Methods for virus inactivation

Abstract

This provides a method for developing an acid-based inactivation protocol for viruses in mixtures. [Solution] A method comprising: preparing a pool of eluate samples of a mixture containing a target molecule purified by a chromatography process; generating a pool of acidified samples having a pH below a first inactivation pH; generating a first data point for the acidified samples; generating a second data point for the inactivated samples; and using the first and second data points, regressing the relationship between the protein concentration of the mixture, the pH of the mixture, the inactivation pH range, and the amount of acid required to bring the mixture into the inactivation pH range.

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 and incorporates its entire disclosure 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. It relates to methods for achieving a target pH in a mixture containing a polypeptide.

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, separation processes such as affinity chromatography are carried out as part of the target molecule preparation process and obtained. After such a separation process, the resulting mixture containing the polypeptide may contain potentially undesirable viruses or other contaminants that are undesirable to be contained in a drug product. Therefore, methods for removing or inactivating such contaminants are desirable. In some commercial - scale target molecule synthesis processes, Process Analytical Technology (PAT) can be implemented. PAT is a system and method involved in the design, analysis, and control of the production process of a target molecule

[0004] ​​​​​​​ This includes. PAT identifies process parameters that affect product quality and regularly checks This includes monitoring the latitudinal meter to ensure product quality is maintained. PAT is the target molecule And to reduce the risks associated with pharmaceutical products in general, as encouraged by regulatory bodies. PAT can improve or maintain the quality of target molecules and / or products. It is possible to provide statistical verification or confirmation that one or more process conditions are met. can. [Overview of the project]

[0005] The methods and systems disclosed herein include polypeptide preparation including virus inactivation. The efficiency and / or productivity of the manufacturing process may be improved. The methods and systems disclosed herein The system can also improve the efficiency and / or productivity of pharmaceutical product preparation methods, as described above. It can address one or more problems identified by this method.

[0006] overview Embodiments of this disclosure describe the inactivation of viruses in a mixture, such as an eluate. This could be about the method of doing so. This method is from a chromatography column, 3.9 This method may involve eluting the mixture at a pH higher than 8.5 or lower. This may further include measuring the protein concentration and measuring the pH of the mixture. Next, based on the protein concentration of the mixture, the pH of the mixture is set to the inactivation pH (inactivation pH). The amount of acid needed to lower the pH to the ivation pH can be calculated. After calculating the amount to be added, the first part of the acid, which is 68% to 99% of the total amount of acid added, The portion may be added to the mixture. This method further ensures that the pH of the mixture is below the inactivation pH. This may include adding an additional portion of acid to the mixture to achieve the following: The mixture is inactivated at an inactivation pH and for an inactivation interval. It can be maintained for l) and configured to inactivate the virus in the mixture.

[0007] Methods for inactivating viruses in a mixture, as described in some embodiments of this disclosure. This may include loading a mixture containing the target molecule into a chromatography column, and the loading is This can be performed at a pH of approximately 5.0 to approximately 8.5. This method further involves chromatography - From the column, the eluted mixture containing the target molecule (el) was obtained at a pH higher than approximately 3.9 and lower than approximately 5.0. This method may include eluting the mixture. In addition, it may include forming a combination of a mixture and an acid, and this combination is effective It is configured to exhibit effective virus inactivation, and this combination is approximately 3.8 or less, approximately 3.0 or less. The above pH is obtained. Using the pH confirmation model, the expected pH of the combination is obtained. The pH can be predetermined. Furthermore, the pH of the combination can be measured and / or You may record it. Calculate the difference between the predicted pH and the recorded pH, and compare the predicted pH and the recorded pH. Corrective measures may be taken based on the calculated difference.

[0008] Further embodiments of this disclosure include an acidic inactivation protocol. This may include a method for developing an elution protocol. This may include creating a pool of liquids, where each eluate in the pool of eluates is a protein Affinity Capture (protein affinity capture) Pro It contains a target molecule purified in Seth. This method may include measuring the pH and / or protein concentration of each eluate in the eluate pool. Further, each eluate in the eluate pool can be titrated to determine the amount of acid required to bring the eluate to the inactivation pH. Then, the relationship between the amount of acid added, the eluate protein concentration, the eluate pH, and the inactivation pH can be regressed.

[0009] In some embodiments of the present 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 the 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 Description of the Drawings The accompanying drawings, which are incorporated herein and constitute a part of this specification, illustrate various exemplary embodiments and, together with the description, serve to explain the principles of the disclosed embodiments. Any feature (e.g., composition, formulation, method, etc.) of the embodiments or examples described herein can be combined with any other embodiment or example, and all such combinations are encompassed by the present disclosure. Further, the described systems and methods are not limited to any single aspect or its embodiment, nor to any combination or permutation of such aspects and embodiments. For the sake of brevity, specific substitutions and combinations are not individually described and / or illustrated herein. tion), method, etc.) can be combined with any other embodiment or example, and all such combinations are encompassed by the present disclosure. Further, the described systems and methods are not limited to any single aspect or its embodiment, nor to any combination or permutation of such aspects and embodiments. For the sake of brevity, specific substitutions and combinations are not individually described and / or illustrated herein.

Brief Description of the Drawings

[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" and "comprises" are used to mean "comprises." "rising)", or other variations thereof, are intended to cover non-exclusive inclusion. A process, method, article, or apparatus that includes a list of elements may only include those elements. Rather than including, as explicitly listed in such processes, methods, articles, or apparatus It may include other elements that are not present or are not unique. (Example) The term does not mean "ideal," but rather "example." It is used with the meaning of "for example". The terms "for example" and "(for example) Regarding "ba) ~nado (such as)" and their grammatical equivalents, there is no particular explicit indication. Unless otherwise specified, the phrase "and without limitation" is understood to follow.

[0015] The term "about" as used in this specification is used to explain variations due to experimental error. This means that. When applied to numbers, the terms "about" and "approximately" are used. "Occasionally" means + / - from the disclosed value unless a different variation is specified. It may show a variation of 5%. When applied to pH values, the terms "about" and "approximately" "(approximately)" may indicate a variation of + / -0.05. When used, the singular forms "a," "an," and "the" are used when the context clearly indicates otherwise. Unless otherwise specified, it includes multiple criteria.

[0016] All numerical data disclosed herein (including all disclosed values, limits, and ranges) are subject to change. Unless otherwise specified, the disclosed figures may vary by + / - 5%. Please note that the pH values ​​disclosed herein may have variations of + / -0.05. All ranges are understood to include the endpoint, for example, 1 cm to 5 cm. This includes lengths of 1 cm, 5 cm, and all distances between 1 cm and 5 cm.

[0017] Detailed explanation This disclosure is intended to protect the manufacturers of specific compositions, formulations, materials, and pharmaceutical products (dr) disclosed herein. (ug product), device, system, experimental conditions, or specific method are not limited to these. Furthermore, many modifications are possible within the scope of those skilled in the art. The terms used herein are specific to those skilled in the art. This is solely for the purpose of explaining the implementation methods, and is not intended to be limiting.

[0018] Unless otherwise defined, all technical and scientific terms used herein are defined in accordance with the provisions of this publication. The terms used in this disclosure have the same meaning as generally understood by those skilled in the art. In the test, any suitable method and material (e.g., those described herein) may be used. You can use something similar or equivalent, but here we will explain a specific method. All publications listed are incorporated herein by reference.

[0019] As used herein, the term “polypeptide” refers to a polypeptide that is covalently bonded via an amide bond. This refers to any amino acid polymer containing more than approximately 20 amino acids. Proteins are not just one amino acid polymer. It contains the amino acid polymer chain (e.g., polypeptide) shown above. Therefore, polypeptide is Proteins are molecules, and to form a single functional biomolecule, multiple proteins are formed from multiple polypeptides. It may contain butylene.

[0020] Post-translational modifications This can modify or alter the structure of polypeptides. For example, disulfide crosslinks (e.g., cis Disulfide bonds (SS bonds) between thein residues can be formed post-translation in some proteins. Some disulfide crosslinks are found in polypeptides, immunoglobulins, proteins, and cofactors. Cofactors are essential for the proper structure, function, and interaction of substrates and other elements. In addition to sulfide bond formation, proteins undergo lipidation (for example) Myristoylation, palmitoylation lation), farnesoylation, geranylgerani Geranylation and glycosylphosphatidylin Nositol (GPI) anchor formation, alkylation (e.g., methylation), acylation, amine Glycosylation (e.g., arginine, asparagine, cysteine, hydroxylysine) , the glycosyl group in serine, threonine, tyrosine and / or tryptophan Addition, and phosphorylation (i.e., serine, threonine, tyrosine, and / or hist It can undergo other post-translational modifications, such as the addition of a phosphate group to thidine. Post-translational modifications include hydrophobicity. It affects electrostatic surface properties, or other properties that determine intersurface interactions involving polypeptides. It is possible.

[0021] As used herein, the term "protein" refers to biotherapy (biotherapeutic). eutic) protein, recombinant protein used in research or therapy, trap Fc fusion proteins and other Fc fusion proteins, chimeric proteins, antibodies, monoclonal proteins Antibodies, human antibodies, bispecific antibodies, antibody fragments, antibody-like molecules, nanobodies, recombinants Includes antibody chimeras, cytokines, chemokines, peptide hormones, etc. Protein-of-interest (POI) is isolated, purified, or It may contain any polypeptide or protein that is preferable to be prepared by other means. Possible. POIs are targeted polypeptides or other polypropylenes produced by cells, including antibodies. It may contain peptides.

[0022] As used herein, the term "antibody" refers to a four-polypeptide chain: connected by disulfide bonds. Immunoglobulins consist of two interconnected heavy chains (H) and two light chains (L). Includes. Typically, antibodies have molecular weights greater than 100 kDa, e.g., 130 kDa~20 For example, 0kDa, or approximately 140kDa, 145kDa, 150kDa, 155kDa It has a molecular weight of 160 kDa, etc. Each heavy chain has a heavy chain variable region (HC in this specification). It includes the heavy chain steady region (abbreviated as VR or VH) and the heavy chain steady region. The heavy chain steady region is CH1, CH2 It consists of three domains, and CH3. Each light chain has a light chain variable region (here, LCVR or It includes the VL (abbreviated as VL) and the light chain constant region. The light chain constant region contains one domain CL. The VH and VL regions are further stored in a more conserved area called the Framework Region (FR). It is further divided into highly variable regions called complementary determination regions (CDRs), which are scattered along with the main region. It can be done. Each VH and VL consists of 3 CDRs and 4 FRs, These are arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1 FR2, CDR2, FR3, CDR3, FR4 (heavy-chain CDRs are HCDR1, HCDR 2 and HCDR3 can be abbreviated as HCDR1 and HCDR2; the light chain CDRs are LCDR1, LCDR2, etc. It can be abbreviated as LCDR3.

[0023] For example, a type of immunoglobulin called immunoglobulin G (IgG) is found in human serum. It is commonly found in cysts and contains four polypeptide chains: two light chains and two heavy chains. Each light chain is cyst One heavy chain is bonded via a disulfide bond, and the two heavy chains are bonded to two cystine disulfides. They are bound to each other via ion bonds. Other classes of human immunoglobulins include IgA, This includes IgM, IgD, and IgE. In the case of IgG, it includes IgG1, IgG2, and IgG 3. There are four subclasses of IgG4. Each subclass has a different constant region. Therefore, it may have different effector functions. In some embodiments described herein, The POI may include a target polypeptide containing IgG. In at least one embodiment... In this case, the target polypeptide contains IgG4.

[0024] As used herein, the term "antibody" also refers to the antigen-binding fragment of a complete antibody molecule. Includes the antigen. Terms such as the "antigen-binding portion" of an antibody and the "antigen-binding fragment" of an antibody are used. When used herein, any natural antigen that specifically binds to an antigen and forms a complex. Enzymatically available, synthetic, or genetically engineered polypeptides or Contains glycoproteins. Antibody antigen-binding fragments contain antibody variable domains and optionally fixed Protein digestion or recombination, including manipulation and expression of DNA encoding constitutive domains. Using any appropriate standard technique, such as genetic engineering, a complete antibody molecule can be derived, for example. Such DNA can be obtained from, for example, commercially available sources, DNA libraries (e.g., farms). (including di-antibody libraries) readily available and / or synthetically available Yes, DNA can be chemically or molecularly derived using techniques, for example, one or more variable DNA molecules. To place the main and / or constant domains in the appropriate configuration, or to introduce codons In order to generate cysteine ​​residues, in order to modify amino acids, to add Sequences may be determined and manipulated for the purpose of, or for, deletion.

[0025] The target molecule (e.g., target polypeptide) is derived from insect baculoviruses, yeast (e.g., P Ichia sp.), or mammalian cells (e.g., CHO cells and CHO-K1 cells) Using recombinant cell-based production systems such as CHO derivatives It can be produced by [unclear]. The term "cell" includes cells suitable for expressing recombinant nucleic acid sequences. Cells include prokaryotes and eukaryotes (unicellular or multicellular), and bacteria. )Cells (e.g., E. coli, Bacillus spp., Streptomyce) strains such as s spp., mycobacteria cells, fungal cells, and yeast. Cells (e.g., S. cerevisiae, S. pombe, P. pastoris, P. (e.g., *methanolica*), plant cells, insect cells (e.g., SF-9, SF-21) baculovirus-infected insect cells (e.g., Trichoplusiani), non-human animal cells Cells, human cells, or cell fusions such as hybridomas or quadromas are included. In some embodiments, the cells are human, monkey, and ape ( APE cells may be hamster, rat, or mouse cells. In some embodiments, odor The cell may be a eukaryote and can be selected from the following cells: CHO (for example, CHO K1, DXB-11 CHO, Veggie-CHO), COS (e.g. COS-7) , retinal cells, Vero, CV1, kidney (e.g., HEK293, 293 EBNA, MS) R 293, MDCK, HaK, BHK), HeLa, HepG2, WI38, MRC 5. Colo205, HB 8065, HL-60, (e.g., BHK21), Jurk at, Daudi, A 431 (epidermis), CV-1, U937, 3T3, L cell, C12 7 cells, SP2 / 0, NS-0, MMT 060562, Sertoli cells, BRL 3A cells, HT1080 cells, myeloma cells, tumor cells, and cells derived from the above-mentioned cells Cellular system. In some embodiments, the cell contains one or more viral genes, for example, a virus It may include retinal cells expressing the S gene (e.g., PER.C6(TM) cells).

[0026] In this specification, the term "target molecule" refers to a target polypeptide (e.g., antibody, antibody fragment, or other proteins or protein fragments), or manufactured, isolated, and purified This is used to refer to and / or other molecules intended to be included in pharmaceutical products. (For example, adeno-associated virus) s)(AAV) or other molecules for therapeutic use. The method according to this disclosure involves the target polypeptide being spoken to. It can be applied to other target molecules as well, for example, AAV This involves appropriate methods (e.g., deep filtration, affinity filtration). It can be prepared according to methods such as A-chromatography, and a mixture containing AAV (for example) The eluate (containing AAV) may be subjected to a method according to this disclosure. One or more methods according to this disclosure Before or after following the procedure, the mixture containing AAV should not undergo any additional steps (e.g., not containing the target sequence). It can be used for "empty cassettes" or removal of AAVs.

[0027] The term "viral content" is a qualitative description of a mixture. This refers to a virus. For example, if a mixture contains a virus or virus-like particles, the mixture is considered a virus. It has a virus content. In one embodiment, the virus content is the amount of virus per unit volume of the mixture. The number of Rus particles or the number of infectious units (i.e., concentration) It can be quantified by [method]. The term "virus concentration" refers to the amount of virus particles (e.g., active This can mean the concentration of (and inactive virus particles) or the concentration of infectious units.

[0028] Exemplary methods for virus inactivation include several viruses and virus-like particles Adding an acid to a mixture to achieve a pH known to inactivate, and This may include holding the mixture at the achieved pH for a predetermined time. For example, several In the embodiments thereof, the methods herein involve retroviruses and retrovirus-like particles It can inactivate the target molecule. In some embodiments, the target molecule can be extracted from a mixture containing the target molecule. A method for preparation may include contacting the mixture with a chromatography apparatus. Such chromatography apparatuses are pre-fabricated devices (for example, Cadence( TM) BioSMB (Pall Biosciences), BioSC (Registered Trademark) (novasep), Varicol(registered trademark)(novasep), Octave( Semba® Biosciences), custom-manufactured equipment, manually Assembled equipment, or simply two or more standard batch chromatographs used in tandem. It may include a tography device.

[0029] In some embodiments, the target molecule is a stripping buffer (strippi Add ng buffer to a chromatography apparatus (e.g., chromatography column) By bringing it into contact and / or equilibration buffer By bringing the buffer into contact with the chromatography apparatus, chromatography It can be eluted from the device. In some embodiments, the stripping buffer is The solution may include water, an alkaline solution, or an alcohol. For example, deionization. Water, such as water, contains less than 5 volume% (vol.%) of dissolved ions, less than 1 volume% of dissolved ions, It may contain less than 0.1% by volume of dissolved ions, or less than 0.01% by volume of dissolved ions. According to several embodiments, the alkaline solution is composed of LiOH, NaOH, KOH, and Ca(OH) )2. Containing one or more alkali ion compounds such as NH4OH or other alkali compounds It is possible. The concentration of the alkaline compound in the stripping buffer is, for example, about 0.1N to about 1.5N, approx. 0.1N to approx. 1N, approx. 0.1N to approx. 1.5N, approx. 0.5N to approx. 1.5N, approx. 0.1N to approx. 0.8N, approx. 0.1N to approx. 0.6N, approx. 0.1N to approx. 0.5N, approx. 0.1N It can be in the range of approximately 0.4N, or approximately 0.1N to approximately 0.3N. For example, stripping The concentrations of the alkali compound in the buffer are approximately 0.1N, 0.2N, 0.3N, and 0. 4N, approximately 0.5N, approximately 0.6N, approximately 0.7N, approximately 0.8N, approximately 0.9N, approximately 1N, approximately 1. It can be 1N, approximately 1.2N, approximately 1.3N, approximately 1.4N, or approximately 1.5N. Stripping buffers containing alcohol include methanol, ethanol, propanol, and Can contain diluent alcohol or other alcohols. Stripping buffer The alcohol concentration inside is approximately 0.1 volume (v) relative to the total weight of the stripping buffer. ol.)% ~ approximately 30 volume%, for example, 0.5 volume% ~ approximately 30 volume%, approximately 0.5 volume% ~ approximately 25% by volume, approximately 0.5% to approximately 25% by volume. Approximately 0.5% to approximately 25% by volume, approximately 1 volume %~approximately 20 volume%, approximately 1 volume%~approximately 15 volume%, approximately 1 volume%~approximately 10 volume%, approximately 10 volume %~approximately 50 volume%, approximately 10 volume%~approximately 40 volume%, approximately 10 volume%~approximately 30 volume%, approximately 10 Volume% to approximately 25% by volume, approximately 15% to approximately 25% by volume, or approximately 20% to approximately 25% by volume It can be %. For example, the alcohol concentration in the stripping buffer is approximately 0.1% by volume. Approximately 0.5 volume%, approximately 1 volume%, approximately 2 volume%, approximately 3 volume%, approximately 5 volume%, approximately 10 volume%, It could be approximately 15% by volume, approximately 20% by volume, or approximately 25% by volume.

[0030] In some embodiments, the equilibration buffer has a composition similar to that of the stripping buffer. They may be similar or identical. In other embodiments, the balancing buffer is The composition may vary compared to the stripping buffer. In some embodiments, The equilibration buffer is, for example, sodium, potassium, magnesium, calcium, quae Contains one or more salts such as phosphates, acetates, phosphates, sulfates, Tris, or other salts. But that's fine.

[0031] In one or more embodiments, a method for inactivating a virus is a mixture containing a target molecule The substance is a chromatography apparatus (e.g., packed bed affinity) Chromatography columns, hydrophobic interaction chromatography columns, ion exchange columns Dissolve from a chromatography column and / or a size exclusion chromatography column. It can be used after being released. Loading the target molecule into the chromatography apparatus (loading g) may vary depending on the upstream process, but the mixture containing the target molecule is constant in volume ( In some embodiments, it can be eluted from the chromatography apparatus in volume. The mixture containing the target molecule is then subjected to a pH of approximately 5.0 to approximately 8.5, for example, approximately 5.5 or higher. pH levels of approximately 8.5 or lower, between approximately 6.0 and 8.5, or between approximately 5.0 and 6.5 are suitable for chloroforms. Load the chromatograph column. As a result of variations in loading, the eluate (e.g., chromatograph) The protein concentration and / or pH of the eluate (eluted from the imaging device) may vary. This fluctuation also causes a change in the amount of acid required to inactivate the virus.

[0032] In conventional manufacturing processes, low pH virus inactivation is achieved through trial and error, Here, a predetermined amount of acid is added to the eluate, and the pH of the eluate and the acid mixture is measured. The measurement step is repeated until an inactivation pH is reached. Due to the potential costs and losses that may arise, such processes are conservative and small in scale. This involves the amount of acid added and often a long inactivation process time on a time scale.

[0033] Aspects of this disclosure provide various advantages to processes for preparing target polypeptides or other target molecules. Points can be provided. For example, one or more methods and / or mathematical models described herein. The process involves determining the amount of acid added, for example, the mixture (target molecule and potentially undesirable virus). Determining the amount of acid needed to bring the pH to an inactivating pH (including viruses or virus-like particles). This can be done. An amount of acid approximately equal to the amount of acid added may be added to the mixture. More details are explained below. As explained, the amount of acid equivalent to the amount of acid added is considered as one bolus. Alternatively, it can be added by administering the acid two or more times. Virus inactivation by this method. The administration of acid for this purpose is more efficient and less susceptible to errors than conventional trial-and-error methods.

[0034] In some aspects of this disclosure, as part of the process, the virus content in the mixture The amount of infectious units present may be minimal or absent, or It can be expected. In some such aspects of this disclosure, the sys disclosed herein The system and method are advantageously incorporated into the manufacturing process as part of the patent, for example. This reduces potential variability within the process and allows for real-time verification of compliance with process standards. It can provide and / or enhance the reliability of the process's 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, the target molecule is chromatographically and / or separated by one or more methods. After preparation using the process, a mixture (e.g., eluate) can be obtained. In the embodiment, for example, protein concentration, target molecule concentration, pH, or combination thereof. One or more measurements of the mixture, including combinations, may be performed. For example, the protein concentration may be: It can be measured by any suitable method, including ultraviolet / visible light spectroscopy. In the embodiment, the protein concentration is determined by the polypeptide target molecule. It is measured using a wavelength that is characteristically absorbed. In such embodiments, the whole protein The concentration of the substance can be approximately equivalent to the concentration of the target molecule (e.g., the target polypeptide). In that embodiment, the mixture containing the target molecule is measured in grams per liter of eluate. (g / L) Approximately 7.0g / L to approximately 35.0g / L, approximately 7.0g / L to approximately 20.0g / L The following ranges are approximately 8.5g / L to 18.5g / L, or approximately 10.0g / L to 17.0g / L. It may have a protein concentration of [value].

[0037] The pH of a mixture can be measured by any suitable method. The measurement performed is important for successfully inactivating viral proteins by low pH. H measurement depends on temperature, the type of pH probe used, and the individual differences between pH probes of the same type. The difference, and / or the physical interaction between the measured medium and the pH probe. It can be affected by the action. Even with a standardized pH measurement process, there can be a variation of ±0.05 pH. This is common. In the fields of polypeptide production or virus inactivation, ±0.05 pH is used. The variation in scale corresponds to approximately 20% of the working pH range, and inactivates the virus and / or processes This can have a detrimental effect on validation. Such fluctuations can occur over time and Both can combine to cause instrument drift and more extreme fluctuations in pH readings. There are even variations in pH measurement. Therefore, in some embodiments, when measuring pH, variations in pH measurement are used. This can be considered. In some embodiments, as suggested, the pH of the eluate This is a standardized method aimed at reducing or eliminating variability in pH measurement. It can be measured by [method]. Standardization of pH measurement methods is a single manufacturer of pH probes. Use of a single lot of pH probes, measurement of pH at a given temperature, measurement sample matrix This may include standardization of risks, etc. In some embodiments, the pH of the eluate is It can be measured by a pH meter, such as a potentiometer or pH meter. Several implementations In this state, the eluate containing the target molecule has a pH of approximately 3.9 to approximately 8.5, for example, approximately 3 0.9~approx. 6.5, approx. 3.9~approx. 5.5, approx. 4.5~approx. 6.5, approx. 4.0~approx. 4.4, approx. 3 It may have a pH of 0.9 to about 4.4, or about 4.0 to about 4.3. In this specification, p The range of the H value or pH value may have variations of ±0.05 pH units.

[0038] Certain viruses and viruses in mixtures (e.g., eluents), formulations, and / or pharmaceutical products. S-like particles (e.g., enveloped viruses, retroviruses, retrovirus-like particles, hypothetical) The presence of sexually transmitted diseases (pseudorabies, herpesviruses, etc.) is such This may affect the ingredients, properties, or usability of mixtures, formulations, and / or pharmaceutical products. For example, if unwanted viruses or virus-like particles are present in a pharmaceutical product, This may affect the stability of the product, shorten its shelf life, or cause the product to violate internal standards, official regulations, or This may result in non-compliance with regulations (e.g., the U.S. Food and Drug Administration). Viruses and virus-like particles Some children experience clinical effects such as immunogenic reactions after being administered a preparation containing the virus. There are some. Embodiments of this disclosure inactivate viruses or virus-like particles, It is useful in reducing or eliminating any or all of the following undesirable effects. For example, embodiments of the present disclosure involve one or more polypeptide purification processes (e.g., pro After the separation process (including the Tein A affinity column), the mixture containing the virus It may be applicable to substances (e.g., eluents).

[0039] In some embodiments, the conductivity of the mixture is measured before virus inactivation. This can be done. In some embodiments, one or more salts are mixed before virus inactivation. It can be added to a compound to adjust its conductivity (for example, to increase its conductivity). One or more such salts are alkali metal salts, alkaline earth metal salts, halides, and It may contain and / or one or more other ionic active compounds. Not limited by theory. The addition of one or more salts to adjust conductivity can affect target molecules, viruses, or retrograde molecules. It may reduce the aggregation of virus-like particles. The composition may influence how the surfaces of these species interact with acids. The addition increases the ionic activity of the mixture, increases the conductivity of the mixture, and targets the molecule or Illus aggregation can be reduced. Therefore, in some embodiments, the conductivity of the mixture This may be related to the degree of aggregation of the target molecule or virus.

[0040] In some cases, embodiments of the present disclosure are used for very low viral content (e.g., 1 (Approximately 0.0001 virus particles or infection units per mL), or no viruses present. It may be applicable to mixtures having a high content. Chromatography and other separation processes. The solution, either alone or in combination, thoroughly purifies and / or separates the target molecule, and the mixture... Unwanted viruses or virus-like particles can be removed in such a situation. In this case, the method described herein is used to inactivate viruses or virus-like particles. In addition, we guarantee the stability of the product, the safety of the product, the effectiveness of the product, and the internal specifications or This may be useful in ensuring compliance with regulatory specifications. Therefore, embodiments of this disclosure For example, ensuring that regulatory guidelines are met and / or redundant quality To provide control, it can also be applied to mixtures without known virus content.

[0041] The virus inactivation protocols and methods described herein are for AAV (e.g., targeted distribution To implement without adversely affecting certain types of viruses, such as AAV (including the column). This is possible. For example, advantageously, the protocols and methods described herein degrade AAVs. It can be carried out without causing any damage. Therefore, the methods described herein target molecules It may be suitable for use in mixtures containing AAV.

[0042] As suggested above, the virus in the mixture will be inactivated at an inactivating pH for an inactivating time. It can be inactivated by maintaining it for a certain period of time. The inactivation pH is 3.8 or lower and 3.0 or higher. pH, for example, 3.35-3.8, 3.75 or less and 3.0 or more, 3.7 or less and 3.0 or more, 3 .65 or less 3.0 or more, 3.6 or less 3.0 or more, 3.55 or less 3.0 or more, 3.5 or less 3 .0 or more, 3.45 or less 3.0 or more, 3.40 or less 3.0 or more, 3.35~3.75, 3 0.5~3.8, 3.5~3.75, 3.5~3.7, 3.5~3.6, or 3.5~3 The pH may be 0.65, for example. As used herein, the pH value or range of pH values ​​is The pH can fluctuate by ±0.05 units. If the inactivation pH is made too high, the inactivation process will be affected. Varying results may occur within the system, potentially leading to insufficient virus inactivation. Lower the inactivation pH. Combing too much can denature target molecules or other proteins, or cause undesirable results. There is a risk that the mixture may be altered by law.

[0043] The inactivation time is the time (interval of) the mixture is held at the inactivation pH. This indicates the inactivation time. The inactivation time is approximately 20 minutes to 90 minutes, for example, approximately 30 minutes, approximately 45 minutes. Approximately 60 minutes, approximately 30-45 minutes, approximately 30-60 minutes, approximately 30-75 minutes, approximately 30 minutes ~90 minutes, 45 minutes to 60 minutes, 45 minutes to 75 minutes, 45 minutes to 90 minutes, 60 minutes This can be approximately 75 minutes, or approximately 60 to 95 minutes. The mixture is deactivated at an inactivating pH. By holding the mixture for an extended period, the viral activity in the mixture is reduced, removed, or ensured. It can be inactivated in a low pH environment, for example, viral envelope proteins. It can denature viral proteins such as those mentioned above. The denatured viral proteins are retroviral It can inactivate sphingovirus and retrovirus-like particles, and eliminate undesirable viral activity in the mixture. It can be reduced.

[0044] The decrease in viral activity in the mixture is expressed as a reduction factor. Therefore, it can be quantified. The reduction coefficient can be calculated according to Equation 1 shown below. ru.

number

[0045] According to one or more embodiments, the amount of acid added is related to the protein concentration of the mixture and the inactivation p It can be calculated based on H. For example, the amount of acid added can be calculated according to formula 2 below. It is possible. 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, and w is the number of moles of acid per kilogram of the mixture (mol / The amount of acid added is expressed in kg, where A, B, and C are constants. The constant A in Equation 2 is the amount of acid added. Units of volume (L·mol / g·kg) of acid per gram-kilogram of substance. It has the constant A, which is 0.0003 L·mol / g·kg or more and 0.0006 L·mol / Less than g·kg, for example, approximately 0.0003 L·mol / g·kg to approximately 0.0005 L·mol l / g·kg, approx. 0.0004L·mol / g·kg ~ approx. 0.0006L·mol / g· kg, approx. 0.00035L·mol / g·kg ~ approx. 0.0005L·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, etc. It is possible. The constants B and C in Equation 2 are units (m) of the number of moles of acid per kilogram of the mixture. It has (ol / kg). The constant B is between -0.1 mol / kg and 0 mol / kg, for example For example, approximately -0.1 mol / kg to approximately 0 mol / kg, and approximately -0.1 mol / kg to approximately -0.0 5 mol / kg, approximately -0.05 mol / kg to approximately 0 mol / kg, or approximately -0.08 m The concentration can be as low as ol / kg, approximately -0.01 mol / kg. The constant C is 0.02 mol / kg. g or more and 0.1 mol / kg or less, for example, approximately 0.02 mol / kg to approximately 0.1 mol / kg g, about 0.02mol / kg~about 0.05mol / kg, about 0.05mol / kg~about 0 This is 0.1 mol / kg, or approximately 0.04 mol / kg to approximately 0.08 mol / kg. obtain.

[0046] In some embodiments, the amount of acid added depends on the protein concentration of the mixture, the inactivation pH, And it can be calculated based on the pH of the mixture. For example, according to Equation 3 shown below. The amount of acid added can be calculated. 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). Yes, y is the inactivation pH, z is the pH of the mixture, and w is the pH per kilogram of the mixture. This is the amount of acid added, expressed as the number of moles of acid (mol / kg), where E, F, G, and H are constants. The constant E in Equation 3 is the amount of liters-moles of acid per gram-kilogram of the mixture. It has units (L·mol / g·kg), and the constants F, G, and H are per kilogram of the mixture. The unit of moles of acid is (mol / kg). The constant E is 0.00005 L·mol. Between 0.0005 L·mol / g·kg and 0.0005 L·mol / g·kg, for example, approximately 0.00005 L ·mol / g·kg~about 0.0005L·mol / g·kg, about 0.0001L·mol / g·kg~0.0005L·mol / g·kg, approximately 0.00005L·mol / g·k g~0.00045L·mol / g·kg, approx. 0.0001L·mol / g·kg~approx. 0 0.00035 L·mol / g·kg, or approximately 0.00035 L·mol / g·kg ~ approximately It can be 0.0005 L·mol / g·kg, etc. The constant F is -0.2 mol / kg or less. Above 0 mol / kg, for example, approximately -0.1 mol / kg to approximately 0 mol / kg, approximately -0. 1mol / kg~approx.-0.05mol / kg, approx.-0.05mol / kg~approximately 0mol / It can be in kg, or approximately -0.08 mol / kg to approximately -0.01 mol / kg. A few Gs is between 0 mol / kg and 0.03 mol / kg, for example, approximately 0 mol / kg to approximately 0.03mol / kg, about 0.001mol / kg~about 0.03mol / kg, about 0.0 0.5 mol / kg to approximately 0.3 mol / kg, or approximately 0.005 mol / kg to approximately 0.0 It can be 25 mol / kg, for example. The constant H is -0.1 mol / kg or greater, or 0.1 mol. Less than / kg, for example, approximately -0.1 mol / kg to approximately 0.1 mol / kg, approximately -0.08 m ol / kg~about 0.08mol-10 / kg, about -0.05mol / kg~about 0.1mo It can be l / kg, or approximately -0.1 mol / kg to approximately 0.05 mol / kg.

[0047] A specific formula relating the concentration of the mixed protein to the amount of acid added and the inactivation pH (as needed) Depending on the mixture's pH, the results may vary depending on the target molecule and / or acid system used. It is possible. The values ​​of the constants defined above that apply to a given target molecule and acid system are as follows: It can be determined by regression according to a defined general formula. Section of the example below As explained in the section, the amount of acid added depends on the protein concentration of the mixture and the strong phase according to the above definition formula. A relationship is unexpectedly found. This unexpectedly strong correlation is described herein. This makes it possible to incorporate the general formulas and their derivatives into PAT.

[0048] In one or more embodiments, the amount of acid added is 0.002 moles (m³) per 1 kg of the mixture. (ol / kg) is approximately 0.025 mol / kg, for example, approximately 0.002 mol / kg. Approx. 0.025mol / kg, approx. 0.01mol / kg to approx. 0.025mol / kg, approx. 0 0.002 mol / kg to approximately 0.020 mol / kg, or approximately 0.005 mol / kg to It is approximately 0.020 mol / kg, for example.

[0049] In some embodiments, after the amount of acid to be added is calculated, the acid is added to the mixture and mixed. It is possible to deactivate substances at pH. For example, in at least one embodiment, acid A bolus of acid equivalent to the amount added is added to the mixture to bring the pH of the mixture below the inactivation pH. This is possible. In other embodiments, the first portion of the acid is added to the mixture, and then the mixture One or more portions of the acid are added to the mixture so that the pH of the mixture falls below the inactivation pH. This may be done. In such embodiments, the first part of the acid is 68% to 9% of the amount of acid added. It is 9%, for example, approximately 75% to 99%, approximately 80% to 99%, approximately 85% to 99%, These range from approximately 90% to 99%, 85% to 95%, or 90% to 99%.

[0050] The first part of the acid is used to add target molecules to a mixture with the lowest possible pH. It can be distributed so as not to be altered by the addition. The additional portion of the acid is added to the first portion of the acid. This includes one or more subsequent additions of acid. For example, the three additions of acid that are carried out after the first addition of the acid (3 additio ns), 4 additions of acid, or 5 additions of acid. This may include ditions, etc. Each of the one or more additions of acid may be 0.1% of the total amount of acid added. A 32% amount, for example, about 0.1% to about 30%, about 0.1% to about 25%, about 0.1% to about 2 0%, approximately 1% to approximately 25%, approximately 0.1% to approximately 15%, approximately 0.1% to approximately 10%, approximately 1% to approximately 1 The amount may be 5%, approximately 1% to 10%, or approximately 0.1% to 5%. One or more applications of acid. Each addition may be in the same amount as one or more other additions of the acid. In other embodiments, Each addition of acid is in the same amount as each other addition of acid.

[0051] In some embodiments, the pH is determined after the first portion of the acid has been added to the mixture. However, this can be measured before the addition of the additional portion of the acid. Some such embodiments In this case, the pH of the mixture measured after the first part of the acid was added was 3.5 or higher. It is 75 or less, for example, approximately 3.5 to 3.75, approximately 3.6 to 3.7, approximately 3.5 to 3 These ranges from 0.65, approximately 3.6 to 3.75, or approximately 3.5 to 3.65. The pH value or pH range may have variations of ±0.02 pH units.

[0052] Acids can be added in the form of one or more acid solutions. The acid solution can be any suitable acid. For example, HCl, HBr, H3PO4, HO2C2O2H, C6H8O7, H2SO3, H3PO4, HNO2, C6H5CO2H, CH3CO2H, HClO, HCN, H3B It may contain O3 or a combination thereof. In addition, or alternatively, an acidic solution may be, for example For example, one of the following: glycine, arginine, sodium acetate, and / or sodium chloride. It may contain the above salts.

[0053] After adding the acid to a mixture containing the target molecule, the resulting mixture has a pH below the inactivating pH. It has H. In some embodiments, the pH of the formed mixture is, for example, It can be measured to confirm that it is within the desired range. As mentioned above, the mixture is inactivated. The mixture may be held at the activation pH for the inactivation time. After retention, the decrease in viral activity is, for example, 2.5 or higher, 3 or higher, 3.5 or higher, or This can occur in equivalent amounts to 4 or more reduction factors. The mixture was held at an inactivating pH for an inactivating time. Furthermore, the pH of the mixture must be between 4.5 and 8.5, for example, between 4.5 and 8.5, or above 5.0. 8.5 or less, 5.8 or more and 8.5 or less, 5.9 or more and 8.5 or less, 6.0 or more and 8.5 or less, 6 .1 to 8.5, 6.2 to 8.5, 6.3 to 8.5, 6.4 to 8.5 The mixture can be titrated by adding an alkaline solution as follows: Yes, it is possible. In this specification, pH values ​​or ranges of pH values ​​may have variations of ±0.02 pH units. It is possible. Alkaline solutions include, for example, NaOH, KOH, LiOH, Ca(OH)2, NH One or more salts such as 4OH, NaCH3CO2 and / or (HOCH2)3CNH2 It may include a base.

[0054] In some embodiments, the mixture is prepared less than one hour after the first portion of the acid is added. For example, less than approximately 50 minutes, less than approximately 45 minutes, less than approximately 40 minutes, less than approximately 35 minutes, or approximately 30 minutes. The titration is performed over a time interval of less than a certain duration.

[0055] Aspects of this disclosure predict the pH of a combination (i.e., a combination of a mixture and an acid). This can also include a method for determining the function. For example, measuring Based on the protein concentration and / or pH of the mixture, and the amount of acid added to the mixture. By doing so, we can determine a function that predicts the pH of the combination (e.g., the inactivation pH). Using such a function, processing errors (e.g., insufficient mixing, insufficient sampling) can be handled. It detects malfunctions (such as errors in the instrument, instrument drift, pH probe abnormalities, etc.) and equipment failures. It is possible.

[0056] In some embodiments, the expected pH of the acidified mixture is H) can be predetermined according to the verification model. The verification model is in the form of Equation 4 shown below. could be. Equation (4) y = Kx + Lw + Mz + N In the formula, y is the inactivation pH, and x is the amount of the mixture expressed in grams per liter (g / L). This is the protein concentration of the mixture, z is the pH of the mixture, and w is the per kilogram of the mixture. This represents the amount of acid added, expressed as the number of moles of acid (mol / kg), where K, L, M, and N are constants. In Equation 4, the constant K has units of liters per gram, and the constant L is the amount of acid per mole. The unit of weight is given in kilograms, while the constants M and N are unitless. The constant K is less than 0 L / g. Above 0.03 L / g, for example, approximately 0 L / g to approximately 0.03 L / g, approximately 0.001 L / g and above. Approximately 0.03 L / g, approximately 0 L / g to approximately 0.025 g / L, or approximately 0.001 g / L to approximately 0 It can be 0.025 L / g, etc. The constant L is between -80 kg / mol and -60 kg / mol. For example, approximately -75 kg / mol to approximately -60 kg / mol, approximately -80 kg / mol to It could be around -65 kg / mol, or around -75 kg / mol to around -65 kg / mol. The constant M is between 0 and 2.0, for example, approximately 0 to approximately 2.0, approximately 0.3 to approximately 2.0, approximately 0. It can be approximately 1.7, or approximately 0.3 to approximately 1.7, etc. The constant N is between -1.0 and 0. The numbers below, for example, approximately -1.0 to approximately 0, approximately -1.0 to approximately -0.1, or approximately -0.9 to approximately - It could be 0.1, for example.

[0057] In some embodiments, the pH and protein concentration of the mixture before the addition of acid are measured. and / or can be recorded: the pH of the mixture, the protein concentration of the mixture, and the acid addition. Based on the amount added, the predicted pH of the acidified mixture is determined in advance using a confirmation model. This allows you to measure, record, and / or predict the actual pH of an acidified mixture. It can be compared with H. Comparing the recorded pH with the predicted pH is possible. This may include calculating the difference (e.g., a percentage difference) between H 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 is, for example, ±0.03 pH from the predicted pH, or from the predicted pH. ±0.05 pH, ±0.07 pH from predicted pH, ±0.09 pH from predicted pH, predicted pH Within ±0.1 pH from the actual pH, ±0.15 pH from the predicted pH, or ±0.2 pH from the predicted pH. Possible. Corrective measures include adjusting the pH meter, adjusting the composition of the mixture, and adjusting one or more environmental factors in the process. This may include, but is not limited to, adjustments to conditions or combinations thereof. pH Me Adjusting the pH meter involves standardizing the pH meter, recalibrating the pH meter, and adjusting the pH profile. Clean, reset, and / or replace the tubing, prepare the reference electrode solution, and adjust the pH. Replacing part of the data, adjusting the position of the pH probe, and / or pH meter This may include other actions that change the signal-to-noise ratio of the data. Adjusting the composition of the mixture is one of the actions that can change the signal-to-noise ratio of the data. Recombining any component solution of the compound or upstream composition, one or more chromatography - or changing the process conditions or equipment components of the separation process, and / or mixing This may include other actions that alter the material composition of the compound, or adjusting one or more environmental conditions of the process. This involves adjusting mixing and / or homogenization processes and systems, and processes. Adjusting the temperature, adjusting the humidity of the process, adjusting the pressure of the process. , or a combination thereof. Deviation from the predicted parameters (devia Such adjustments based on (tion) can be incorporated as part of PAT.

[0059] The above formulas and mathematical models are part of the method for developing an acid-inactivation protocol. It can be prepared as follows. A method for developing an acidic inactivation protocol is a mixture (e.g., This may include preparing a pool of eluates, where each mixture in the pool is a protein Contains target molecules purified in the affinity capture process. For example, sample The pool is collected from the eluate of the protein affinity chromatography column. This method allows you to determine the pH and protein concentration (e.g., target molecule) of each sample. This may further include measuring the concentration of each sample. After determining the pH, each sample is titrated to determine the amount of acid needed to inactivate the sample. It can be determined. In some embodiments, the inactivation pH is set for each sump of the pool. It is possible to collect multiple data points from the titration, and to perform two or more titration repetitions. It can be defined as a sufficiently broad range. In other embodiments, a single data from each sample You can collect only points.

[0060] In some embodiments, relationships (e.g., mathematical models) ) are the amount of acid added, the concentration of eluate protein, the pH of the mixture, and / or inactivation. This relationship can be regressed between pH levels. As mentioned above, this relationship can be regressed according to Equation 2 or Equation 3. This is possible. In some embodiments, a method for developing an acidic inactivation protocol. This further includes regression on the confirmation model. The confirmation model is regression according to Equation 4 described above. It is possible.

[0061] Figure 1 shows an exemplary process 1 for inactivating a virus in a mixture according to the present disclosure. Step 00 is shown in flowchart format. According to step 101, the first pH (for example, 3.9) The mixture (highly concentrated) can be eluted from the chromatography column. Step 10 According to 2, the protein concentration of the mixture (e.g., target molecule concentration) is measured using, for example, ultraviolet / visible light. It can be measured by spectroscopy or other methods. Optionally, the conductivity and / or conductivity of the mixture. Alternatively, the pH can be measured, for example, using a potentiometer or other method. Step 1 Add one or more salts to the mixture in an amount sufficient to adjust the conductivity of the mixture according to 03. This can be done. According to step 104, the pH of the mixture can be changed to a second pH (e.g., an inactivating pH). The amount of acid needed to reduce the protein content of the mixture can be calculated. This can be done based on the concentration of the substance, the pH of the mixture, or a combination thereof. According to 105, the first portion of the acid can be added to the mixture. Several embodiments In this case, the first part of the acid is a bolus of acid corresponding to the calculated amount of acid added. In the embodiment, the first part of the acid is the volume or amount of the acid as the calculated amount of acid added. It can be 68% to 99%. According to step 105, optionally, a second (secondary ) Add the acid so that the combination of the mixture and the acid is below a second pH (e.g., inactivation pH). It may be added to the mixture. According to step 106, the combination of the mixture and the acid is adjusted to the second pH Maintain the inactivation pH (e.g., the inactivation pH) for the duration of the inactivation interval to inactivate the virus in the eluate. It is possible. In some embodiments,

[0062] Figure 2 shows an exemplary process 2 for inactivating a virus in a mixture according to this disclosure. Step 00 is shown in flowchart format. According to step 201, the mixture containing the target molecule is first It can be loaded into the chromatography column at a pH of approximately 5.0 to 8.5. . According to step 202, elute the mixture to a second pH (for example, higher than approximately 3.9 and lower than or equal to approximately 5.0). The eluted mixture may be eluted from the chromatography column. . In accordance with step 203, the protein concentration (e.g., target molecule concentration) of the eluted mixture is adjusted, for example, , can be measured and / or recorded by ultraviolet / visible light spectroscopy. Step 20 In accordance with 4, the pH and / or conductivity of the eluted mixture may be optionally measured. The pH is, For example, it can be measured and / or recorded using a potentiometer. According to step 205 This means that adding one or more salts to the mixture in an amount sufficient to adjust the conductivity of the mixture is Yes, it is possible. Add the acid to the elution mixture according to step 206, and combine the elution mixture and the acid. It is possible to form a set and configure the combination to exhibit effective virus inactivation. Furthermore, the combination has a pH lower than the second pH (for example, about 3.8 or lower and about It may have a pH of 3.0 or higher. In accordance with step 207, the combination is obtained using a pH confirmation model. The predicted pH may be predetermined. The pH of the combination is measured according to step 208. It can be called and / or recorded. According to step 209, the predicted pH of the combination and measurement The difference from the recorded pH can be calculated. According to step 210, the combination Correction measures are taken based on the calculated difference between the measured pH and the measured and / or recorded pH. It may be taken.

[0063] Figure 3 shows an exemplary process 3 for developing an acidic inactivation protocol according to this disclosure. Step 00 is shown in flowchart format. Prepare a pool of eluate samples according to step 301. This can be done, and here each eluate sample in the pool of eluate samples has affinity - Contains the target molecule purified by the capture process. Eluten with Sun according to step 302. The protein concentration (e.g., target molecule concentration) of each eluate sample in the pool of pulls is, for example, Measurement can be performed using ultraviolet / visible light spectroscopy. Optionally, the pool of eluate samples The pH of each eluate sample may be measured, for example, using a potentiometer. (According to step 303) The amount of acid needed to lower the pH of each eluate sample to the inactivation pH is determined by the eluate sample This can be determined by titrating each eluate sample in the sample pool. In accordance with 304, the amount of acid added, the eluate protein concentration, the eluate pH, and / or inactivation. The relationship with pH can be regressed. According to step 305, a confirmation model can be created as needed. You can use regression.

[0064] Figures 1-3 each show a specific sequence of steps, but the steps that are performed and their Please understand that the order in which the steps are performed may be modified. Furthermore, the steps (e.g., measurement) and / or one or more of the recording steps) can be added from any method disclosed herein. It can be removed. Furthermore, each of Figures 1-3 shows the process with respect to the eluate. However, it should be understood that the process is applicable to any mixture containing the target molecule. be. [Examples]

[0065] The following embodiments are intended to illustrate the present disclosure without being substantially limiting. This disclosure includes further aspects and embodiments consistent with the above description and the following examples. This is understood.

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

[0067] Example 1 The target polypeptide was eluted from a protein A affinity column, and multiple samples of the mixture were taken. Samples were obtained. The protein concentration of each mixture sample was obtained by ultraviolet / visible spectroscopy. The units of protein in grams per liter of mixture are shown in Table 1 below. Furthermore, the pH of each mixture sample was measured and is shown in Table 1.

[0068] The eluate pool contains 40 eluate samples (i.e., 40 mixture samples), Each sample was inactivated using a 0.25 M phosphoric acid (H3PO4) solution at pH 3.35-3. Titrate to 0.86 and add the amount of acid needed (for example, the amount needed to deactivate the mixture to pH 0.86). The amount of acid present was determined. Six out of 40 samples (Sample 1~ shown in Table 1) were selected. Regarding 6), multiple data points were obtained by repeating the titration multiple times. For example, sample 30.89g of acid per kilogram of the mixture that deactivates No. 1 to pH 3.70 is One data point, and an additional 5.35 g of acid per kilogram of mixture was added. This results in 36 units per kilogram of the mixture used to inactivate sample No. 1 to pH 3.59. 24g of acid is a separate data point. These titration data are mixed with the amount of acid added. The unit of grams of acid per kilogram of mixture and the unit of grams of acid per kilogram of mixture The unit of measurement is 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 mixture protein concentration and inactivation pH were determined according to Equation 2. The relationship between the amount of acid added and the coefficient of determination was regression-examined. The regression equation below (Equation 5) is the coefficient of determination (R 2 )but It was determined to be 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 mixture protein concentration and mixture according to Equation 3 are calculated. The relationship between the pH of the substance, the inactivation pH, and the amount of acid added was regression-examined. The regression equation (Equation 6) below is , coefficient of determination (R 2 ) was determined to be 0.97. Unexpectedly, the amount of acid added was approximately 2. The relationship between protein concentration, mixture pH, and inactivation pH shown in 3 is highly correlated. . w=0.0001986x-0.01162y+0.01510z-0.01692 Formula (6)

[0071] Example 4 The confirmation model was regression-tested using the titration data shown in Table 1, according to General Equation 4. The regression equation (Equation 7) is the coefficient of determination (R 2 ) was determined to be 0.93. Also, unexpectedly, The activation pH is highly correlated with the relationship between protein concentration, mixture pH, and inactivation pH. Ta. 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.

Claims

1. A method for inactivating viruses in a mixture, The mixture was eluted from the chromatography column at a pH higher than 3.9 and lower than 8.

5. To make someone do something; 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.

2. Maintaining the aforementioned combination at the 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 above combination further comprises titrating until the pH is between 4.5 and 8.

5. The method described in item 1.

3. Before adding the first portion of the acid to the mixture, the pH of the mixture is measured. The method according to claim 1, further comprising:

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 according to claim 3, which is calculated as follows. 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.

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 according to claim 4.

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 claim 3, wherein the amount of acid required to lower the pH to the inactivation pH is calculated. 。

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 claim 1, which is approximately 0.002 moles to approximately 0.025 moles per kilogram. Law.

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

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 claim 1, further comprising the following:

10. The chromatography column performs the protein affinity capture process. The method according to claim 1, configured to perform the action described above.

11. After the first portion of the acid is added, and before the additional portion of the acid is added, The method according to claim 1, wherein the pH of the mixture is approximately 3.5 to approximately 3.

75.

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 rhombus column; From the chromatography column, an elution mixture containing the target molecule is obtained, approximately 3. To elute a elution 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...

13. If the difference between the predicted pH and the recorded pH is approximately 0.15 or more, the correction measure The method according to claim 12, wherein the arrangement is taken.

14. The aforementioned corrective measures include adjusting the pH meter, adjusting the composition of the mixture, and one or more other such measures. The method according to claim 12, which includes adjusting the environmental conditions, or a combination thereof. 。

15. The combination is maintained at a pH of approximately 3.8 or less and approximately 3.0 or more for approximately 30 minutes, according to the claim. Method 12.

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 one hour. The method according to claim 12, further comprising titrating to a pH of 5 or less.

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 according to claim 12, further comprising the above.

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.

19. The pH confirmation model according to claim 18, comprising 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 is the amount of acid added to the compound, where K, L, M, and N are constants.)

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, 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.

21. The method according to claim 20, further comprising regressing on a confirmation model.

22. Multiple data points are determined using at least one eluate from the pool of eluates. The method according to claim 20, further comprising doing the following.

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.

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 claim 23, further comprising determining H in advance.

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 according to claim 24, further comprising the following:

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 according to claim 23, further comprising the above.

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