Method to inactivate viruses

Abstract

Embodiments of the present disclosure describe a system and method for controlling the pH of a polypeptide-containing mixture. The pH may be controlled for any suitable purpose, for example, to inactivate a virus in the mixture. The method may include the step of eluting a mixture (e.g., an eluent) having a pH greater than, for example, 3.9 and, for example, less than, 8.5 from a chromatography column. The method may further include one or more of the steps of measuring the protein concentration of the mixture and measuring the pH of the mixture. Subsequently, the amount of acid required to reduce the pH of the mixture to a target pH may 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 may be added to the mixture, wherein said portion of the acid is sufficient to achieve the target pH.

Description

Technology Field

[0001] Cross-reference of related applications

[0002] This application claims priority to U.S. provisional patent application No. 62 / 881,692 filed on August 1, 2019, the full disclosure thereof is incorporated herein by reference.

[0003] Technology field

[0004] The present disclosure generally relates to a method for achieving a target pH in a polypeptide-containing mixture. More particularly, the present disclosure relates to a method for achieving a target pH in a polypeptide-containing mixture to help ensure that enveloped viruses or virus-like particles are inactivated. Background Technology

[0005] background

[0006] In the preparation of polypeptides, target molecules (e.g., target polypeptide components of drug products) can be separated from the culture medium. A separation process, such as affinity chromatography, can be performed as part of the target molecule preparation process. After such a separation process, the resulting mixture containing polypeptides may potentially contain unwanted viruses or other contaminants that are undesirable to be included in the drug product. Therefore, a method for removing or inactivating such contaminants is desirable.

[0007] In some commercial-scale target molecule synthesis processes, Process Analysis Technology (PAT) may be implemented. PAT includes systems and methods related to the design, analysis, and control of the manufacturing process of target molecules. PAT involves identifying process parameters that affect product quality and periodically monitoring parameters that ensure product quality is maintained. PAT is generally recommended by regulatory agencies to reduce risks associated with target molecules and drug products. PAT may provide statistical validity or confirmation that one or more process conditions are met to improve or maintain the quality of the target molecule and / or product.

[0008] The methods and systems disclosed herein can improve the efficiency and / or productivity of polypeptide manufacturing methods including virus inactivation. The methods and systems disclosed herein can improve the efficiency and / or productivity of drug product manufacturing methods and can solve one or more of the identified problems.

[0009] substance

[0010] Embodiments of the present disclosure may describe a method for inactivating a virus in a mixture, for example, an eluent. The method may include the step of eluting the mixture from a chromatography column at a pH greater than 3.9 and less than 8.5. The method may further include the steps of measuring the protein concentration of the mixture and measuring the pH of the mixture. Subsequently, the amount of acid required to reduce the pH of the mixture to an inactivation pH may be calculated based on the protein concentration of the mixture. After calculating the amount of acid to be added, a first portion of the acid may be added to the mixture, wherein the first portion of the acid is 68% to 99% of the amount of acid to be added. The method may further include the step of adding an additional portion of acid to the mixture so that the pH of the mixture becomes inactivation pH or lower. In the method of the present disclosure, the mixture may be maintained at an inactivation pH for an inactivation interval configured to inactivate the virus in the mixture.

[0011] In some embodiments of the present disclosure, a method for inactivating a virus in a mixture may include the step of loading a mixture containing a target molecule onto a chromatography column, said loading occurring at a pH of approximately 5.0 or higher and approximately 8.5 or lower. The method may further include the step of eluting the eluted mixture containing the target molecule from the chromatography column at a pH of approximately 3.9 or higher and approximately 5.0 or lower. The method may also include the step of adding an acid to the mixture to form a mixture of the mixture and the acid, wherein the mixture is configured to demonstrate effective virus inactivation and the mixture has a pH of approximately 3.8 or lower and approximately 3.0 or higher. The expected pH of the mixture may be predetermined using a pH verification model. Additionally, the pH of the mixture may be measured and / or recorded. The difference between the expected pH and the recorded pH may be calculated, and a correction measure may be performed based on the calculated difference between the expected pH and the recorded pH.

[0012] Further embodiments of the present disclosure may include a method for developing an acid inactivation protocol. The method may include the step of preparing a pool of eluents, wherein each eluent of the pool of eluents contains a target molecule purified in a protein affinity capture process. The method may include the step of measuring the pH and / or protein concentration of each eluent of the pool of eluents. Additionally, each eluent of the pool of eluents may be titrated to determine the amount of acid required to make the eluent reach an inactivation pH. Subsequently, a relationship may be established between the amount of added acid, the protein concentration of the eluent, the pH of the eluent, and the inactivation pH.

[0013] In some embodiments of the present disclosure, a method for inactivating a virus in a mixture may include the step of measuring the protein concentration of the mixture. Subsequently, the amount of acid required to reduce the pH of the mixture to an inactivation pH may be calculated based on the protein concentration of the mixture. After calculating the amount of acid to be added, the amount of acid required to reduce the pH of the mixture may be added to the mixture. Brief explanation of the drawing

[0014] The accompanying drawings, which are included in the specification and constitute part thereof and illustrate various exemplary embodiments together with the specification, explain the principles of the disclosed embodiments. Any feature of the above-described embodiments or examples (e.g., compositions, formulations, methods, etc.) may be combined with any other embodiments or examples, and all such combinations are included in the disclosure herein. Furthermore, the described systems and methods are not limited to any single aspect or embodiment thereof, or to any combination or permutation of such aspects and embodiments. For the sake of brevity, specific permutations and combinations are not discussed or / or illustrated separately in this specification. FIG. 1 illustrates an exemplary process for inactivating a virus in an eluent according to the present disclosure in the form of a flowchart; FIG. 2 illustrates an exemplary process for inactivating a virus in an eluent in accordance with the present disclosure in the form of a flowchart; FIG. 3 illustrates an exemplary process for developing an acid inactivation protocol in accordance with the present disclosure in the form of a flowchart. Specific details for implementing the invention

[0015] As used herein, the terms “comprises,” “comprising,” or any other variation thereof, intended to include a non-exclusive inclusion, may mean that a process, method, article, or device comprising a list of elements may not include only these elements, but may include other elements explicitly enumerated or inherent in such process, method, article, or device. The term “exemplary” is used in the sense of “example” rather than “ideal.” In the case of the terms “for example” and “such as,” and their grammatical synonyms, the phrase “and without limitation” is understood to follow, unless otherwise clearly stated.

[0016] As used herein, the term “about” means to describe variations due to experimental error. When applied to numerical values, the terms “about” and “approximately” may indicate a variation of + / - 5% from the disclosed numerical value unless a different variation is specified. When applied to pH values, the terms “about” and “approximately” may indicate a variation of + / - 0.05. One singular form (“a”, “an”) and the above (“the”) as used herein include a plurality of references unless explicitly indicated by the context.

[0017] Unless different variations are specified, 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 numerical values. pH values ​​disclosed herein may have a variation of + / - 0.05. Additionally, all ranges are understood to include endpoints, for example, 1 centimeter (cm) to 5 cm includes lengths of 1 cm and 5 cm, and all distances between 1 cm and 5 cm.

[0018] details

[0019] The present disclosure is not limited to the specific compositions, formulations, manufacturers of materials, drug products, devices, systems, experimental conditions, or specific methods disclosed herein, as numerous variations are possible within the authority of those skilled in the art. The terms used herein are intended to describe only specific embodiments and are not intended to be limiting.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure pertains. Any suitable method and material (e.g., similar or equivalent to that disclosed herein) may be used in the practice or testing of this disclosure, but specific methods are described herein. All mentioned publications are incorporated herein by reference.

[0021] As used herein, the term “polypeptide” refers to any amino acid polymer having more than about 20 amino acids covalently linked by amide bonds. Proteins comprise one or more amino acid polymer chains (e.g., polypeptides). Thus, polypeptides may be proteins, and proteins may comprise multiple polypeptides forming a single functional biomolecule.

[0022] Post-translational modifications can alter or change the structure of polypeptides. For example, disulfide bridges (e.g., SS bonds between cysteine ​​residues) can be formed post-translationally in some proteins. Some disulfide bridges are essential for the appropriate structure, function, and interactions of polypeptides, immunoglobulins, proteins, cofactors, substrates, etc. In addition to disulfide bond formation, proteins may undergo other post-translational modifications, e.g., lipidation (e.g., myristoylation, palmitoylation, farnesoylation, geranylgeranylation, and glycosylphosphatidylinositol (GPI) anchor formation), 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 surface-to-surface interactions in which the polypeptide participates.

[0023] As used herein, the term "protein" includes biotherapeutic proteins, recombinant proteins used in irradiation 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, etc. Protein-of-interest (POI) may comprise any polypeptide or protein that is preferably isolated, purified, or otherwise prepared. The POI may comprise other polypeptides prepared by cells containing the target polypeptide or antibody.

[0024] As used herein, the term “antibody” comprises an immunoglobulin composed of four polypeptide chains interconnected by disulfide bonds: two heavy (H) chains and two light (L) chains. Typically, the antibody has a molecular weight greater than 100 kDa, e.g., 130 kDa to 200 kDa, e.g., about 140 kDa, 145 kDa, 150 kDa, 155 kDa, or 160 kDa. 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, CL. The VH and VL regions may be further subdivided into a supervariable region referred to as the complementarity determining region (CDR) and placed into a more conservative region referred to as the framework region (FR). Each VH and VL consists of 3 CDRs and 4 FRs, arranged from the amino-terminus to the carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (heavy chain CDRs may be abbreviated as HCDR1, HCDR2, and HCDR3; light chain CDRs may be abbreviated as LCDR1, LCDR2, and LCDR3).

[0025] The class of immunoglobulins designated as immunoglobulin G (IgG) is common in human serum, for example, and comprises four polypeptide chains—two light chains and two heavy chains. Each light chain is linked to one heavy chain via one cystine disulfide bond, and the two heavy chains are linked to each other via two cystine disulfide bonds. Other classes of human immunoglobulins include IgA, IgM, IgD, and IgE. In the case of IgG, four subclasses exist: IgG 1, IgG 2, IgG 3, and IgG 4. Each subclass differs in their invariant regions and, consequently, may have different effector functions. In some embodiments described herein, the POI may comprise a target polypeptide comprising IgG. In at least one embodiment, the target polypeptide comprises IgG 4.

[0026] As used herein, the term "antibody" also includes antigen-binding fragments of complete antibody molecules. As used herein, terms such as "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, etc., include any naturally occurring, enzymatically available, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Antigen-binding fragments of antibodies may be derived, for example, from complete antibody molecules using any suitable standard technique, such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and optionally constant domains. Such DNA may be known and / or readily available, for example, from commercial sources, DNA libraries (including, for example, phage-antibody libraries), or synthesized. DNA may be sequenced and manipulated chemically or using molecular biological techniques to, for example, arrange one or more variable and / or constant domains into a suitable batch, introduce codons, create cysteine ​​residues, modify, add, or delete amino acids, etc.

[0027] Target molecules (e.g., target polypeptides) are produced in recombinant cell-based production systems, e.g., insect baculovirus systems, yeast systems (e.g., Pichia species ( Pichia It can be prepared using sp.)), or mammalian cells (e.g., CHO cells and CHO derivative-like CHO-K1 cells). The term "cell" includes any cell suitable for expressing the recombinant nucleic acid sequence. Cells include prokaryotic and eukaryotic cells (single-cell or multi-cell), bacterial cells (e.g., E. coli ( E. coli ), Basilos species ( Bacillus spp.), Streptomyces species ( Streptomyces Strains of spp.), mycobacterial cells, fungal cells, yeast cells (e.g., S. cerevisiae ( S. cerevisiae), Es Pombe ( S. pombe ), P. Pastoris( P. pastoris ), P. Methanolica( P. methanolica ) etc.), plant cells, insect cells (e.g., SF-9, SF-21, baculovirus-infected insect cells, trichoplucyanis( Trichoplusiani Includes non-human animal cells, human cells, or cell fusions, e.g., hybridomas or quadromas. In some embodiments, the cells may be human, monkey, ape, hamster, rat, or mouse cells. In some embodiments, the cells may be eukaryotic cells 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 cells (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, A431 (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 cells derived from the aforementioned cells Cell line. In some embodiments, the cell may comprise one or more viral genes, for example, retinal cells (e.g., PER.C6™ cells) expressing viral genes.

[0028] The term “target molecule” may be used herein to refer to a target polypeptide (e.g., an antibody, an antibody fragment, or other protein or protein fragment) or to refer to other molecules intended to be produced, isolated, purified, and / or included in a drug product (e.g., an adeno-associated virus (AAV) or other molecules for therapeutic use). The method according to the present disclosure may refer to a target polypeptide and may also be applicable to other target molecules. AAV may be prepared, for example, according to a suitable method (e.g., deep filtration, affinity chromatography, etc.), and a mixture containing AAV (e.g., an eluent containing AAV) may be applied to the method according to the present disclosure. Before or after following one or more methods of the present disclosure, the mixture containing AAV may be applied to an additional procedure (e.g., “empty cassettes” or removal of AAV not containing the target sequence).

[0029] The term "viral content" refers to a qualitative description of a mixture. For example, if the mixture contains viruses or virus-like particles, said mixture has a viral content. In some embodiments, the viral content may be quantified in terms of the number of viral particles or the number of infective units (i.e., concentration) per volume of the mixture. The term "viral concentration" may refer to the concentration of viral particles (e.g., active and inactive viral particles) or the concentration of infective units.

[0030] An exemplary method for inactivating a virus may include adding an acid to a mixture to achieve a pH known to inactivate some viruses and virus-like particles, and maintaining the mixture at the achieved pH for a predetermined period of time. For example, in some embodiments, the method of the present invention 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 chromatography device. Such a chromatography device may include a pre-fabricated device (e.g., Cadence™ BioSMB (Pall Biosciences), BioSC® (novasep), Varicol® (novasep), or Octave (Semba® Biosciences)), a custom-made device, a manual-assembled device, or just two or more standard batch chromatography devices used in a tandem.

[0031] In some embodiments, the target molecule may be eluted from the chromatography apparatus by contacting the stripping buffer to the chromatography apparatus (e.g., a chromatography column) and / or by contacting the equilibrium buffer to the chromatography apparatus. In some embodiments, the stripping buffer may comprise water, an alkaline solution, or a solution containing an alcohol. Water, for example, deionized water, may have, for example, less than 5 volume percent (vol.%) of dissolved ions, less than 1 vol.% of dissolved ions, less than 0.1 vol.% of dissolved ions, or even less than 0.01 vol.% of dissolved ions. According to some embodiments, the alkaline solution may comprise one or more alkaline ionic compounds, LiOH, NaOH, KOH, Ca(OH)2, NH4OH, or other alkaline compounds. The concentration of the alkaline compound in the stripping buffer may be, for example, in the range of about 0.1 N to about 1.5 N, about 0.1 N to about 1 N, about 0.1 N to about 1.5 N, about 0.5 N to about 1.5 N, about 0.1 N to about 0.8 N, about 0.1 N to about 0.6 N, about 0.1 N to about 0.5 N, about 0.1 N to about 0.4 N, or about 0.1 N to about 0.3 N. For example, the concentration of an alkaline compound in the stripping buffer may be about 0.1 N, about 0.2 N, about 0.3 N, about 0.4 N, about 0.5 N, about 0.6 N, about 0.7 N, about 0.8 N, about 0.9 N, about 1 N, about 1.1 N, about 1.2 N, about 1.3 N, about 1.4 N, or about 1.5 N. The stripping buffer containing alcohol may include methanol, ethanol, propanol, benzyl alcohol, or other alcohols. The concentration of alcohol in the stripping buffer is about 0.1 vol.% to about 30 vol.%, for example, about 0.5 vol.% to about 30 vol.%, or about 0.5 vol.% to about 25 vol.%, based on the total weight of the stripping buffer.The range may be %, about 0.5 vol.% to about 25 vol.%, about 0.5 vol.% to about 25 vol.%, about 1 vol.% to about 20 vol.%, about 1 vol.% to about 15 vol.%, about 1 vol.% to about 10 vol.%, about 10 vol.% to about 50 vol.%, about 10 vol.% to about 40 vol.%, about 10 vol.% to about 30 vol.%, about 10 vol.% to about 25 vol.%, about 15 vol.% to about 25 vol.%, or about 20 vol.% to about 25 vol.%. For example, the concentration of alcohol in the stripping buffer may be about 0.1 vol.%, about 0.5 vol.%, about 1 vol.%, about 2 vol.%, about 3 vol.%, about 5 vol.%, about 10 vol.%, about 15 vol.%, about 20 vol.%, or about 25 vol.%.

[0032] In some embodiments, the equilibrium buffer may have a composition similar or identical to that of the stripping buffer. In other embodiments, the composition of the equilibrium buffer may be variable compared to that of the stripping buffer. In some embodiments, the equilibrium buffer may contain one or more salts, for example, sodium, potassium, magnesium, calcium, citrate, acetate, phosphate, sulfate, Tris, or other salts.

[0033] In one or more embodiments, a method for inactivating a virus may be used after a mixture containing a target molecule is eluted from a chromatography device (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). While the loading of the target molecule into the chromatography device may be variable depending on the upstream process, the mixture containing the target molecule may be eluted from the chromatography device in a constant volume. In some embodiments, the mixture containing the target molecule is loaded onto the chromatography column at a pH of approximately 5.0 or higher and approximately 8.5 or lower, for example, approximately 5.5 or higher and approximately 8.5 or lower, approximately 6.0 or higher and approximately 8.5 or lower, or approximately 5.0 to approximately 6.5. As a result of modifications in loading, the protein concentration and / or pH of the eluent (e.g., eluent eluted from a chromatography device) may be variable. Due to these modifications, the amount of acid required to be added for virus inactivation is also variable.

[0034] In conventional manufacturing processes, low-pH virus inactivation is performed by trial and error, wherein a predetermined amount of acid is added to the eluent, the pH of the mixture of eluent and acid is measured, and the addition and measurement steps are repeated until the inactivation pH is reached. Due to the potential costs and losses that can result from the addition of excessive acid, this process is conservative and involves the addition of small amounts of acid and long inactivation process times, often on the scale of several hours.

[0035] Aspects of the present disclosure may provide various advantages to a method for preparing a target polypeptide or other target molecule. For example, by implementing one or more of the above methods and / or mathematical models, an amount of acid to be added—for example, the amount of acid required to bring a mixture (containing the target molecule and, potentially, an unwanted virus or virus-like particle) to an inactivation pH—can be determined. An amount of acid approximately equivalent to the amount of acid to be added to the mixture may be added. As described in more detail below, said amount of acid equivalent to the amount of acid to be added may be added as a single bolus of acid or in two or more doses. Acid administration for virus inactivation may be more efficient and less sensitive in this manner than conventional trial-and-error methods.

[0036] In some embodiments of the present disclosure, as part of the process, the content of the virus or the content of the infection-causing unit in the mixture may be known or expected to be minimal or non-existent. In some such embodiments of the present disclosure, the system and method disclosed herein may advantageously be introduced into a manufacturing process as part of a PAT, for example, to reduce potential variability within the process, provide real-time verification of compliance with process standards and / or increase confidence in the integrity of the process.

[0037] The additional benefits and advantages of the embodiments of the present disclosure will be apparent to those skilled in the art.

[0038] As mentioned above, after preparing the target molecule using one or more chromatography and / or separation processes, a mixture (e.g., an eluent) may be obtained. In some embodiments, one or more measurements may be performed on the mixture, including, for example, protein concentration, target molecule concentration, pH, or a combination thereof. Protein concentration may be measured by any suitable method, including, for example, by ultraviolet / visible spectroscopy. In some embodiments, protein concentration is measured using a wavelength that is uniquely absorbed by the target molecule, which may be a polypeptide. In these embodiments, the total protein concentration may be approximately equivalent to the concentration of the target molecule (e.g., a target polypeptide). In some embodiments, the mixture containing the target molecule contains approximately 7.0 grams of protein per liter of eluent ( g / L ) to approximately 35.0 g / L , 7.0 g / L That is, approximately 20.0 g / L Below, approximately 8.5 g / L to approximately 18.5 g / L , or approximately 10.0 g / L to approximately 17.0 g / L It can have a protein concentration of

[0039] The pH of the mixture can be measured by any suitable method. Accurate and consistent pH measurement is important for the successful inactivation of viral proteins down to low pH. pH measurements 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 measured medium and the pH probe. Even in standardized pH measurement processes, a variation of ± 0.05 pH can be common. In the field of polypeptide manufacturing or viral inactivation, a variation of ± 0.05 pH can represent about 20% of the operating pH range and can adversely affect viral inactivation and / or process validity. Such variation can even become complicated over time and can cause instrument drift and more extreme variation in pH measurements. Therefore, in some embodiments, variation in pH measurements may be taken into account when measuring pH. In some embodiments, as mentioned, the pH of the eluent may be measured by a standardized method for the purpose of reducing or eliminating variability in pH measurements. 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 eluent may be measured by a pH meter, for example, a potentiometric pH meter. In some embodiments, the eluent containing the target molecule may have a pH of approximately 3.9 or higher and approximately 8.5 or lower, for example, approximately 3.9 to approximately 6.5, approximately 3.9 to approximately 5.5, approximately 4.5 to approximately 6.5, approximately 4.0 to approximately 4.4, approximately 3.9 to approximately 4.4, or approximately 4.0 to approximately 4.3. The pH values, or ranges of pH values ​​used herein, may have a variation of ±0.05 pH units.

[0040] The presence of certain viruses and virus-like particles (e.g., enveloped viruses, retroviruses, retrovirus-like particles, pseudorabies, herpes viruses, etc.) in mixtures (e.g., eluents), formulations, and / or drug products may affect the composition, properties, or utility of such mixtures, formulations, and / or drug products. For example, the presence of unwanted viruses or virus-like particles in drug products may affect product stability, reduce the shelf life of the product, or fail to meet internal, overview, or regulatory (e.g., FDA) specifications. Some viruses or virus-like particles may cause clinical effects, such as immunogenic reactions, upon administration of drug products containing viruses. Embodiments of the present disclosure may be useful for reducing or eliminating some or all of these unwanted effects by inactivating viruses or virus-like particles. For example, embodiments of the present disclosure may be applied to a mixture (e.g., eluent) having a virus content after one or more polypeptide purification processes (e.g., a separation process including a protein A affinity column).

[0041] In some embodiments, the conductivity of the mixture may be measured prior to virus inactivation. In some embodiments, one or more salts may be added to the mixture prior to virus inactivation to adjust its conductivity (e.g., increase conductivity). 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 to theory, adding 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 affects the way the surfaces of these species interact with acid. Adding salts to the mixture may increase the ionic activity of the mixture, increase the conductivity of the mixture, 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.

[0042] In some cases, embodiments of the present disclosure may be applicable to mixtures having extremely low viral content (e.g., about 0.0001 or less of viral particles or infection-causing units per mL) or even non-existent viral content. Chromatography and other separation processes, either alone or in combination, may appropriately purify or separate target molecules and remove unwanted viruses or virus-like particles from the mixture. In some of these cases, the method according to the present disclosure serves to further ensure that the viruses or virus-like particles are inactivated and may be advantageous in ensuring product stability, product safety, product efficacy, and compliance with internal or regulatory specifications. Accordingly, embodiments of the present disclosure may also be applicable to any known virus-free mixtures to ensure regulatory guidelines and to meet and / or provide unnecessary quality controls.

[0043] The above-described virus inactivation protocol and method can be implemented without causing adverse effects to specific types of viruses, e.g., AAV (e.g., AAV containing a target sequence). For example, advantageously, the above-described protocol and method can be performed without degrading the AAV. Therefore, the above-described method may be suitable for use in mixtures containing AAV as a target molecule.

[0044] As previously mentioned, viruses in a mixture can be inactivated by maintaining the mixture at an inactivation pH during an inactivation interval. The inactivation pH may be pH 3.8 or lower and 3.0 or higher, for example, 3.35 to 3.8, 3.75 or lower and 3.0 or higher, 3.7 or lower and 3.0 or higher, 3.65 or lower and 3.0 or higher, 3.6 or lower and 3.0 or higher, 3.55 or lower and 3.0 or higher, 3.5 or lower and 3.0 or higher, 3.45 or lower and 3.0 or higher, 3.4 or lower and 3.0 or higher, 3.35 to 3.75, 3.5 to 3.8, 3.5 to 3.75, 3.5 to 3.75, 3.5 to 3.7, 3.5 to 3.6, or 3.5 to 3.65. The pH values, or ranges of pH values ​​used herein, may have a variation of ±0.05 pH units. If the inactivation pH is set too high, there is a risk of insufficient virus inactivation due to deformation within the process. If the inactivation pH is set too low, there is a risk of denaturing target molecules or other proteins, or otherwise altering the mixture in an undesirable way.

[0045] The inactivation interval describes the time interval during which the mixture is maintained at an inactivation pH. The inactivation interval may be approximately 20 minutes to approximately 90 minutes, for example, approximately 30 minutes, approximately 45 minutes, approximately 60 minutes, approximately 30 minutes to approximately 45 minutes, approximately 30 minutes to approximately 60 minutes, approximately 30 minutes to approximately 75 minutes, approximately 30 minutes to approximately 90 minutes, approximately 45 minutes to approximately 60 minutes, approximately 45 minutes to approximately 75 minutes, approximately 45 minutes to approximately 90 minutes, approximately 60 minutes to approximately 75 minutes, or approximately 60 minutes to approximately 95 minutes. Maintaining the mixture at an inactivation pH during the inactivation interval can reduce or eliminate viral activity in the mixture, or ensure its absence. A low pH environment can denature viral proteins, for example, viral envelope proteins. Denatured viral proteins can inactivate retroviruses and retrovirus-like particles, thereby reducing unwanted viral activity in the mixture.

[0046] The reduction in viral activity in a mixture can be quantified by a reduction factor. The reduction factor can be calculated according to Equation 1 shown below:

[0047] Equation (1)

[0048]

[0049] In the above formula, V1 is the volume of the mixture before virus inactivation, C1 is the virus concentration or infection-inducing unit per volume of the mixture before virus inactivation, V2 is the volume of the mixture after virus inactivation, and C2 is the virus concentration of the mixture after virus inactivation. In various embodiments of the present disclosure comprising a method for inactivating viruses, a reduction factor of 2.5 or more is achieved, for example, a reduction factor of 3 or more, 3.5 or more, 4 or more, 4.5 or more, or 5 or more. As used herein, "effective virus inactivation" may refer to virus inactivation associated with a reduction factor of 2.5 or more, 3 or more, 3.5 or more, 4 or more, 4.5 or more, or 5 or more.

[0050] 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 shown below:

[0051] Equation (2)

[0052] w = Ax + By + C

[0053] In the above formula, x is grams per liter ( g / L ) is the protein concentration of the mixture in units, y is the inactivating pH, and w is the mole of acid per kilogram of mixture ( mol / kg ) is the amount of acid added in units, and A, B, and C are constants. Constant A in Equation 2 is the liter-molar of acid per gram-kilogram of mixture ( Lㆍmol / gㆍkg It has the unit of ). Constant A is 0.0003 Lㆍmol / gㆍkg Above and 0.0006 Lㆍmol / gㆍkg Below, for example, approximately 0.0003 Lㆍmol / gㆍkg to approximately 0.0005 Lㆍmol / gㆍkg , approximately 0.0004 Lㆍmol / gㆍkg to approximately 0.0006 Lㆍmol / gㆍkg , approximately 0.00035 Lㆍmol / gㆍkg to approximately 0.0005 Lㆍmol / gㆍkg , approximately 0.00035 Lㆍmol / gㆍkg to approximately 0.0006 Lㆍmol / gㆍkg , or approximately 0.0004 Lㆍmol / gㆍkg to approximately 0.00055 Lㆍmol / gㆍkg It can be. Constants B and C in Equation 2 are the moles of acid per kilogram of mixture ( mol / kg It has the unit of ). Constant B is -0.1 mol / kg Anomalies and 0 mol / kg Below, 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 It can be. The constant C is 0.02 mol / kg Above and 0.1 mol / kg Below, 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 It could be.

[0054] 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:

[0055] Equation (3)

[0056] w = Ex + Fy + Gz + H

[0057] In the above formula, x is grams per liter ( g / L ) is the protein concentration of the mixture in units, y is the inactivation pH, z is the pH of the mixture, and w is the mole of acid per kilogram of the mixture ( mol / kg ) is the amount of acid added, and E, F, G, and H are constants. The constant E in Equation 3 is the liter-molar of acid per gram-kilogram of the mixture ( Lㆍmol / gㆍkg It has ) units, and the constants F, G, and H are the moles of acid per kilogram of mixture ( mol / kg It has the unit of ). The constant E is 0.00005 Lㆍmol / gㆍkg Above and 0.0005 Lㆍmol / gㆍkg Below, for example, approximately 0.00005 Lㆍmol / gㆍkg to approximately 0.0005 Lㆍmol / gㆍkg , approximately 0.0001 Lㆍmol / gㆍkg to 0.0005 Lㆍmol / gㆍkg , approximately 0.00005 Lㆍmol / gㆍkg to 0.00045 Lㆍmol / gㆍkg, approximately 0.0001 Lㆍmol / gㆍkg to approximately 0.00035 Lㆍmol / gㆍkg , or approximately 0.00035 Lㆍmol / gㆍkg to approximately 0.0005 Lㆍmol / gㆍkg It can be. The constant F is -0.2 mol / kg Anomalies and 0 mol / kg Below, 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 It can be. The constant G is 0 mol / kg Above and 0.03 mol / kg Below, 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 It can be. The constant H is -0.1 mol / kg Above and 0.1 mol / kgBelow, for example, approximately -0.1 mol / kg to approximately 0.1 mol / kg , approximately -0.08 mol / kg to approximately 0.08 mol / kg , approximately -0.05 mol / kg to approximately 0.1 mol / kg , or approximately -0.1 mol / kg to approximately 0.05 mol / kg It could be a numerical value.

[0058] A specific formula relating the mixture protein concentration versus the amount of acid added and the inactivation pH (which may, optionally, depend on the mixture pH) may vary depending on the target molecule and / or acid system used. The values ​​of the above-defined constants applied to the provided target molecule and acid system can be measured by regression according to the above-defined general formula. As described in the examples section below, the amount of acid added is found to have an unexpectedly strong correlation with the mixture protein concentration according to the above-defined formula. This unexpectedly strong correlation can be attributed to the above-described general formula and their derivatives introduced into the PAT.

[0059] In one or more embodiments, the amount of acid added is approximately 0.002 moles of acid per kilogram of mixture ( mol / kg ) to approximately 0.025 mol / kg , for example, approximately 0.002 mol / kg to approximately 0.025 mol / kg , approximately 0.01 mol / kg to approximately 0.025 mol / kg , approximately 0.002 mol / kgto approximately 0.020 mol / kg , or approximately 0.005 mol / kg to approximately 0.020 mol / kg am.

[0060] In some embodiments, after calculating the amount of acid to be added, the acid may be added to the mixture so that the mixture reaches an inactivated pH. For example, in at least one embodiment, a bolus of acid equivalent to the amount of acid to be added may be added to the mixture so that the pH of the mixture becomes inactivated pH or lower. In other embodiments, a first portion of acid may be added to the mixture, and then subsequently, one or more additional portions of acid may be added to the mixture so that the pH of the mixture becomes inactivated pH or lower. In such embodiments, the first portion of acid is 68% to 99% of the amount of acid to be added, for example, approximately 75% to approximately 99%, approximately 80% to approximately 99%, approximately 85% to approximately 99%, approximately 90% to approximately 99%, approximately 85% to approximately 95%, or approximately 90% to approximately 99%.

[0061] The first portion of acid may be added in a proportion such that the target molecule is not denatured in the mixture having the lowest possible pH by the addition of the first portion. The additional portion of acid may include one or more additions of acid occurring after the addition of the first portion of acid, for example, three additions of acid, four additions of acid, or five additions of acid occurring after the addition of the first portion of acid. Each addition among the one or more additions of acid may be an amount of 0.1% to 32% of the amount of acid added, for example, approximately 0.1% to approximately 30%, approximately 0.1% to approximately 25%, approximately 0.1% to approximately 20%, approximately 1% to approximately 25%, approximately 0.1% to approximately 15%, approximately 0.1% to approximately 10%, approximately 1% to approximately 15%, approximately 1% to approximately 10%, or approximately 0.1% to approximately 5%. Each of the one or more additions of acid may be the same amount as one or more other additions of acid. In other embodiments, each addition of acid may be the same amount as each other addition of acid.

[0062] In some embodiments, the pH may be measured after the first portion of the acid is added to the mixture, but before the addition of the additional portion of the acid. In some of these embodiments, the pH of the mixture measured after the addition of the first portion of the acid is 3.5 or greater and 3.75 or less, for example, approximately 3.5 to approximately 3.75, approximately 3.6 to approximately 3.7, approximately 3.5 to approximately 3.65, approximately 3.6 to approximately 3.75, or approximately 3.5 to approximately 3.65. The pH values, or ranges of pH values ​​used herein, may have a variation of ±0.02 pH units.

[0063] The acid may be added in the form of one or more acidic solutions. The acidic solutions may contain any suitable acid, for example, HCl, HBr, H3PO4, HO2C2O2H, C6H8O7, H2SO3, H3PO4, HNO2, C6H5CO2H, CH3CO2H, HClO, HCN, H3BO3, or a combination thereof. Additionally, or alternatively, the acidic solutions may contain one or more salts, for example, glycine, arginine, sodium acetate, and / or sodium chloride.

[0064] After adding an amount of acid to a mixture containing a target molecule, the resulting mixture has a pH that is below the inactivation pH. In some embodiments, the pH of the resulting mixture may be measured to determine, for example, whether it is within a desired range. As described above, the mixture may be maintained at the inactivation pH for an inactivation interval. After maintaining the mixture at the inactivation pH for an inactivation interval, a decrease in viral activity may occur equivalent to, for example, a reduction factor of 2.5 or higher, 3 or higher, 3.5 or higher, or 4 or higher. After maintaining the mixture at an inactivating pH for an inactivation interval, an alkaline solution may be added to the mixture to titrate the pH of the mixture to 4.5 or higher and 8.5 or lower, for example, 4.5 or higher and 8.5 or lower, 5.0 or higher and 8.5 or lower, 5.8 or higher and 8.5 or lower, 5.9 or higher and 8.5 or lower, 6.0 or higher and 8.5 or lower, 6.1 or higher and 8.5 or lower, 6.2 or higher and 8.5 or lower, 6.3 or higher and 8.5 or lower, or 6.4 or higher and 8.5 or lower. The pH values, or ranges of pH values ​​used herein, may have a variation of ±0.02 pH units. The alkaline solution may include one or more bases, for example, NaOH, KOH, LiOH, Ca(OH)2, NH4OH, NaCH3CO2 and / or (HOCH2)3CNH2.

[0065] In some embodiments, the mixture is titrated within less than 1 hour after the first part of the acid is added, for example, within approximately 50 minutes, approximately 45 minutes, approximately 40 minutes, approximately 35 minutes, or approximately 30 minutes after the first part of the acid is added.

[0066] Aspects of the present disclosure may also include a method for measuring a function that predicts the pH of a mixture (i.e., a mixed mixture and an acid). For example, a function that predicts the pH of the mixture (e.g., an inactivated pH) may be measured based on the measured protein concentration and / or pH of the mixture and the amount of acid added to the mixture. Using this function, process errors (e.g., insufficient mixing, poor sampling, etc.) or equipment defects (mechanical error, instrument drift, pH probe malfunction, etc.) may be detected.

[0067] In some embodiments, the expected pH of the oxidized mixture can be predetermined according to a verification model. The verification model may be in the form of Equation 4 shown below:

[0068] Equation (4)

[0069] y = Kx + Lw + Mz + N

[0070] In the above formula, y is the inactivating pH, and x is grams per liter ( g / L ) is the protein concentration of the mixture in units, z is the pH of the mixture, and w is the mole of acid per kilogram of the mixture ( mol / kg ) is the amount of acid added, and K, L, M, and N are constants. In Equation 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 is 0 L / g Above and 0.03 L / g Below, for example, approximately 0 L / gto approximately 0.03 L / g , approximately 0.001 L / g to approximately 0.03 L / g , approximately 0 L / g to approximately 0.025 L / g , or approximately 0.001 L / g to approximately 0.025 L / g It can be. The constant L is -80 kg / mol Above and -60 kg / mol Below, 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 It may be. The constant M may be 0 or greater and 2.0 or less, 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 numeric value of -1.0 or greater and 0 or less, for example, approximately -1.0 to approximately 0, approximately -1.0 to approximately -0.1, or approximately -0.9 to approximately -0.1.

[0071] In some embodiments, the pH and protein concentration of the mixture may be measured and / or recorded before the addition of acid. Based on the pH of the mixture, the protein concentration of the mixture, and the amount of acid added, the expected pH of the oxidized mixture may be pre-determined using a verification model. The actual pH of the oxidized mixture may be measured, recorded, and / or compared with the expected pH. Comparing the recorded pH with the expected pH may include calculating the difference (e.g., percentage difference) between the recorded pH and the expected pH.

[0072] In some embodiments, if the difference between the recorded pH and the expected pH exceeds a threshold amount, a calibration measure may be performed. The threshold may be, for example, ±0.03 pH from the expected pH, ±0.05 pH from the expected pH, ±0.07 pH from the expected pH, ±0.09 pH from the expected pH, ±0.1 pH from the expected pH, ±0.15 pH from the expected pH, or ±0.2 pH from the expected pH. Calibration measures may include, but are not limited to, adjusting the pH meter, adjusting the composition of the mixture, adjusting one or more process environment conditions, or a combination thereof. Adjusting the pH meter may include standardization, the pH meter, recalibrating the pH meter, cleaning, resetting, and / or repositioning the pH probe, adjusting the reference electrode solution, repositioning a part of the pH meter, adjusting the position of the pH probe, and / or other measures to change the signal-to-noise ratio of the pH meter. Adjustment of the composition of the mixture may include recombining any component solution or upstream composition of the mixture, changing process conditions or device components of one or more chromatography or separation processes, and / or other measures to change the material composition of the mixture. Adjustment of one or more environmental conditions of the process may include adjustment of the mixing and / or homogenization process and system, adjustment of the temperature of the process, adjustment of the humidity of the process, adjustment of the pressure of the process, or a combination thereof. Such adjustments based on deviations from expected parameters may be introduced as part of the PAT.

[0073] The above-described equations and mathematical models may be generated as part of a method for developing an acid inactivation protocol. A method for developing an acid inactivation protocol may include the step of preparing a pool of mixtures (e.g., eluents), wherein each mixture in the pool contains a target molecule purified in a protein affinity capture process. For example, a pool of samples may be collected from the eluent of a protein affinity chromatography column. The method may further include the step of measuring the pH and protein concentration (e.g., concentration of the target molecule) of each sample. After measuring the pH and protein concentration of each sample, each sample may be titrated to determine the amount of acid required to bring the sample to an inactivation pH. In some embodiments, the inactivation pH may be limited to a broadly sufficient range to allow for two or more repeated titrations, which enables 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.

[0074] In some embodiments, a relationship (e.g., a mathematical model) may be regressed between the amount of added acid, the eluent protein concentration, the mixture pH, and / or the inactivation pH. The relationship may be regressed according to Equation 2 or Equation 3 mentioned above. In some embodiments, the method for developing an acid inactivation protocol further includes regressing a verification model. The verification model may be regressed according to Equation 4 mentioned above.

[0075] FIG. 1 illustrates, in the form of a flowchart, an exemplary process 100 for inactivating a virus in a mixture according to the present disclosure. According to step 101, the mixture may be eluted from a chromatographic column at a first pH (e.g., greater than 3.9). According to step 102, the protein concentration of the mixture (e.g., target molecule concentration) may be measured, for example, by UV / visible spectroscopy or other methods. Optionally, the conductivity and / or pH of the mixture may be measured, for example, by a potentiometric pH meter or other methods. According to step 103, one or more salts may 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 reduce the pH of the mixture to a second pH (e.g., inactivation pH) may be calculated. This calculation may 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 acid may be added to the mixture. In some embodiments, the first portion of acid is a bolus of acid equivalent to the calculated amount of acid added. In other embodiments, the first portion of acid may be 68% to 99% of the volume or amount of acid as the calculated amount of acid added. According to step 105, optionally, a secondary acid may be added to the mixture so that the mixture of acid and the mixture becomes a second pH (e.g., inactivation pH) or lower. According to step 106, the mixture of acid and the mixture may be maintained at the second pH (e.g., inactivation pH) during an inactivation interval to inactivate the virus in the eluent. In some embodiments,

[0076] FIG. 2 illustrates, in the form of a flowchart, an exemplary process 200 for inactivating a virus in a mixture according to the present disclosure. According to step 201, a mixture containing a target molecule may be loaded onto a chromatography column at a first pH (e.g., approximately 5.0 or higher and approximately 8.5 or lower). According to step 202, the eluted mixture may be eluted from the chromatography column at a second pH (e.g., approximately 3.9 or higher and approximately 5.0 or lower). 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 spectroscopy. According to step 204, optionally, the pH and / or conductivity of the eluted mixture may be measured. The pH may be measured and / or recorded, for example, by a potentiometric pH meter. According to step 205, one or more salts may be added to the mixture in sufficient amounts to adjust the conductivity of the mixture. According to step 206, an acid may be added to the eluted mixture to form a mixture of the eluted mixture and the acid, and thus the mixture is configured to demonstrate effective virus inactivation. Additionally, the mixture may have a pH of two pH values ​​(e.g., approximately 3.8 or lower and approximately 3.0 or higher). According to step 207, the expected pH of the mixture may be pre-determined using a pH verification model. According to step 208, the pH of the mixture may be measured and / or recorded. According to step 209, the difference between the expected pH of the mixture and the measured / recorded pH may be calculated. According to step 210, a calibration measure may be taken based on the calculated difference between the expected pH of the mixture and the measured and / or recorded pH.

[0077] FIG. 3 illustrates, in the form of a flowchart, an exemplary process 300 for developing an acid inactivation protocol according to the present disclosure. According to step 301, a pool of eluent samples may be prepared, wherein each eluent sample of the pool of eluent samples contains a target molecule purified in an affinity capture process. According to step 302, the protein concentration (e.g., target molecule concentration) of each eluent sample of the pool of eluent samples may be measured, for example, by UV / visible spectroscopy. Optionally, the pH of each eluent sample of the pool of eluent samples may be measured, for example, by a potentiometric pH meter. According to step 303, the amount of acid required to reduce the pH of each eluent sample to an inactivation pH may be measured by titrating each eluent sample of the pool of eluent samples. According to step 304, the relationship between the amount of added acid, the eluent protein concentration, the eluent pH, and / or the inactivation pH can be regressed. According to step 305, optionally, a verification model can also be regressed.

[0078] Although each of FIGS. 1-3 illustrates a specific sequence of steps, it should be understood that the steps performed and the order in which they are performed may be modified. Additionally, steps (e.g., one or more of measurement and / or recording steps) may be added or removed from any of the methods disclosed herein. Additionally, although each of FIGS. 1-3 illustrates steps in relation to an eluent, it should be understood that said steps may be applied to any mixture containing a target molecule.

[0079] Examples

[0080] The following examples are intended to illustrate the present disclosure without factual limitation. It will be understood that the present disclosure includes additional aspects and embodiments consistent with the description and the following examples.

[0081] In the following examples, the target polypeptide was prepared from a mixture containing the target polypeptide, host cell protein, virus, and other contaminants, impurities, and components. The target polypeptide was prepared from Chinese hamster ovary cells grown in a suspension culture.

[0082] Example 1

[0083] The target polypeptide was eluted from a protein A affinity column, and multiple samples of the mixture were obtained. The protein concentration of each mixture sample was determined by UV / visible spectroscopy and is shown in grams of protein per liter of mixture in Table 1 below. Additionally, the pH of each mixture sample was measured and is shown in Table 1.

[0084] The eluent pool contained 40 eluent samples (i.e., 40 samples of the mixture), and each sample was titrated with a 0.25 M solution of phosphoric acid (H3PO4) to an inactivating pH of 3.35 to 3.86 to determine the amount of acid added (e.g., the amount of acid that needs to be added to make the mixture inactivated pH). For 6 of the 40 samples (Samples 1-6, shown in Table 1), the titration was performed in multiple replicates to generate multiple data points. For example, 30.89 grams of acid per kilogram of mixture, which makes Sample No. 1 inactivated pH 3.70, is one data point, and when 5.35 additional grams of acid per kilogram of mixture are added, 36.24 grams of acid per kilogram of mixture, which makes Sample No. 1 inactivated pH 3.59, is another data point. Data for these titrations are shown in Table 1 below, and the amount of acid added is expressed in units of grams of acid per kilogram of mixture and moles of acid per kilogram of mixture.

[0085]

[0086]

[0087] Example 2

[0088] Using the titration data shown in Table 1, the mixture protein concentration, inactivation pH, and amount of acid added were regressed according to Equation 2. The regression equation shown below (Equation 5) has a measured coefficient (R²) of 0.84. 2 It was measured to have ).

[0089] Equation (5)

[0090] w = 0.0004532x - 0.01135y + 0.04213

[0091] Example 3

[0092] In addition, regarding the titration data shown in Table 1, the relationship between the mixture protein concentration, mixture pH, inactivation pH, and amount of acid added is regressed according to Equation 3. The regression equation shown below (Equation 6) has a measured coefficient (R²) of 0.97. 2 It was measured to have ). Unexpectedly, as shown in general formulas 2 and 3, the amount of acid added is significantly related to the relationship between protein concentration, mixture pH, and inactivation pH.

[0093] Equation (6)

[0094] w = 0.0001986x - 0.01162y + 0.01510z - 0.01692

[0095] Example 4

[0096] Using the titration data shown in Table 1, the verification model was regressed according to General Equation 4. With the regression equation (Equation 7) shown below, the measured coefficient (R²) was 0.93. 2 It was measured to have ). Also, unexpectedly, inactivation pH is strongly correlated with the relationship between protein concentration, mixture pH, and inactivation pH.

[0097] Equation (7)

[0098] y = 0.01488x - 71.65w + 1.094z - 0.6727

[0099] Those skilled in the art will recognize that the concepts on which the present disclosure is based can be readily used as a basis for designing other methods and systems to achieve several of the purposes of the present disclosure. Additionally, while aspects of the present disclosure describe specific steps in a specific process (e.g., inactivation of a virus in a mixture), those skilled in the art will recognize that the systems and methods disclosed herein may be available in other contexts (e.g., inactivation of a virus in other mixtures containing polypeptides, such as before a chromatography process or after combining additional components and eluents in a process for manufacturing a formulation of a drug substance). Accordingly, the claims are not to be considered limited by the description above.

Claims

Claim 1 A method for inactivating a virus in a mixture, the method comprising: a step of eluting the mixture from a chromatography column at a pH greater than 3.9 and less than 8.5; a step of measuring the protein concentration of the mixture; a step of calculating the amount of acid required to reduce the pH of the mixture to an inactivation pH based on the protein concentration of the mixture; and a step of adding a first portion of acid to the mixture, wherein the first portion of acid is 68% to 99% of the amount of acid required to reduce the pH of the mixture to an inactivation pH. A method comprising the step of adding an additional portion of acid to the mixture such that the pH of the mixture and the acid mixture becomes less than or equal to the inactivating pH, and calculating the amount of acid required to reduce the pH of the mixture to the inactivating pH according to the following formula (2): Formula (2) w = Ax + By + C In the above formula (2), w is the amount of acid in molar units of acid per kilogram of mixture, x is the protein concentration of the mixture in grams per liter, y is the inactivating pH, and A, B, and C are constants. Claim 2 A method according to claim 1, further comprising the step of maintaining the mixture at the inactivating pH during an inactivation interval; and, after the inactivation interval, titrating the mixture to a pH of 4.5 or higher and 8.5 or lower, wherein the mixture is titrated within less than 1 hour after adding the first portion of the acid to the mixture. Claim 3 A method according to claim 1, further comprising the step of measuring the pH of the mixture before adding the first portion of the acid to the mixture. Claim 4 delete Claim 5 In paragraph 1, A is approximately 0.0003 Lㆍmol / gㆍkg Above and approximately 0.0006 Lㆍmol / gㆍkg Below; B is approximately -0.1 mol / kg Above and approximately 0.0 mol / kg A method in which C is approximately 0.02 or greater and approximately 0.1 or less. Claim 6 A method according to paragraph 3, wherein the amount of acid required to reduce the pH of the mixture to an inactivated pH is calculated based on the protein concentration of the mixture and the pH of the mixture. Claim 7 A method according to claim 1, wherein the amount of acid required to reduce the pH of the mixture to the inactivating pH is about 0.002 moles to about 0.025 moles of acid per kilogram of mixture. Claim 8 A method according to claim 1, wherein the inactivating pH is approximately 3.8 or less and approximately 3.0 or more. Claim 9 A method according to claim 1, further comprising the steps of: measuring the conductivity of the mixture; and adding one or more salts to the mixture in an amount sufficient to adjust the conductivity of the mixture. Claim 10 A method according to claim 1, wherein the chromatography column is configured to perform a protein affinity capture process. Claim 11 A method according to claim 1, wherein the pH of the mixture is between approximately 3.5 and approximately 3.75 after adding the first portion of the acid and before adding the additional portion of the acid. Claim 12 A method for inactivating a virus in a mixture, the method comprising: loading a mixture containing a target molecule onto a chromatography column, wherein the mixture has a pH of approximately 5.0 or higher and approximately 8.5 or lower; eluting an eluted mixture containing the target molecule from the chromatography column, wherein the eluted mixture has a pH of approximately 3.9 or higher and approximately 5.0 or lower; adding a predetermined amount of acid to the eluted mixture to form a mixture of the eluted mixture and the acid, wherein the mixture is configured to exhibit effective virus inactivation and the mixture has a pH of approximately 3.8 or lower and approximately 3.0 or higher; and pre-determining an expected pH of the mixture using a pH verification model, wherein the pH verification model comprises the following equation (4): equation (4) y = Kx + Lw + Mz + N, wherein y is the pH of the mixture and x is the amount in grams per liter A protein concentration of the mixture, where z is the pH of the mixture before adding the amount of acid to the mixture, w is the amount of acid added to the mixture in moles of acid per kilogram of eluent, and K, L, M, and N are constants; a method comprising the steps of: recording the pH of the mixture; calculating the difference between the expected pH and the recorded pH; and performing a corrective action based on the calculated difference between the expected pH and the recorded pH. Claim 13 A method in which, in the case of claim 12, the correction measure is performed when the difference between the above-mentioned pH and the above-mentioned recorded pH is approximately 0.15 or greater. Claim 14 A method according to claim 12, wherein the calibration measure comprises adjusting a pH meter, adjusting the composition of the mixture, adjusting one or more environmental conditions, or a combination thereof. Claim 15 In claim 12, the method wherein the above mixture is maintained at a pH of approximately 3.8 or lower and approximately 3.0 or higher for approximately 30 minutes. Claim 16 A method according to claim 12, further comprising the step of titrating the mixture to a pH of approximately 4.5 or higher and approximately 8.5 or lower within less than 1 hour after adding the amount of acid to the mixture. Claim 17 The method of claim 12 further comprises the steps of: measuring the conductivity of the mixture; and adding one or more salts to the mixture in an amount sufficient to adjust the conductivity of the mixture. Claim 18 A method according to claim 12, further comprising the steps of: measuring the pH of the mixture before adding the amount of acid to the mixture; and measuring the protein concentration of the mixture. Claim 19 delete Claim 20 A method for developing an acidic virus inactivation protocol for a mixture, wherein the method comprises: a step of preparing a pool of eluent samples of said mixture, wherein each eluent sample of the pool of eluent samples comprises a target molecule purified in a protein affinity capture process; a step of measuring the pH and protein concentration of each eluent sample; a step of titrating each eluent sample of said pool of eluent samples to determine the amount of acid required to make each eluent sample reach an inactivation pH; and a step of measuring data points using each eluent sample of said pool of eluent samples. A method comprising the step of using the above data points to regress the relationship between the amount of acid required to make the mixture inactivated pH, the protein concentration of the mixture, the pH of the mixture, and the inactivated pH, wherein the relationship comprises the following equation (3): equation (3) w = Ex + Fy + Gz + H, where in equation (3), w is the amount of acid added in molar units of acid per kilogram of mixture, x is the protein concentration of the mixture in grams per liter, y is the inactivated pH, z is the pH of the mixture, and E, F, G, and H are constants. Claim 21 A method, wherein, in paragraph 20, the verification model additionally includes regression. Claim 22 A method according to claim 20, further comprising the step of measuring more than one data point using at least one eluent of the pool of eluents. Claim 23 A method for inactivating a virus in a mixture, wherein the method comprises: a step of measuring the protein concentration of the mixture; a step of calculating the amount of acid required to reduce the pH of the mixture to an inactivating pH based on the protein concentration of the mixture; and a step of adding the amount of acid required to reduce the pH of the mixture to the inactivating pH to the inactivating pH to the mixture, and calculating the amount of acid required to reduce the pH of the mixture to the inactivating pH according to the following formula (3): formula (3) w = Ex + Fy + Gz + H In the above formula (3), w is the amount of acid added in molar units of acid per kilogram of mixture, x is the protein concentration of the mixture in grams per liter, y is the inactivating pH, z is the pH of the mixture, and E, F, G, and H are constants. Claim 24 A method according to claim 23, further comprising the step of pre-determining the expected pH of the mixture after adding the acid to the mixture using a pH verification model. Claim 25 A method according to claim 24, further comprising the steps of: recording the pH of the mixture of the acid and the mixture; calculating the difference between the expected pH and the recorded pH; and performing a correction measure based on the calculated difference between the expected pH and the recorded pH. Claim 26 A method according to claim 23, further comprising: a step of measuring the conductivity of the mixture before adding the amount of acid; and a step of adding one or more salts to the mixture in an amount sufficient to adjust the conductivity of the mixture.

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