pH. Comparing the recorded pH to the predicted pH may include calculating the difference (e.g., percentage difference) between the recorded pH and the predicted pH.

[0058] In some embodiments, if the difference between the recorded pH and the predicted pH is greater than a threshold, corrective measures may be taken. The threshold may be, for example, ±0.03 pH from the predicted pH, ±0.05 pH from the predicted pH, ±0.07 pH from the predicted pH, ±0.09 pH from the predicted pH, ±0.1 pH from the predicted pH, ±0.15 pH from the predicted pH, or ±0.2 pH from the predicted pH. Corrective measures may include, but are not limited to, adjusting the pH meter, adjusting the composition of the mixture, adjusting one or more environmental conditions of the process, or a combination thereof. Adjusting the pH meter may include standardizing the pH meter, recalibrating the pH meter, washing, resetting and / or replacing the pH probe, adjusting the reference electrode solution, replacing a part of the pH meter, adjusting the position of the pH probe, and / or other actions that change the signal-to-noise ratio of the pH meter. Adjusting the composition of the mixture may include recombining any component solutions of the mixture or upstream composition, changing process conditions or instrument components of one or more chromatography or separation processes, and / or other actions that change the material composition of the mixture. Adjusting one or more environmental conditions of a process may include adjusting the mixing and / or homogenization process and system, adjusting the process temperature, adjusting the process humidity, adjusting the process pressure, or a combination thereof. Such adjustments based on deviations from predicted parameters may be incorporated as part of the PAT.

[0059] The above formulas and mathematical models may be derived as part of a method for developing an acidic inactivation protocol. A method for developing an acidic inactivation protocol may include preparing a pool of mixtures (e.g., eluents), where each mixture in the pool contains a target molecule purified in a protein affinity capture process. For example, a pool of samples can be collected from the eluents of a protein affinity chromatography column. This method may further include measuring the pH and protein concentration (e.g., the concentration of the target molecule) of each sample. After determining the pH and protein concentration of each sample, each sample can be titrated to determine the amount of acid required to bring the sample to an inactivating pH. In some embodiments, the inactivating pH may be defined as a sufficiently broad range that allows for two or more titrations to be repeated, enabling the collection of multiple data points from each sample in the pool. In other embodiments, only a single data point may be collected from each sample.

[0060] In some embodiments, a relationship (e.g., a mathematical model) can be regressiond between the amount of added acid, the eluate protein concentration, the mixture pH, and / or the inactivation pH. This relationship can be regressiond according to Equation 2 or Equation 3, as described above. In some embodiments, the method for developing an acid inactivation protocol further includes regression of a confirmation model. The confirmation model can be regressiond according to Equation 4, as described above.

[0061] Figure 1 shows an exemplary process 100 for inactivating a virus in a mixture according to the present disclosure in flowchart form. According to step 101, the mixture can be eluted from a chromatographic column to a first pH (e.g., higher than 3.9). According to step 102, the protein concentration (e.g., target molecule concentration) of the mixture can be measured, for example, by ultraviolet / visible light spectroscopy or other methods. Optionally, the conductivity and / or pH of the mixture can be measured, for example, by a potentiometer or other methods. According to step 103, one or more salts can be added to the mixture in an amount sufficient to adjust the conductivity of the mixture. According to step 104, the amount of acid required to lower the pH of the mixture to a second pH (e.g., inactivation pH) can be calculated. This calculation can be performed based on the protein concentration of the mixture, the pH of the mixture, or a combination thereof. According to step 105, a first portion of the acid can be added to the mixture. In some embodiments, the first portion of the acid is a bolus of acid corresponding to the calculated amount of acid added. In other embodiments, the first portion of the acid may be 68% to 99% of the volume or amount of the acid as the calculated acid addition amount. Optionally, according to step 105, a second (secondary) acid may be added to the mixture such that the combination of the mixture and the acid is below a second pH (e.g., inactivation pH). According to step 106, the combination of the mixture and the acid may be held at the second pH (e.g., inactivation pH) for an inactivation interval to inactivate the virus in the eluate. In some embodiments,

[0062] Figure 2 shows an exemplary process 200 for inactivating a virus in a mixture according to this disclosure in flowchart form. According to step 201, a mixture containing the target molecule may be loaded onto a chromatography column at a first pH (e.g., between about 5.0 and about 8.5). According to step 202, an eluted mixture at a second pH (e.g., between about 3.9 and about 5.0) may be eluted from the chromatography column. According to step 203, the protein concentration (e.g., target molecule concentration) of the eluted mixture may be measured and / or recorded, for example, by ultraviolet / visible light spectroscopy. According to step 204, the pH and / or conductivity of the eluted mixture may optionally be measured. The pH may be measured and / or recorded, for example, using a potentiometer. According to step 205, one or more salts may be added to the mixture in an amount sufficient to adjust the conductivity of the mixture. According to step 206, an acid may be added to the eluted mixture to form a combination of the eluted mixture and the acid, configured such that the combination exhibits effective virus inactivation. Furthermore, the combination may have a pH lower than or equal to the second pH (e.g., approximately 3.8 or lower and approximately 3.0 or higher). The predicted pH of the combination may be predetermined using a pH verification model according to step 207. The pH of the combination can be measured and / or recorded according to step 208. The difference between the predicted pH of the combination and the measured / recorded pH can be calculated according to step 209. Corrective measures may be taken according to step 210 based on the calculated difference between the predicted pH of the combination and the measured and / or recorded pH.

[0063] Figure 3 shows an exemplary process 300 for developing an acid inactivation protocol according to this disclosure in flowchart form. According to step 301, a pool of eluate samples can be prepared, where each eluate sample in the pool contains a target molecule purified in an affinity capture process. According to step 302, the protein concentration (e.g., target molecule concentration) of each eluate sample in the pool can be measured, for example, by ultraviolet / visible light spectroscopy. Optionally, the pH of each eluate sample in the pool can be measured, for example, by a potentiometer. According to step 303, the amount of acid required to lower the pH of each eluate sample to the inactivation pH can be determined by titrating each eluate sample in the pool. According to step 304, the relationship between the amount of acid added, the eluate protein concentration, the eluate pH, and / or the inactivation pH can be regressiond. According to step 305, a confirmation model may be regressiond as needed.

[0064] Figures 1-3 each illustrate a specific sequence of steps, but it should be understood that the steps performed and the order in which they are performed may be modified. Furthermore, steps (e.g., one or more measurement and / or recording steps) may be added to or removed from any method disclosed herein. In addition, although each of Figures 1-3 illustrates a step with respect to an eluate, it should be understood that the steps are applicable to any mixture containing the target molecule. [Examples]

[0065] The following embodiments are intended to illustrate the disclosure without being inherently limiting. It is understood that the disclosure includes further aspects and embodiments consistent with the foregoing description and the following embodiments.

[0066] In the following examples, the target polypeptide was prepared from a mixture containing the target polypeptide, host cell proteins, viruses, and other contaminants, impurities, and components. The target polypeptide was prepared in Chinese hamster ovary cells grown in suspension culture.

[0067] Example 1 The target polypeptide was eluted from a protein A affinity column to obtain multiple samples of the mixture. The protein concentration of each mixture sample was obtained by UV / Vis spectroscopy and is shown in Table 1 below in grams of protein per liter of mixture. Furthermore, the pH of each mixture sample was measured and is shown in Table 1.

[0068] The eluate pool contained 40 eluate samples (i.e., 40 mixture samples), and each sample was titrated with a 0.25 M solution of phosphoric acid (H3PO4) to an inactivation pH of 3.35–3.86 to determine the amount of acid to add (e.g., the amount of acid that needs to be added to bring the mixture to an inactivation pH). For 6 of the 40 samples (Samples 1–6 shown in Table 1), the titration was repeated multiple times to obtain multiple data points. For example, 30.89 g of acid per kilogram of the mixture to inactivate Sample No. 1 to pH 3.70 is one data point, and adding an additional 5.35 g of acid per kilogram of the mixture to inactivate Sample No. 1 to pH 3.59 results in 36.24 g of acid per kilogram, which is another data point. The data for these titrations, in terms of the amount of acid added in grams of acid per kilogram of the mixture and in moles of acid per kilogram of the mixture, are shown in Table 1 below. Table 1 - Mixture Titration Data [Table 1-1] [Table 1-2]

[0069] Example 2 Using the titration data shown in Table 1, the relationship between the protein concentration in the mixture, the inactivation pH, and the amount of acid added was regression-treated according to Equation 2. The regression equation below (Equation 5) is given by the coefficient of determination (R 2 It was determined that the value is 0.84. w=0.0004532x-0.01135y+0.04213 Equation (5)

[0070] Example 3 Furthermore, referring to the titration data shown in Table 1, the relationship between the protein concentration of the mixture, the pH of the mixture, the inactivation pH, and the amount of acid added was regression-treated according to Equation 3. The regression equation below (Equation 6) is given by the coefficient of determination (R 2 The ratio was determined to be 0.97. Unexpectedly, the amount of acid added correlated highly with the relationship between protein concentration, mixture pH, and inactivation pH shown in general formulas 2 and 3. w=0.0001986x-0.01162y+0.01510z-0.01692 Formula (6)

[0071] Example 4 The confirmation model was regressioned using the titration data shown in Table 1, according to General Equation 4. The regression equation below (Equation 7) is the coefficient of determination (R 2 The inactivation pH was determined to be 0.93. Unexpectedly, the inactivation pH was highly correlated with the relationship between protein concentration, mixture pH, and inactivation pH. y=0.01488x-71.65w+1.094z-0.6727 Formula (7)

[0072] Those skilled in the art will realize that the concepts underlying this disclosure are not other concepts for carrying out some of the purposes of this disclosure. You will understand that it can be easily used as a basis for designing methods and systems. Furthermore, aspects of this disclosure relate to specific steps in a particular process (e.g., in a mixture) Although it is described regarding virus inactivation, those skilled in the art will understand that the system disclosed herein The method and its application may be used in other contexts (for example, in the process of manufacturing prescribed drugs). Before the chromatography process or after combining the eluate with other components, etc. It is also understood that this is applicable to virus inactivation in other mixtures containing Virus. Therefore, the scope of the claims is not limited by the foregoing description. It will not be done. <Note> Item 1 A method for inactivating viruses in a mixture, Elute the mixture from the chromatography column at a pH higher than 3.9 and less than 8.5. To cause; To measure the protein concentration of the aforementioned mixture; Based on the protein concentration of the mixture, the pH of the mixture is lowered to an inactivation pH. Calculate the amount of acid needed; The first part of the acid, which is necessary to lower the pH of the mixture to an inactivating pH. Adding a first portion of the acid, which is 68% to 99% of the total amount of acid, to the mixture; and The pH of the mixture and the acid combination is set to be below the inactivation pH. A method comprising adding an additional portion of acid. Section 2 Maintaining the aforementioned combination at the aforementioned inactivation pH for the inactivation time; and After the aforementioned inactivation time, less than one hour after adding the first portion of the acid to the mixture, The method according to claim 1, further comprising titrating the combination until the pH is between 4.5 and 8.5. Section 3 Before adding the first portion of the acid to the mixture, measure the pH of the mixture. The method described in item 1, including the method described in item 1. Section 4 The amount of acid required to lower the pH of the mixture to the inactivating pH is given by the following formula The method described in item 3 is used to calculate this. w = Ax + By + C (In the formula, w is the amount of acid expressed as the number of moles of acid per kilogram of the mixture, and x is 1 The protein concentration of the mixture expressed in grams per liter, where y is the same as the un This is the activation pH, where A, B, and C are constants. Section 5 A is approximately 0.0003 L·mol / g·kg or more, or approximately 0.0006 L·mol / g·kg The following; B is approximately -0.1 mol / kg or more and approximately 0.0 mol / kg or less; and C is between approximately 0.02 and approximately 0.1. The method described in item 4. Section 6 Based on the protein concentration of the mixture and the pH of the mixture, the pH of the mixture is The method according to item 3, wherein the amount of acid required to lower the pH to the aforementioned inactivation pH is calculated. Section 7 The amount of acid required to lower the pH of the mixture to the inactivating pH is the amount of the mixture The method according to item 1, wherein the amount is approximately 0.002 moles to approximately 0.025 moles per kilogram. Section 8 The method according to item 1, wherein the inactivation pH is approximately 3.8 or less and approximately 3.0 or more. Section 9 To measure the electrical conductivity of the mixture; and To adjust the conductivity of the mixture, add one or more salts in sufficient quantities to the mixture. The method according to item 1, further comprising the above. Section 10 The chromatography column performs the protein affinity capture process. The method described in item 1, configured to perform the action. Section 11 After the first portion of the acid is added, and before the additional portion of the acid is added, The method according to item 1, wherein the pH of the mixture is approximately 3.5 to approximately 3.75. Section 12 A method for inactivating viruses in a mixture, A mixture containing a target molecule, having a pH of approximately 5.0 to approximately 8.5, Loading into a romagraphic column; From the chromatography column, an elution mixture containing the target molecule is obtained, approximately 3. To elute a mixture having a pH higher than 9 and approximately 5.0 or lower; A certain amount of acid is added to the elution mixture, and the combination of the elution mixture and the acid is effective. It is configured to exhibit effective virus inactivation, with a pH of approximately 3.8 or lower and approximately 3.0 or higher. To form a combination of having; The predicted pH of the above combination is determined in advance using a pH verification model; Record the pH of the aforementioned combination; Calculating the difference between the predicted pH and the recorded pH; and Based on the calculated difference between the predicted pH and the recorded pH, corrective measures are taken. Methods that include... Section 13 If the difference between the predicted pH and the recorded pH is approximately 0.15 or more, the correction measure The method described in item 12, wherein the arrangement is taken. Section 14 The aforementioned correction measures include adjusting the pH meter, adjusting the composition of the mixture, and one or more other such measures. The method described in paragraph 12, which includes adjusting the environmental conditions, or a combination thereof. Section 15 The method according to item 12, wherein the combination is maintained at a pH of approximately 3.8 or less and approximately 3.0 or more for approximately 30 minutes. Section 16 After adding the aforementioned amount of acid to the aforementioned mixture, the combination is reduced to approximately 4.5 to approximately 8 in less than 1 hour. The method according to item 12, further comprising titrating to a pH of 0.5 or less. Section 17 To measure the electrical conductivity of the mixture; and To adjust the conductivity of the mixture, add one or more salts in sufficient quantities to the mixture. The method described in paragraph 12, further including the following. Section 18 Measure the pH of the mixture before adding the aforementioned amount of acid to the mixture; and The method according to claim 12, further comprising measuring the protein concentration of the mixture. Section 19 The pH confirmation model is the method described in item 18, which includes the following formula. y = Kx + Lw + Mz + N (In the formula, y is the pH of the above combination, and x is expressed in grams per liter.) The protein concentration of the mixture is the amount of the mixture before adding the amount of acid. The pH of the mixture is given, where w is the number of moles of acid per kilogram of the eluate. This represents the amount of acid added to the compound, where K, L, M, and N are constants. Item 20 A method for developing an acidic deactivation protocol for a mixture, A pool of eluate samples of the aforementioned mixture, and the pool of eluate samples!! Elution The liquid sample contains target molecules purified by the protein affinity capture process. To prepare a pool of eluent samples; Measure the pH and protein concentration of each eluate sample; Each eluate sample in the pool of eluate samples is titrated to inert each eluate sample. Determining the amount of acid needed to achieve the desired pH; Determine data points using each eluate sample from the pool of eluate samples. thing; and Using the aforementioned data points, determine the amount of acid required to bring the mixture to an inactivating pH. The relationship between the protein concentration of the mixture, the pH of the mixture, and the inactivation pH is Methods that involve regression. Section 21 The method described in section 20, further including regression on the confirmation model. Section 22 Multiple data points are determined using at least one eluate from the pool of eluates. The method described in paragraph 20, further including the following: Section 23 A method for inactivating viruses in a mixture, To measure the protein concentration of the aforementioned mixture; Based on the protein concentration of the mixture, the pH of the mixture is reduced to an inactivation pH. Calculating the amount of acid needed to do so; and This includes adding an amount of acid to the mixture necessary to bring the pH of the mixture to an inactivating pH. Hmm, a method. Section 24 Using a pH confirmation model, the predicted p of the mixture after the acid is added to the mixture is used. The method according to paragraph 23, further comprising determining H in advance. Section 25 Record the pH of the combination of the acid and the aforementioned mixture; Calculating the difference between the predicted pH and the recorded pH; and Based on the calculated difference between the predicted pH and the recorded pH, corrective measures are taken. The method described in paragraph 24, further including the following: Section 26 Before adding the aforementioned amount of acid: To measure the electrical conductivity of the mixture; and To adjust the conductivity of the mixture, add one or more salts in sufficient quantities to the mixture. The method described in paragraph 23, further including the following.

Claims

1. A method for inactivating viruses in a mixture, Elute the mixture from the chromatography column at a pH higher than 3.9 and less than 8.5; To measure the protein concentration of the aforementioned mixture; Based on the protein concentration of the mixture, calculate the amount of acid required to lower the pH of the mixture to an inactivating pH; Adding to the mixture a first portion of the acid, which is 68% to 99% of the amount of acid necessary to lower the pH of the mixture to an inactivating pH; and This includes adding an additional portion of the acid to the mixture such that the pH of the mixture and the acid combination becomes less than or equal to the inactivation pH. The amount of acid required to lower the pH of the mixture to the inactivating pH is given by the following formula (2): w=Ax+By+C Formula (2) A method calculated according to (formula (2)), where w is the amount of acid expressed as the number of moles of acid per kilogram of the mixture, x is the protein concentration of the mixture expressed as grams per liter, y is the inactivation pH, A, B and C are constants, and the values ​​of A, B and C are regression-driven according to formula (2).

2. Maintaining the aforementioned combination at the inactivation pH for the inactivation time; and The method according to claim 1, further comprising adding the first portion of the acid to the mixture after the inactivation time, and then titrating the combination for less than one hour until the pH is 4.5 or higher and 8.5 or lower.

3. The method according to claim 1, further comprising measuring the pH of the mixture before adding the first portion of the acid to the mixture.

4. A is 0.0003 L / mol / g / kg or more and 0.0006 L / mol / g / kg or less; B is -0.1 mol / kg or more and 0.0 mol / kg or less; and C is between 0.02 and 0.

1. The method according to claim 1.

5. The method according to claim 1, wherein the amount of acid required to lower the pH of the mixture to the inactivation pH is 0.002 moles to 0.025 moles per kilogram of the mixture.

6. The method according to claim 1, wherein the inactivation pH is 3.8 or less and 3.0 or more.

7. To measure the electrical conductivity of the mixture; and The method according to claim 1, further comprising adding a sufficient amount of one or more salts to the mixture to adjust the conductivity of the mixture.

8. The method according to claim 1, wherein the chromatography column is configured to perform a protein affinity capture process.

9. The method according to claim 1, wherein the pH of the mixture is 3.5 to 3.75 after the first portion of the acid is added and before the additional portion of the acid is added.

10. A method for inactivating viruses in a mixture, A mixture containing the target molecule, with a pH between 5.0 and 8.5, is loaded into a chromatography column; Elute from the chromatography column an ​​elution mixture containing the target molecule, having a pH higher than 3.9 and less than or equal to 5.0; Based on the protein concentration of the mixture, calculate the amount of acid required to lower the pH of the mixture to an inactivating pH; Adding the aforementioned amount of acid to the elution mixture to form a combination of the elution mixture and the acid that is configured to exhibit effective virus inactivation and has a pH of 3.8 or less and 3.0 or more; The predicted pH of the above combination is determined in advance using a pH verification model; Record the pH of the aforementioned combination; Calculating the difference between the predicted pH and the recorded pH; and This includes taking corrective measures based on the calculated difference between the predicted pH and the recorded pH, The pH confirmation model is given by the following formula (4): y=Kx+Lw+Mz+N Formula (4) A method comprising (4) (wherein y is the pH of the combination, x is the protein concentration of the mixture expressed in grams per liter, z is the pH of the mixture before the amount of acid is added to the mixture, w is the amount of acid added to the mixture expressed in moles of acid per kilogram of eluate, K, L, M and N are constants, and the values ​​of K, L, M and N are regressions according to (4).)

11. The method according to claim 10, wherein the correction measure is taken when the difference between the predicted pH and the recorded pH is 0.15 or more.

12. The method according to claim 10, wherein the corrective measures include adjusting a pH meter, adjusting the composition of the mixture, adjusting one or more environmental conditions, or a combination thereof.

13. The method according to claim 10, wherein the combination is maintained at a pH of 3.8 or less and 3.0 or more for 30 minutes.

14. The method according to claim 10, further comprising adding the aforementioned amount of acid to the mixture and then titrating the combination to a pH of 4.5 or more and 8.5 or less in less than one hour.

15. To measure the electrical conductivity of the mixture; and The method according to claim 10, further comprising adding a sufficient amount of one or more salts to the mixture to adjust the conductivity of the mixture.

16. Measure the pH of the mixture before adding the aforementioned amount of acid to the mixture; and The method according to claim 10, further comprising measuring the protein concentration of the mixture.

17. A method for developing an acid inactivation protocol for viruses in a mixture, A pool of eluate samples of the mixture, wherein each eluate sample in the pool contains a target molecule purified by a protein affinity capture process; Measure the pH and protein concentration of each eluate sample; To determine the amount of acid required to bring each eluate sample to an inactivating pH by titrating each eluate sample in the pool of eluate samples; Determining data points using each eluate sample in the pool of eluate samples; and This includes using the aforementioned data points to regress the relationship between the amount of acid required to bring the mixture to an inactivating pH, the protein concentration of the mixture, the pH of the mixture, and the inactivating pH. A method wherein the data points represent the amount of acid required to bring each eluate sample in the pool of eluate samples to the inactivation pH.

18. The method according to claim 17, further comprising regressing a confirmation model using the data points.

19. Determining data points using each eluate sample from the pool of eluate samples includes determining a first data point using a first eluate from the pool of eluate samples, and determining a second data point using a second eluate from the pool of eluate samples. The method according to claim 17, further comprising determining a third data point using the first eluent and determining a fourth data point using the second eluent.

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