Methods for inactivating viruses

Abstract

The present invention provides a method for inactivating viruses using high temperature transient (HTST) treatment, and adjusting multiple parameters to reduce and / or minimize precipitate generation and sedimentation.

Description

[0001] This application is a divisional application of Chinese patent application 201380031879.X. The application date of the original application is June 20, 2013, and the name of the invention is “Method for inactivating viruses and other exogenous factors”.

[0002] Cross-references to related applications

[0003] This application claims priority to U.S. Patent Application No. 13 / 844,051, filed March 15, 2013, and U.S. Provisional Patent Application No. 61 / 662,349, filed June 20, 2012, which are incorporated herein by reference in their entireties. Technical Field

[0004] The present invention provides a method for inactivating viruses using high temperature transient (HTST) treatment and adjusting various parameters to reduce and / or minimize the generation and deposition of precipitates. Background Art

[0005] Viruses are potential contaminants in drug production processes, particularly when biopharmaceuticals are derived from mammalian cell cultures. The source of viral contaminants can be the culture medium used for cell culture or the cell line that produces the intended biological product. Current methods for preventing viral contamination of biopharmaceuticals during production processes include high temperature transient (HTST) cell culture medium treatment for inactivating viruses, which may be introduced into the cell culture medium through raw materials and amplified during the culture process (Schleh, M. et al. 2009. Biotechnol. Prog. 25 (3): 854-860 and Kiss, R. 2011. PDA JPharm Sci and Tech. 65: 715-729). It has been reported that temperatures above about 85°C required for HTST are effective methods for inactivating viruses, wherein temperatures above about 95°C are required for inactivating parvoviruses, which are common cell culture virus contaminations that have been documented to exist in cell culture processes, and which resist many chemical and physical inactivators (inactivating agents) (Schleh et al.).

[0006] Although HTST treatment has been shown to be highly effective in the inactivation of viruses, precipitation or precipitate formation occurs in a variety of cell culture media when the treatment is performed. The precipitation causes residues to accumulate on surfaces within the HTST system and causes equipment scaling, so that it can no longer heat the culture medium to the target temperature for completely inactivating viral contaminants. In addition, the precipitation may also scale the filter, which is usually used downstream of the HTST system for the final treatment of microorganisms such as bacteria from the culture medium. The filter scaling may result in the inability to complete the culture medium treatment step before the cell culture process. In some instances, the precipitate may also affect the performance of the cell culture medium and hinder the efficient production of biopharmaceuticals from the cultured cell lines. In order to prevent precipitation, the temperature can be lowered, but successful viral inactivation may be adversely affected. In addition, the generation of precipitation during the HTST cell culture medium treatment may result in frequent cleaning or maintenance of the equipment used for the HTST treatment during the production process, which significantly increases the cost of the process. Therefore, there is a need to prevent precipitation generation during the HTST treatment of removing or inactivating viral contaminants, and not adversely affect the efficiency of the treatment.

[0007] The invention described herein meets these needs by providing a method for effectively inactivating viral contaminants in cell culture media using HTST treatment that adjusts process parameters that result in reduced or prevented precipitate formation.

[0008] All references cited herein, including patent applications and publications, are hereby incorporated by reference in their entirety. Summary of the invention

[0009] The present invention provides methods, processes, systems and compositions for inactivating viral contaminants and / or other contaminants in cell culture media by using high temperature transient (HTST) treatment and adjusting multiple parameters in the culture media, such as pH and / or calcium and / or phosphate concentration combinations. In addition, methods, processes, systems and compositions for reducing fouling of equipment and filters used for HTST treatment are also provided.

[0010] Thus, in one aspect, the present invention provides a method for inactivating viruses or adventitious agents in a cell culture medium while maintaining the medium suitable for cell culture, the method comprising (a) subjecting the cell culture medium to high temperature transient (HTST) treatment, and (b) adjusting one or more parameters selected from pH, calcium level and phosphate level.

[0011] In other aspects, the present invention provides a method for inactivating viruses in a cell culture medium, comprising subjecting the cell culture medium to a high temperature transient (HTST) treatment, wherein the culture medium has a pH between about pH 5.0 and about pH 6.9 during the HTST treatment. On the other hand, the present invention provides a method for inactivating viruses in a cell culture medium, comprising subjecting the cell culture medium to a high temperature transient (HTST) treatment, wherein the culture medium has a pH between about pH 5.0 and about pH 7.2 during the HTST treatment. In some embodiments, the culture medium has a pH between about pH 5.3 and about pH 6.3 during the HTST treatment. In other embodiments, the culture medium has a pH of about pH 6.0 during the HTST treatment. In any embodiment, the HTST treatment includes raising the temperature of the culture medium to at least about 85°C and for a sufficient time to inactivate viruses or possible viruses in the culture medium. In some embodiments, the temperature of the culture medium is raised to at least about 93°C and for a sufficient time to inactivate viruses or possible viruses in the culture medium. In some embodiments, the temperature of the culture medium is raised to at least about 95, 97, 99, 101 or 103° C. for a sufficient time to inactivate viruses or potential viruses in the culture medium. In some embodiments, the pH of the culture medium is lowered to between about pH 5.0 and about pH 6.9 during the HTST treatment prior to the polypeptide production stage. In some embodiments, the pH of the culture medium is then brought to between about 6.9-7.2 for the polypeptide production stage.

[0012] In another aspect, the invention provides a method for inactivating viruses in a cell culture medium, comprising limiting the total amount of phosphate and calcium in the culture medium to less than about 10 mM during HTST treatment. In some embodiments, the total concentration of phosphate and calcium in the culture medium is limited to less than about 9, 8, 7, 6, 5, 4, 3, 2, or 1 mM during HTST treatment. In some embodiments, prior to the polypeptide production stage, the total amount of phosphate and calcium in the culture medium is limited to less than about 10 mM during HTST treatment. In some embodiments, during the protein production stage, the total amount of phosphate and calcium in the culture medium is then increased to a level sufficient for polypeptide production.

[0013] On the other hand, the present invention provides a method for reducing fouling of equipment for HTST treatment, the method comprising subjecting a cell culture medium used in the equipment to a high temperature transient (HTST) treatment, wherein the culture medium has a pH between about pH 5.0 and about pH 6.9 during the HTST treatment. In some embodiments, during the HTST treatment, the culture medium has a pH between about pH 5.3 and about pH 6.3. In some embodiments, during the HTST treatment, the culture medium has a pH of about pH 6.0. In some embodiments, the fouling comprises precipitation on the equipment for HTST treatment. In any embodiment, the HTST treatment comprises raising the temperature of the culture medium to at least about 85°C and for a sufficient time to inactivate viruses in the culture medium. In some embodiments, the temperature of the culture medium is raised to at least about 95, 97, 99, 101 or 103°C and for a sufficient time to inactivate viruses in the culture medium.

[0014] In another aspect, the present invention provides a method for reducing fouling of equipment used for HTST treatment to inactivate viruses, the method comprising limiting the total amount of phosphate and calcium in the culture medium used in the equipment to less than about 10 mM during the HTST treatment. In some embodiments, the total concentration of phosphate and calcium in the culture medium during the HTST treatment is limited to less than about 9, 8, 7, 6, 5, 4, 3, 2 or 1 mM. In some embodiments, the fouling comprises precipitation on the equipment used for HTST treatment. In any embodiment, the virus is selected from the family parvoviridae, paramyoxviridae, orthomyxoviridae, bunyaviridae, rhabdoviridae, reoviridae, togaviridae, caliciviridae and picornaviridae. In any embodiment, the virus is an enveloped virus. In any embodiment, the virus is a non-enveloped virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 is a schematic diagram of a representative HTST skid used in production.

[0016] Figure 2 is a heating profile depicting a treatment using a sand bath method and HTST to a target set point of 102° C. The traces show the heating profile in minutes, with the endpoint of each trace described by the time and the temperature at the endpoint (e.g., endpoint value of "6.2,102" = endpoint value at 6.2 minutes is 102° C.).

[0017] Figure 3 are graphs of media samples known to precipitate during HTST treatment after heat treatment by sand bath method. A) Uncentrifuged samples of Media 1 at pH 7.0 or pH 6.4 and Media 2 at pH 7.0 or pH 6.7 in heat treated pressure vessels. B) Centrifuged aliquots of Media 1 at pH 7.0 or pH 6.4 and Media 2 at pH 7.0 or pH 6.7 from treated pressure vessels.

[0018] Figure 4 Categorical parameter estimates for the media formulation (based on Medium 4) are described for parameter terms associated with higher levels of turbidity for sand bath treated samples.

[0019] Fig. 5 is a series of figures describing the precipitation response surface measured by turbidity during the sand bath heat treatment, and this turbidity comes from 3 aspects (different calcium, phosphate and pH levels) based on 4 formulations of culture medium. The perspective plane part visible in each grid shows the area where the turbidity is lower than 5NTU, which is related to the invisible precipitation in the culture medium sample tested and represents the safe operation scheme. A) side view, showing the turbidity measurement in the culture medium with different concentrations of phosphate and calcium. The top view shows the turbidity measurement in the culture medium, wherein B) has different concentrations of phosphate and calcium, C) has different phosphate concentrations and pH levels, and D) different calcium concentrations and pH levels.

[0020] Figure 6 is a graph depicting medium formulations located on the Medium 4 response surface at pH 7.0 for known and estimated calcium and phosphate concentrations. Despite differences in other components across all formulations, calcium and phosphate concentrations strongly correlate with possible precipitation following heat treatment. Arrows indicate that the medium shifts to a precipitation regime due to addition of hydrolysate containing additional levels of phosphate and / or calcium.

[0021] Figure 7 Depicted are the average heating profiles for four media formulations in a sand bath system. Five different temperature endpoints were obtained (represented by two numbers, e.g. "2.5, 75.9" = sample taken at 2.5 minutes, resulting in an average temperature of 75.9°C). The solid or open circles associated with the visible precipitate observed in the uncentrifuged and centrifuged samples are superimposed in the right hand photo. Open circles = no precipitate was detected by visual inspection in either the uncentrifuged or centrifuged sample. Solid circles = precipitate was detected by visual inspection in both or one sample.

[0022] Figure 8is a graph depicting turbidity (NTU) values ​​obtained from 5 different temperature endpoint samples from 4 different media formulations. Turbidity values ​​greater than -8 NTU are associated with visible precipitation identified by direct visual inspection or inspection of centrifuged samples.

[0023] Fig. 9 is a series of graphs showing iron (left) and copper (right) losses after HTST treatment, where both the HTST process and the filtration operation were successful.

[0024] FIG. 10 is a graph showing the changes in A) pH level, B) calcium (Ca) concentration, C) phosphate (PO 4 Main effects plot of A) concentration, D) iron (Fe) concentration, and E) copper (Cu) concentration on iron recovery.

[0025] Fig.11 Described is a series of graphs showing interaction plots from results of statistically designed experiments (Design of Experiments (DoE)) showing the relationship between pH, Ca, PO 4 The interactive effects between Fe and Fe on iron recovery.

[0026] Fig.12 is a graph showing the dependence of the final Fe concentration on the initial Fe concentration in heat treated medium. All other medium components were at normal 1.5× Medium 4 levels.

[0027] FIG. 13 is a graph showing the dependence of Fe compensation on adjustment of several parameters in heat-treated medium. A) Dependence of Fe recovery on pH level, and B) Dependence of Fe recovery on concentrations of calcium and phosphate in heat-treated medium. All other medium components were at normal 1.5× Medium 4 levels. On the x-axis scale, the standard concentration of Medium 4 is represented by 1.

[0028] Fig.14 is a graph showing the relationship between calcium and phosphate recovery and Fe recovery after heat treatment. The data points within the red circle indicate samples that showed visible precipitation and increased turbidity (NTU). The straight line represents the relationship between Fe recovery and calcium data points.

[0029] Fig.15 is a graph showing iron levels in various cell culture medium formulations before and after HTST treatment. For a particular cell culture medium (e.g., Medium 14), the left bar graph represents the expected level of iron in the cell culture medium after HTST treatment, the middle bar graph represents the actual level of iron in the cell culture medium before HTST treatment (Pre-HTST), and the right bar graph represents the actual level of iron in the cell culture medium after HTST treatment (Post-HTST).

[0030] Fig.16 is a graph showing that the addition of iron to HTST-treated medium is beneficial for the growth of the NS0 myeloma cell line in cell culture. HTST+ indicates that the cell culture medium was HTST-treated. HTST- indicates that the cell culture medium was not HTST-treated. Fe indicates the presence of supplemented iron. DETAILED DESCRIPTION

[0031] The present inventors have unexpectedly discovered that adjusting the pH of a cell culture medium, adjusting the calcium concentration, adjusting the phosphate concentration, adjusting both the calcium and phosphate concentrations and / or limiting the total amount of phosphate and calcium in the culture medium, or adjusting the pH, calcium concentration, and phosphate concentration in combination, and subjecting the cell culture medium to HTST treatment at a specific temperature range and for a sufficient period of time can effectively inactivate viruses (or other infectious and / or exogenous factors) in the culture medium, and can also reduce equipment fouling by minimizing or preventing precipitate formation.

[0032] The present invention provides a method for reducing precipitate on a device for high temperature transient (HTST) treatment to inactivate viruses, the method comprising subjecting a cell culture medium used in the device to HTST treatment, wherein the culture medium has a pH between about pH 5.0 and about pH 6.9 or between about pH 5.0 and about pH 7.2 during the HTST treatment. On the other hand, the present invention provides a method for reducing precipitate on a device for high temperature transient (HTST) treatment to inactivate viruses, the method comprising subjecting a cell culture medium used in the device to HTST treatment, wherein the culture medium has a pH between about pH 5.0 and about pH 7.2 during the HTST treatment. In other aspects, the present invention provides a method for reducing precipitate on a device for HTST treatment to inactivate viruses, the method comprising limiting the total amount of phosphate and calcium in the cell culture medium used in the device to less than about 10 mM during the HTST treatment.

[0033] In another aspect, the present invention provides a method for inactivating viruses in a cell culture medium, comprising subjecting the cell culture medium to HTST treatment, wherein the culture medium has a pH between about pH 5.0 and about pH 6.9 during the HTST treatment. In another aspect, the present invention provides a method for inactivating viruses in a cell culture medium, comprising subjecting the cell culture medium to HTST treatment, wherein the culture medium has a pH between about pH 5.0 and about pH 7.2 during the HTST treatment. In other aspects of the present invention, prior to the polypeptide production phase of the cell culture, during the HTST treatment, the pH of the culture medium is lowered to between about pH 5.0 and about pH 6.9. In some aspects, the pH of the culture medium is then brought to between about pH 6.9 and about pH 7.2. In other aspects, the present invention provides a method for inactivating viruses in a cell culture medium, comprising limiting the total amount of phosphate and calcium in the cell culture medium to less than about 10 mM during the HTST treatment.

[0034] I. General Technology

[0035] The techniques and methods described or referenced herein are conventional methods generally known and commonly used by those skilled in the art, such as Sambrook et al., Molecular Cloning: A Laboratory Manual 3d edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Current Protocols in Molecular Biology (FM Ausubel, et al. eds., (2003)); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (MJ MacPherson, BD Hames and GR Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Animal Cell Culture (RI Freshney, ed. (1987)); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Manual, Humana Press; Notebook(JECellis,ed.,1998)Academic Press;Animal Cell Culture(RIFreshney),ed.,1987);Introduction toCell and Tissue Culture(JPMather and PERoberts,1998)Plenum Press;Cell andTissue Culture:Laboratory Procedures(A.Doyle,JBGriffiths,and DGNewell,eds.,1993-8)J.Wiley and Sons; Handbook of Experimental Immunology (DMWeir and C.C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JMMiller and M.P. Calos, eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (JEColigan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (CAJanewayand P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and JD Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (VT DeVita et al., eds., JB Lippincott Company, 1993). .

[0036] II. Definitions

[0037] "Culturing" cells means contacting the cells with a cell culture medium under conditions suitable for cell survival and / or growth and / or cell proliferation.

[0038] "Batch culture" refers to a culture in which all components for cell culture (including cells and all culture nutrients) are provided to the culture vessel at the start of the culture.

[0039] As used herein, the phrase "fed-batch cell culture" refers to a batch culture in which cells and culture medium are initially provided to a culture vessel and during the culture period, other culture nutrients are fed to the culture continuously or non-continuously in increments, with or without periodic cell and / or product harvests, prior to termination of the culture.

[0040] "Flow-through culture" refers to a culture in which cells are confined in culture by, for example, filtration, encapsulation, anchoring to microcarriers, etc., and culture medium is continuously or intermittently introduced into and removed from the culture vessel.

[0041] "Culture vessel" refers to a vessel for culturing cells. The culture vessel may be of any size as long as it can be used for culturing cells.

[0042] The terms "culture medium" and "cell culture medium" refer to a nutrient source used to grow or maintain cells. As will be appreciated by those skilled in the art, a nutrient source may contain components required for cell growth and / or survival or may contain components that aid cell growth and / or survival. Vitamins, essential or non-essential amino acids, and trace elements are examples of culture medium components. It should be understood that in this specification, "culture medium" and "culture fluid" are used interchangeably.

[0043] "Chemically defined cell culture medium" or "CDM" is a culture medium of a specified composition that is free of animal-derived or undefined products such as animal serum and peptone. As will be appreciated by those skilled in the art, a CDM can be used in a polypeptide production process such that cells are contacted with the CDM and the polypeptide is secreted into the CDM. Thus, it should be understood that a composition can contain a CDM and a polypeptide product, and the presence of the polypeptide product does not render the CDM chemically undefined.

[0044] "Chemically undefined cell culture medium" refers to a culture medium whose chemical composition is not specified, and which may contain one or more animal-derived or undefined products such as animal serum and peptone. As will be appreciated by those skilled in the art, a chemically undefined culture medium may contain animal-derived products as a nutrient source.

[0045] The terms "polypeptide" and "protein" used interchangeably herein refer to polymers of amino acids of any length. The polymer may be linear or branched, it may contain modified amino acids, and it may be interrupted by non-amino acids. The term also includes amino acid polymers that are natural or modified by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphodiesterase, or any other manipulation or modification, such as conjugation with a labeling component. The definition also includes, for example, polypeptides containing one or more amino acid analogs (including, for example, non-natural amino acids, etc.), as well as other modifications known in the art. As used herein, the terms "polypeptide" and "protein" particularly include antibodies.

[0046] "Isolated polypeptide" means a polypeptide that is recovered from a cell or cell culture in which the polypeptide is expressed.

[0047] As used interchangeably herein, "nucleic acid" refers to a polymer of nucleotides of any length, and includes DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase or synthesis reaction. Polynucleotides can include modified nucleotides, such as methylated nucleotides and their analogs. If present, modifications based on nucleotide structure can be made before or after assembling the polymer.

[0048] "Isolated nucleic acid" means and includes a non-naturally occurring, recombinant or naturally occurring sequence that is not present in or separated from its usual context.

[0049] A "purified" polypeptide means a polypeptide that has been increased in purity so that the polypeptide exists in a more pure form than it exists in its natural environment and / or when initially produced and / or synthesized and / or amplified under laboratory conditions. Purity is a relative term and does not necessarily mean absolute purity.

[0050] The term "antibody" is used in its broadest sense and specifically encompasses, for example, monoclonal antibodies (including agonists, antagonists and neutralizing antibodies), antibody compositions with multiple epitope specificities, polyclonal antibodies, single-chain antibodies, multispecific antibodies (e.g., bispecific antibodies), immunoadhesins, and antibody fragments, as long as the fragment exhibits the desired biological or immunological activity. The term "immunoglobulin (Ig)" is used interchangeably with antibody herein.

[0051] "Antibody fragments" include a portion of a full-length antibody, typically its antigen binding or variable region. Examples of antibody fragments include Fab, Fab', F(ab') 2and Fv fragments; single-chain antibody molecules; diabodies; linear antibodies; and multispecific antibodies formed from antibody fragments.

[0052] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, i.e., except for mutations that may occur naturally in a minimum amount, the individual antibodies contained in the population are identical. Monoclonal antibodies are highly specific and target a single antigenic site. In addition, in contrast to polyclonal antibody preparations comprising different antibodies targeting different determinants (epitopes), each monoclonal antibody targets a single determinant on an antigen. In addition to its specificity, monoclonal antibodies are advantageous because they can be synthesized without being contaminated by other antibodies. The modifier "monoclonal" should not be interpreted as requiring antibodies to be produced by any particular method. For example, it can be prepared by the hybridoma method described by Kohler et al., Nature, 256:495 (1975) for the first time or in bacteria, eukaryotic animals or plant cells using recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567) to prepare monoclonal antibodies for the present invention. "Monoclonal antibodies" can also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature, 352:624-628 (1991) and Mark et al., J. Mol. Biol., 222:581-597 (1991).

[0053] The monoclonal antibodies herein include "chimeric" antibodies (wherein a portion of the heavy chain and / or light chain is identical or homologous to the corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, and the remainder of the chain is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass), as well as fragments of such antibodies, as long as the fragment exhibits the desired biological activity (see U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984)). The chimeric antibodies of interest herein include "primatized" antibodies, which comprise variable domain antigen-binding sequences derived from non-human primates (e.g., Old World Monkeys, Great Apes, etc.) and human constant region sequences.

[0054] "Humanized" antibodies are forms of non-human (e.g., rodent) antibodies, which are chimeric antibodies containing minimal sequences derived from non-human antibodies. In most cases, humanized antibodies are human immunoglobulins (receptor antibodies), wherein the residues from the hypervariable regions of non-human species (donor antibodies) such as mice, rats, rabbits or non-human primates are substituted for the residues from the hypervariable regions of the receptor. In some instances, the framework region (FR) residues of human immunoglobulins are substituted with corresponding non-human residues. In addition, humanized antibodies may be included in the receptor antibody or in residues that do not exist in the donor antibody. These modifications are prepared to further improve antibody performance. In a word, humanized antibodies substantially all include at least one, and usually two variable regions, wherein all or substantially all hypervariable loops correspond to the hypervariable loops of non-human immunoglobulins, and all or substantially all FRs are FRs of human immunoglobulin sequences. Optionally, humanized antibodies also at least include a part of an immunoglobulin constant region (Fc) (usually a human immunoglobulin constant region). For additional details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).

[0055] "Contaminants" refer to substances other than the desired polypeptide product. Contaminants include, but are not limited to: host cell substances, such as CHOP; leached protein A; nucleic acids; variants, fragments, aggregates or derivatives of the desired polypeptide; another polypeptide; endotoxins; viral contaminants; cell culture medium components, etc.

[0056] As used herein, the term "precipitate" refers to solid or insoluble particles generated in a solution. There are many forms of precipitates, and exemplary precipitates are described herein. Non-limiting examples include: calcium phosphate precipitation, insoluble white phosphate, oxides, iron phosphate and calcium iron phosphate precipitation. Calcium phosphate precipitation can be a process in which calcium and phosphate in a solution form insoluble particles, i.e., precipitation. The insoluble particles can be referred to as calcium phosphate. Calcium phosphates include, but are not limited to: monocalcium phosphate, monocalcium phosphate, tricalcium phosphate, white phosphate and hydroxyapatite. Insoluble particles can include other components, i.e., polypeptides, nucleic acids, lipids, ions, chelating agents and metals. This definition also includes such particles produced by further precipitation or by aggregation, flocculation and / or rearrangement.

[0057] As used herein, the term "scaling" refers to the accumulation and formation of unwanted materials on the surface of processing equipment. Scaling can be characterized as a complex, non-steady-state, momentum, mass and heat transfer problem, with chemical, solubility, corrosion and biological processes occurring. Scaling can be attributed to precipitation, i.e., calcium phosphate precipitation.

[0058] As used herein, "exogenous agents" include viruses and bacteria (including bacteria that can pass through sterilizing grade filters). An "infectious agent" is a type of "exogenous agent."

[0059] It should be understood that aspects and embodiments of the present invention described herein include "comprising," "consisting of," and "consisting essentially of" aspects and embodiments.

[0060] As used herein, the use of the terms "a," "an," etc. means one or more unless expressly stated otherwise.

[0061] Reference to a value or parameter as "about" herein includes (and describes) embodiments referring to the value or parameter itself. For example, description referring to "about X" includes description "X". Numerical ranges include the values ​​defining the range.

[0062] III. Cell Culture Medium

[0063] The methods of inactivating viruses, infectious agents, and / or adventitious agents in culture media can be applied to any type of cell culture media that is involved in viral contamination, potential viral contamination, or contaminated with other infectious agents or with adventitious agents. It should be understood that the present invention contemplates compositions and methods that can be applied to cell cultures and cell culture media that may be involved in viral contamination as well as actual viral contamination.

[0064] The methods of inactivating viruses, reducing precipitate formation, and reducing fouling of equipment used for HTST treatment can be used to produce compositions of cell culture media in which viruses, exogenous factors, and other infectious agents have been inactivated. Thus, any composition of cell culture media and intermediates thereof treated by a system of viral inactivation, exogenous factor inactivation, and / or infectious agent inactivation is contemplated and detailed herein. It has been demonstrated that adjusting specific cell culture media parameters (pH, calcium levels, and phosphate levels) can reduce or prevent the formation of precipitates in the culture media after HTST treatment at a temperature that is effective to inactivate viral contaminants and other contaminants in the culture media.

[0065] Independent adjustment of cell culture medium parameters such as pH, calcium concentration or amount, and phosphate concentration or amount (and any combination thereof) can be used with HTST treatment to reduce precipitation (e.g., complexes comprising calcium and phosphate), reduce HTST equipment fouling, reduce filter fouling, while retaining cell culture suitability. Cell culture medium that maintains cell culture suitability allows cells to proliferate, grow, survive, produce any polypeptides, proteins, or compounds, secrete any such products into the culture medium, and any other characteristics included in the purpose of suitability for cell culture as understood by those skilled in the art.

[0066] Independent adjustment of the cell culture medium parameters described herein can be applied to any cell culture method, and can be beneficial to the production of polypeptides and / or proteins, as well as the production of other products such as cells, culture medium and other components in the culture medium. Adjusting these cell culture medium parameters for reducing or preventing precipitates in the cell culture medium is beneficial to the production of biopharmaceuticals such as polypeptide drug products. Using a cell culture medium with adjusted parameters or with adjusted components can effectively remove viral contaminants from biopharmaceutical products, and reduce or prevent precipitates, which can affect cell culture medium performance, hinder biopharmaceuticals from being effectively produced from cultured cell lines, and cause HTST equipment fouling. Any culture medium described in detail herein can be used for any stage in cell growth, maintenance and biopharmaceutical production, and can be used for basal culture medium and / or feed medium. In a variation, the culture medium as described herein results in an acceptable turbidity or precipitation level of a composition comprising a biopharmaceutical separated from a cell culture cultured in a culture medium treated with HTST.

[0067] A cell culture medium comprising one or more of the following components is provided: (a) calcium and (b) phosphate. In some variations, the cell culture medium comprises component (a) or (b) or components (a) and (b). In other variations, the cell culture medium does not comprise components (a) and (b). In some aspects, the cell culture medium is a chemically defined cell culture medium. In other aspects, the cell culture medium is a chemically undefined cell culture medium. In either aspect, the cell culture medium is used to inactivate viruses during HTST treatment. In either aspect, the cell culture medium is treated with HITST to inactivate viruses that may come from the raw materials and the operations used to prepare the culture medium.

[0068] The culture medium components can be added to the composition in a form known in the art. For example, calcium can be provided as, but not limited to, calcium chloride, anhydrous calcium chloride, calcium carbonate, calcium phosphate, tricalcium phosphate, calcium L-lactate hydrate, calcium folinate, and calcium nitrate tetrahydrate. For example, phosphate can be provided as, but not limited to, sodium phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, phosphate buffered saline, calcium phosphate, and calcium hydrogen phosphate.

[0069] The ratio of phosphate to calcium can be adjusted for HTST treatment so that the complex containing calcium and phosphate (e.g., calcium phosphate (CaPO 4 In one embodiment, the total amount of phosphate and calcium is adjusted or limited so that a complex comprising calcium and phosphate (e.g., calcium phosphate (CaPO 4 In another variation, the total amount of phosphate and calcium can be limited so that CaPO 4The formation of the complex enables the HTST operation to proceed successfully (e.g., the HTST and the filtration equipment are not fouled). Methods for detecting problematic conditions involve indirect measurements of precipitation including turbidity, operational observation of the HTST and the filtration equipment, and visual observation of the HTST and the filtration equipment (e.g., by using an inner pore surface inspection instrument). In one aspect, the culture medium is a cell culture medium containing a total amount of phosphate and calcium limited to less than about 10 mM. In another variation, the concentration of total phosphate and calcium in the culture medium is less than about 9, 8, 7, 6, 5, 4, 3, 2, or 1 mM. In another variation, the concentration of total phosphate and calcium in the culture medium is from about 1 mM to about 9 mM; from about 2 mM to about 8 mM; from about 3 mM to about 7 mM; from about 4 mM to about 6 mM; from about 1 mM to about 8 mM; from about 1 mM to about 7 mM; from about 1 mM to about 6 mM; from about 1 mM to about 5 mM; from about 1 mM to about 4 mM; from about 1 mM to about 3 mM; from about 1 mM to about 2 mM; from about 2 mM to about 9 mM; from about 3 mM to about 9 mM; from about 4 mM to about 9 mM; from about 5 mM to about 9 mM; from about 6 mM to about 9 mM; from about 7 mM to about 9 mM; from about 8 mM to about 9 mM; one of about 9 or 8 or 7 or 6 or 5 or 4 or 3 or 2 or 1 mM; at least about one of 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 and not more than about 9 mM. In one variation, prior to the polypeptide production phase of the cell culture, during HTST treatment, the concentration of total phosphate and calcium in the culture medium is from about 1 mM to about 9 mM; from about 2 mM to about 8 mM; from about 3 mM to about 7 mM; from about 4 mM to about 6 mM; from about 1 mM to about 8 mM; from about 1 mM to about 7 mM; from about 1 mM to about 6 mM; from about 1 mM to about 5 mM; from about 1 mM to about 4 mM; from about 1 mM to about 3 mM; from about 1 mM to about 2 mM; from about 2 mM to about 9 mM; from about 3 mM to about 9 mM; from about 4 mM to about 9 mM; from about 5 mM to about 9 mM; from about 6 mM to about 9 mM; from about 7 mM to about 9 mM; from about 8 mM to about 9 mM; one of about 9 or 8 or 7 or 6 or 5 or 4 or 3 or 2 or 1 mM; at least about one of 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 and no more than about 9 mM. In any aspect of the invention, prior to the polypeptide production phase of the cell culture, during the HTST treatment, the total amount of calcium and phosphate in the culture medium is limited to less than about 10 mM. In other aspects of the invention, during the polypeptide production phase of the cell culture, the total amount of calcium and phosphate in the culture medium is then increased to a level sufficient to produce the polypeptide. In some aspects, the cell culture medium is free of calcium and phosphate.

[0070] When the culture medium contains phosphate, the level of calcium in the cell culture medium can be adjusted. The adjustment can be to increase or decrease the calcium level. In some embodiments, the calcium level is reduced (including removing calcium). In other embodiments, the calcium level is reduced so that the generation of a complex comprising calcium and phosphate is inhibited. In other embodiments, calcium is removed from the culture medium before HTST treatment. In any of these embodiments, the pH is adjusted so that the generation of a complex comprising calcium and phosphate is inhibited (for example, the amount of the complex formed is reduced compared to the complex formed if these adjustments are not made). In other embodiments, the calcium level can be further adjusted to a suitable level for cell culture after HTST treatment. The time between HTST treatment and adjusting the calcium level to a suitable level for cell culture after HTST can be changed. Time intervals of seconds, minutes, days, weeks, months or years are contemplated within the scope of the present invention.

[0071] In another variation, the culture medium is a cell culture medium containing a total amount of calcium limited to less than about 10 mM. In another variation, the concentration of total calcium in the culture medium is less than about 9, 8, 7, 6, 5, 4, 3, 2, or 1 mM. In another variation, the concentration of total calcium in the culture medium is from about 1 mM to about 9 mM; from about 2 mM to about 8 mM; from about 3 mM to about 7 mM; from about 4 mM to about 6 mM; from about 1 mM to about 8 mM; from about 1 mM to about 7 mM; from about 1 mM to about 6 mM; from about 1 mM to about 5 mM; from about 1 mM to about 4 mM; from about 1 mM to about 3 mM; from about 1 mM to about 2 mM; from about 2 mM to about 9 mM; from about 3 mM to about 9 mM; from about 4 mM to about 9 mM; from about 5 mM to about 9 mM; from about 6 mM to about 9 mM; from about 7 mM to about 9 mM; from about 8 mM to about 9 mM; one of about 9 or 8 or 7 or 6 or 5 or 4 or 3 or 2 or 1 mM; at least about one of 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 and no more than about 9 mM. In another variation, prior to the polypeptide production phase of the cell culture, during the HTST treatment, the concentration of total calcium in the culture medium is from about 1 mM to about 9 mM; from about 2 mM to about 8 mM; from about 3 mM to about 7 mM; from about 4 mM to about 6 mM; from about 1 mM to about 8 mM; from about 1 mM to about 7 mM; from about 1 mM to about 6 mM; from about 1 mM to about 5 mM; from about 1 mM to about 4 mM; from about 1 mM to about 3 mM; from about 1 mM to about 2 mM; from about 2 mM to about 9 mM; from about 3 mM to about 9 mM; from about 4 mM to about 9 mM; from about 5 mM to about 9 mM; from about 6 mM to about 9 mM; from about 7 mM to about 9 mM; from about 8 mM to about 9 mM; one of about 9 or 8 or 7 or 6 or 5 or 4 or 3 or 2 or 1 mM; at least about one of 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 and no more than about 9 mM. In any aspect of the invention, prior to the polypeptide production phase of cell culture, during HTST treatment, the total amount of calcium in the culture medium is limited to less than about 10 mM. In other aspects of the invention, during the polypeptide production phase of cell culture, the total amount of calcium in the culture medium is then increased to a level sufficient to produce the polypeptide. In some aspects, the cell culture medium does not contain phosphate.

[0072] On the other hand, when the culture medium contains calcium, the phosphate level can be adjusted. The adjustment can be to increase or decrease the phosphate level. In some embodiments, the phosphate level is reduced. In some embodiments, the phosphate level is reduced so that the formation of a complex comprising calcium and phosphate is inhibited. In some embodiments, phosphate is removed from the culture medium before HTST treatment. In some embodiments, the pH is adjusted so that the generation of a complex comprising calcium and phosphate is inhibited. In some embodiments, the phosphate level is further adjusted to a suitable level for cell culture after HTST treatment. The time between HTST treatment and the adjustment of the phosphate level to a suitable level for cell culture after HTST can be varied. Time intervals of seconds, minutes, days, weeks, months or years are contemplated within the scope of the present invention.

[0073] In another variation, the culture medium is a cell culture medium comprising a total amount of phosphate limited to less than about 10 mM. In another variation, the concentration of total phosphate in the culture medium is less than about 9, 8, 7, 6, 5, 4, 3, 2, or 1 mM. In another variation, the concentration of total phosphate in the culture medium is from about 1 mM to about 9 mM; from about 2 mM to about 8 mM; from about 3 mM to about 7 mM; from about 4 mM to about 6 mM; from about 1 mM to about 8 mM; from about 1 mM to about 7 mM; from about 1 mM to about 6 mM; from about 1 mM to about 5 mM; from about 1 mM to about 4 mM; from about 1 mM to about 3 mM; from about 1 mM to about 2 mM; from about 2 mM to about 9 mM; from about 3 mM to about 9 mM; from about 4 mM to about 9 mM; from about 5 mM to about 9 mM; from about 6 mM to about 9 mM; from about 7 mM to about 9 mM; from about 8 mM to about 9 mM; one of about 9 or 8 or 7 or 6 or 5 or 4 or 3 or 2 or 1 mM; at least about one of 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 and no more than about 9 mM. In another variation, prior to the polypeptide production phase of the cell culture, during HTST treatment, the concentration of total phosphate in the culture medium is from about 1 mM to about 9 mM; from about 2 mM to about 8 mM; from about 3 mM to about 7 mM; from about 4 mM to about 6 mM; from about 1 mM to about 8 mM; from about 1 mM to about 7 mM; from about 1 mM to about 6 mM; from about 1 mM to about 5 mM; from about 1 mM to about 4 mM; from about 1 mM to about 3 mM; from about 1 mM to about 2 mM; from about 2 mM to about 9 mM; from about 3 mM to about 9 mM; from about 4 mM to about 9 mM; from about 5 mM to about 9 mM; from about 6 mM to about 9 mM; from about 7 mM to about 9 mM; from about 8 mM to about 9 mM; one of about 9 or 8 or 7 or 6 or 5 or 4 or 3 or 2 or 1 mM; at least about one of 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 and no more than about 9 mM. In any aspect of the invention, prior to the polypeptide production phase of cell culture, during HTST treatment, the total amount of phosphate in the culture medium is limited to less than about 10 mM. In other aspects of the invention, during the polypeptide production phase of cell culture, the total amount of phosphate in the culture medium is then increased to a level sufficient to produce the polypeptide. In some aspects, the cell culture medium does not contain calcium.

[0074] In one variation, the cell culture medium comprises one or both of components (a) and (b), wherein the total amount of (a) or (b) or (a) and (b) is limited to less than about 10 mM in the concentration of each component (a) and (b) described herein. It should be understood that the cell culture medium can contain any combination of components (a) and (b) in the concentration ranges provided herein, just as if each concentration were clearly listed separately. For example, it should be understood that the culture medium in one variation comprises components (a) and (b), wherein calcium is 0.5 mM and phosphate is about 2.5 mM. In some aspects, the cell culture medium is a chemically defined cell culture medium. In other aspects, the cell culture medium is a chemically undefined culture medium. In either aspect, the cell culture medium is used to inactivate viruses during HTST treatment. In either aspect, the culture medium does not contain calcium during HTST treatment. In another aspect, calcium is added to the cell culture medium after HTST treatment. In either aspect, the culture medium does not contain phosphate during HTST treatment. In another aspect, phosphate is added to the cell culture medium after HTST treatment.

[0075] In one variation, the culture medium is a cell culture medium comprising a phosphate concentration of about 0mM to about 1mM and a calcium concentration of about 0.5mM to about 3mM. In another variation, the phosphate concentration is about 1mM to about 1.25mM, and the calcium concentration is about 0mM to about 2.5mM. In another variation, the phosphate concentration is about 1.25mM to about 1.5mM, and the calcium concentration is about 0mM to about 2.25mM. In another variation, the phosphate concentration is about 1.5mM to about 1.75mM, and the calcium concentration is about 0mM to about 2mM. In one variation, the phosphate concentration is about 1.75mM to about 2mM, and the calcium concentration is about 0mM to about 1.75mM. In another variation, the phosphate concentration is about 2mM to about 2.25mM, and the calcium concentration is about 0mM to about 1.5mM. In another variation, the phosphate concentration is about 2mM to about 2.25mM, and the calcium concentration is about 0mM to about 1.5mM. In another variation, the phosphate concentration is about 2.25mM to about 2.5mM, and the calcium concentration is about 0mM to about 1.25mM. Still in another variation, the phosphate concentration is about 2.5mM to about 2.75mM, and the calcium concentration is about 0mM to about 1.15mM. In another variation, the phosphate concentration is about 2.75mM to about 3mM, and the calcium concentration is about 0mM to about 1mM. In another variation. The phosphate concentration is about 3mM to about 3.5mM, and the calcium concentration is about 0mM to about 0.8mM. In another variation, the phosphate concentration is about 3.5mM to about 4.5mM, and the calcium concentration is about 0mM to about 0.6mM. In one variation, the phosphate concentration is about 4.5 mM to about 5 mM, and the calcium concentration is about 0 mM to about 0.5 mM. In another variation, the phosphate concentration is about 5 mM to about 5.5 mM, and the calcium concentration is about 0 mM to about 0.25 mM. In another variation, the phosphate concentration is about 5.5 mM to about 6 mM, and the calcium concentration is about 0 mM to about 0.1 mM. In another aspect, as shown in exemplary FIG. 5 (e.g. Figure 5B , Figure 5C and Figure 5D), the parameters of pH, calcium, and phosphate can be adjusted independently to keep turbidity (as an indirect measure of phosphate-based precipitation) within the grid area (in this example, the grid area represents turbidity values ​​equal to or below the gross-failure turbidity threshold of 5 NTU) and avoid the precipitation range shown in the non-grid area (where the non-grid area represents turbidity responses above the gross-failure turbidity threshold of 5 NTU). The response surface shown in Figure 5 shows the multi-factorial effect of pH, calcium concentration, and phosphate concentration on turbidity (as an indirect measure of calcium phosphate-based precipitation). Therefore, the response surface shows how to adjust these factors in combination rather than just one factor at a time to find an acceptable HTST treatment operation set point for the combination of pH and acceptable calcium and phosphate concentrations in the culture medium to be treated.

[0076] Another independent parameter that can be adjusted (in addition to calcium and phosphate, which are other independent parameters) is pH. Therefore, when the culture medium contains calcium and phosphate, the pH can be adjusted. In some embodiments, the pH of the prepared culture medium is reduced to an appropriate level before HTST treatment. In some embodiments, the pH is adjusted by reducing it to an appropriate level. In some embodiments, the pH is adjusted to less than about 7.2. In some embodiments, the pH is adjusted to about 5.0–7.2. In some embodiments, the pH is further adjusted to a suitable level for cell culture after HTST treatment. In some embodiments, the pH is adjusted to about 6.9–7.2. The time interval between HTST treatment and adjusting the pH level to a suitable level for cell culture after HTST can be changed. Time intervals of seconds, minutes, days, weeks, months, or years are contemplated within the scope of the present invention.

[0077] In other aspects, cell culture media comprising a pH between about pH 5.0 and about pH 7.2 are provided. In one aspect, various types of cell culture media comprising a pH between about pH 5.0 and about pH 6.9 are provided. In some aspects, the cell culture media is a chemically defined cell culture media. In other aspects, the cell culture media is a chemically undefined cell culture media. In either aspect, the cell culture media is used to inactivate viruses during HTST treatment. In either aspect, the pH of the culture media during HTST treatment is between about pH 5.0 and about pH 7.2. This can be before the polypeptide production stage of cell culture. Optionally, one skilled in the art can adjust the pH of the culture media after HTST treatment so that the culture media is at a pH suitable for the cell culture process. In either aspect, before the polypeptide production stage of cell culture, the pH of the culture media is reduced to between about pH 5.0 and about pH 6.9 during HTST treatment. On the other hand, the pH of the culture media is then brought to between about pH 6.9 and about pH 7.2 for use in the polypeptide production stage of cell culture. In one aspect, following HTST treatment, the pH of the culture medium is then brought to between about pH 6.9 and about pH 7.2 for the polypeptide production phase of cell culture.

[0078] In one variation, the culture medium is a cell culture medium comprising a pH between about pH 5.0 and about pH 7.2. In another variation, the culture medium is a cell culture medium comprising a pH between about pH 5.0 and about pH 6.9. In another variation, the pH of the culture medium is pH about 5.0 to about 7.2; about 5.0 to about 6.9; about 5.2 to about 6.7; about 5.4 to about 6.5; about 5.6 to about 6.3; about 5.8 to about 6.1; about 5.9 to about 6.0; about 5.0 to about 6.7; about 5.0 to about 6.5; about 5.0 to about 6.3; about 5.0 to about 6.1; about 5.0 to about 5.9; about 5.0 to about 5.7; about 5.0 to about 5.5; about 5.0 to about 5.3; about 5.0 to about 5.1; about 5.2 to about 6. 9; about 5.4 to about 6.9; about 5.6 to about 6.9; about 5.8 to about 6.9; about 6.0 to about 6.9; about 6.0 to about 6.9; about 6.2 to about 6.9; about 6.4 to about 6.9; about 6.6 to about 6.9; about one of 5.0 or 5.2 or 5.4 or 5.6 or 5.8 or 6.0 or 6.2 or 6.4 or 6.6 or 6.8 or 6.9; at least about one of 5.0 or 5.2 or 5.4 or 5.6 or 5.8 or 6.0 or 6.2 or 6.4 or 6.6 or 6.8 and no more than about 6.9. In one variation, the culture medium is a cell culture medium comprising a pH between about pH 5.0 and about pH 7.2. In another variation, prior to the polypeptide production phase of the cell culture, during the HTST treatment, the pH of the culture medium is at a pH of about 5.0 to about 6.9; about 5.2 to about 6.7; about 5.4 to about 6.5; about 5.6 to about 6.3; about 5.8 to about 6.1; about 5.9 to about 6.0; about 5.0 to about 6.7; about 5.0 to about 6.5; about 5.0 to about 6.3; about 5.0 to about 6.1; about 5.0 to about 5.9; about 5.0 to about 5.7; about 5.0 to about 5.5; about 5.0 to about 5.3; about 5.0 to about 5.1; about In one aspect, the pH of the culture medium is between about pH 5.0 and about pH 7.2 during the HTST treatment. This can be prior to the polypeptide production stage of the cell culture. After the HTST treatment, the culture medium can be adjusted to a pH suitable for the cell culture process as desired.In either aspect, prior to the polypeptide production phase of cell culture, during the HTST treatment, the pH of the culture medium is lowered to between about pH 5.0 and about pH 6.9. In either aspect, prior to the polypeptide production phase of cell culture, during the HTST treatment, the pH of the culture medium is between about pH 5.0 and about pH 6.9.

[0079] In one variation, the culture medium is a cell culture medium comprising a pH between about pH 6.9 and about pH 7.2. In another variation, the pH of the culture medium is a pH of about 6.9 to about 7.2; about 7.0 to about 7.1; about 6.9 to about 7.1; about 6.9 to about 7.0; about 7.0 to about 7.2; about 7.1 to about 7.2; about one of 6.9 or 7.0 or 7.1 or 7.2; at least about one of 6.9 or 7.0 or 7.1 and no more than about 7.2. In one variation, the culture medium is a cell culture medium comprising a pH of about pH 6.9 to about pH 7.2. In another variation, the pH of the culture medium is from pH about 6.9 to about 7.2; from about 7.0 to about 7.1; from about 6.9 to about 7.1; from about 6.9 to about 7.0; from about 7.0 to about 7.2; from about 7.1 to about 7.2; one of about 6.9 or 7.0 or 7.1 or 7.2; at least about 6.9 or 7.0 or 7.1 and no more than about 7.2, for use in the polypeptide production phase of cell culture. In either aspect, the pH of the culture medium is brought to between about pH 6.9 and about pH 7.2 for use in the polypeptide production phase of cell culture. In either aspect, after HTST treatment, the pH of the culture medium is brought to between about pH 6.9 and about pH 7.2 for use in the polypeptide production phase of cell culture. In either aspect, after HTST treatment, the pH of the culture medium is between about pH 6.9 and about pH 7.2 for use in the polypeptide production phase of cell culture.

[0080] In one variation, the culture medium is a cell culture medium comprising a pH between about pH 5.0 and about pH 6.9, and the total amount of phosphate and calcium is less than about 10 mM. In one variation, during HTST treatment, the culture medium is a cell culture medium comprising a pH between about pH 5.0 and about pH 6.9, and the total amount of phosphate and calcium is less than about 10 mM. In either variation, the pH and the amount of calcium and phosphate are any amount described herein. In either aspect, the culture medium is calcium-free during HTST treatment. In another aspect, calcium is added to the cell culture medium after HTST treatment. In another aspect, the culture medium is phosphate-free during HTST treatment. In another aspect, phosphate is added to the cell culture medium after HTST treatment. In another aspect, the pH of the culture medium is brought to a pH between about pH 6.9 and about pH 7.2 for use in the polypeptide production stage of cell culture. In another aspect, the pH of the culture medium is brought to a pH between about pH 6.9 and about pH 7.2 for use in the polypeptide production stage of cell culture after HTST treatment.

[0081] In one variation, the culture medium is a cell culture medium comprising a phosphate concentration of about 0 mM to about 0.5 mM and a pH of about 6.4 to about 7.4. In another variation, the phosphate concentration is about 0.5 mM to about 0.75 mM, and the pH is about 6.4 to about 7.35. In another variation, the phosphate concentration is about 0.75 mM to about 1 mM, and the pH is about 6.4 to about 7.25. In another variation, the phosphate concentration is about 1 mM to about 1.25 mM, and the pH is about 6.4 to about 7.2. In one variation, the phosphate concentration is about 1.25 mM to about 1.5 mM, and the pH is about 6.4 to about 7.1. In another variation, the phosphate concentration is about 1.5 mM to about 1.75 mM, and the pH is about 6.4 to about 7.05. In another variation, the phosphate concentration is about 1.75mM to about 2mM, and the pH is about 6.4 to about 7. In yet another variation, the phosphate concentration is about 2mM to about 2.25mM, and the pH is about 6.4 to about 6.9. In one variation, the phosphate concentration is about 2.25mM to about 2.5mM, and the pH is about 6.4 to about 6.85. In another variation, the phosphate concentration is about 2.5mM to about 2.75mM, and the pH is about 6.4 to about 6.75. In another variation, the phosphate concentration is about 2.75mM to about 3mM, and the pH is about 6.4 to about 6.7. In another variation, the phosphate concentration is about 3mM to about 3.25mM, and the pH is about 6.4 to about 6.6. In one variation, the phosphate concentration is about 3.25mM to about 3.5mM, and the pH is about 6.4 to about 6.5. In another variation, the phosphate concentration is from about 3.5 mM to about 3.75 mM, and the pH is from about 6.4 to about 6.45.

[0082] In one variation, the calcium concentration is from about 0 mM to about 1 mM, and the pH is from about 6.65 to about 7.4. In another variation, the calcium concentration is from about 0.1 mM to about 0.25 mM, and the pH is from about 6.55 to about 7.4. In another variation, the calcium concentration is from about 0.25 mM to about 0.5 mM, and the pH is from about 6.5 to about 7.4. In another variation, the calcium concentration is from about 0.5 mM to about 0.6 mM, and the pH is from about 6.5 to about 7.2. In one variation, the calcium concentration is from about 0.6 mM to about 0.75 mM, and the pH is from about 6.5 to about 7. In another variation, the calcium concentration is from about 0.75 mM to about 1 mM, and the pH is from about 6.4 to about 6.9. In another variation, the calcium concentration is from about 1 mM to about 1.1 mM, and the pH is from about 6.4 to about 6.8. In another variation, the calcium concentration is about 1.1mM to about 1.25mM, and the pH is about 6.4 to about 6.7. In one variation, the calcium concentration is about 1.25mM to about 1.5mM, and the pH is about 6.4 to about 6.65. In another variation, the calcium concentration is about 1.5mM to about 1.75mM, and the pH is about 6.4 to about 6.55. In another variation, the calcium concentration is about 1.75mM to about 2mM, and the pH is about 6.4 to about 6.5. In one variation, the calcium concentration is about 2mM to about 2.25mM, and the pH is about 6.4 to about 6.45. In another variation, the calcium concentration is about 2.25mM to about 2.5mM, and the pH is about 6.4 to about 6.43. In another variation, the calcium concentration is about 2.5mM to about 2.6mM, and the pH is about 6.4 to about 6.41.

[0083] Each culture medium component can be present in an amount that produces one or more favorable properties, such as viral inactivation, reduced precipitate generation, and reduced fouling of HTST processing equipment. In one variation, the cell culture medium provided herein contains culture medium parameters as described in Table 1. It should be understood that the culture medium composition can include any one or more culture medium components of Table 1 (e.g., any one or more components in (a)-(c)), such as a culture medium containing each component in (a), (b) and (c) in any amount listed in Table 1, or a culture medium composition containing (a), (b) and (d), or a culture medium composition containing components (a) and (b), or a culture medium composition containing components (a) and (c), or a culture medium composition containing components (b) and (c), or a culture medium composition containing components (a) and (d), or a culture medium composition containing components (b) and (d), as if each combination and amount of components are clearly listed separately. In some aspects, the cell culture medium is a chemically determined cell culture medium. In other aspects, the cell culture medium is a chemically undetermined cell culture medium. In either aspect, the cell culture medium is used to inactivate viruses during the HTST treatment. In either aspect, the culture medium is free of calcium during the HTST treatment. In another aspect, calcium is added to the cell culture medium after the HTST treatment. In either aspect, the culture medium is free of phosphate during the HTST treatment. In another aspect, phosphate is added to the cell culture medium after the HTST treatment. In another aspect, the pH of the culture medium is brought to between about pH 6.9 and about pH 7.2 for the polypeptide production phase of the cell culture. In either aspect, after the HTST treatment, the pH of the culture medium is brought to between about pH 6.9 and about pH 7.2 for the polypeptide production phase of the cell culture.

[0084] Table 1. Exemplary levels of media components or parameters

[0085]

[0086]

[0087] In one variation, the culture medium provided herein comprises calcium and phosphate. In one variation, the culture medium comprising calcium and phosphate is a feed culture medium. In another variation, the culture medium comprising calcium and phosphate is a basal culture medium. In some aspects, the feed culture medium is a production culture medium. In some aspects, the cell culture medium is a chemically defined cell culture medium. In other aspects, the cell culture medium is a chemically undefined cell culture medium. In either aspect, during the HTST treatment, the cell culture medium is used to inactivate viruses. In either aspect, before the polypeptide production stage of the cell culture, during the HTST treatment, the total amount of phosphate and calcium in the culture medium is limited to less than about 10mM. In either aspect, during the polypeptide production stage of the cell culture, the total amount of phosphate and calcium in the culture medium is increased to a level sufficient for protein expression. In either aspect, during the HTST treatment, the pH of the culture medium is between about pH 5.0 and about pH 6.9. In either aspect, before the polypeptide production stage of the cell culture, during the HTST treatment, the pH of the culture medium is reduced to between about pH 5.0 and about pH 6.9. In another aspect, the pH of the culture medium is then brought to about pH 6.9 to about pH 7.2 for the polypeptide production phase of cell culture. In one aspect, after HTST treatment, the pH of the culture medium is then brought to about pH 6.9 to about pH 7.2 for the polypeptide production phase of cell culture.

[0088] In one variation, the culture medium provided herein comprises a cell culture medium, wherein the culture medium has a pH between about pH 5.0 and about pH 6.9. In one variation, the culture medium comprising calcium and phosphate is a feed culture medium. In one variation, the culture medium comprising calcium and phosphate is a basal culture medium. In some aspects, the cell culture medium is a chemically defined cell culture medium. In other aspects, the cell culture medium is a chemically undefined cell culture medium. In either aspect, during the HTST treatment, the cell culture medium is used to inactivate viruses. In either aspect, before the polypeptide production stage of the cell culture, during the HTST treatment, the pH of the culture medium is reduced to between about pH 5.0 and about pH 6.9. On the other hand, the pH of the culture medium is then brought to between about pH 6.9 and about pH 7.2 for the polypeptide production stage of the cell culture. On the other hand, after the HTST treatment, the pH of the culture medium is brought to between about pH 6.9 and about pH 7.2 for the polypeptide production stage of the cell culture. In either aspect, during the HTST treatment, the total amount of phosphate and calcium in the culture medium is limited to less than about 10 mM. In either aspect, prior to the polypeptide production phase of cell culture, during HTST treatment, the total amount of phosphate and calcium in the culture medium is limited to less than about 10 mM. In either aspect, during the polypeptide production phase of cell culture, the total amount of phosphate and calcium in the culture medium is increased to a level sufficient for polypeptide production.

[0089] In one variation, the invention provides a culture medium in which the cell culture medium components are adjusted using the response surface described in detail in Example 2. In another variation, the invention provides a culture medium in which the cell culture medium components are adjusted using the response surface described in detail in Example 2. Figure 6 Adjusting cell culture medium composition using a response surface.

[0090] In one variation, the invention provides such a culture medium, wherein a trace metal is added to the culture medium after the culture medium is subjected to HTST treatment, in one aspect, the trace metal is at least one or more trace metals selected from iron or copper. In one variation, the invention provides such a culture medium, wherein iron is added to the culture medium in an amount between about 1 μM and about 125 μM after the culture medium is subjected to HTST treatment. In another variation, after the medium is subjected to HTST treatment, the concentration of iron in the medium is from about 1 μM to about 125 μM; from about 10 μM to about 120 μM; from about 20 μM to about 110 μM; from about 30 μM to about 100 μM; from about 40 μM to about 90 μM; from about 50 μM to about 80 μM; from about 60 μM to about 70 μM; from 1 μM to about 120 μM; from 1 μM to about 110 μM; from 1 μM to about 100 μM; from 1 μM to about 90 μM; from 1 μM to about 80 μM; from 1 μM to about 70 μM; from 1 μM to about 60 μM; from 1 μM to about 50 μM; from 1 μM to about 40 μM; from 1 μM to about 30 μM; from 1 μM to about 20 μM; from 1 μM to about 10 μM; from 10 μM to about 125 μM; or about 1 or 10 or 20 or 30 or 40 or 50 or 60 or 70 or 80 or 90 or 100 or 110 or 120 or 125 μM; at least about 1 or 10 or 20 or 30 or 40 or 50 or 60 or 70 or 80 or 90 or 100 or 110 or 120 and no more than about 125 μM.

[0091] In one aspect, when the medium provided herein is used for the method for inactivating viruses during HTST treatment, it leads to one or more favorable properties, compared with the properties when different mediums are used for the inactivation of viruses during HTST treatment. The precipitation generated in the HTST treatment for inactivating viruses of the cell culture medium used to produce biopharmaceuticals (e.g., antibody products) may affect the quality properties of biopharmaceuticals, such as the activity of biopharmaceuticals. In addition, the formation of precipitates in the cell culture medium during HTST treatment may cause scaling of HTST equipment. The scaling of HTST equipment may affect the ability of HTST treatment to effectively inactivate viruses. In one aspect, the fouling includes precipitation on the equipment for HTST treatment. In a variation, compared with the precipitation formed in different mediums used for inactivation of viruses during HTST treatment, when the medium provided herein is used for the method for inactivating viruses during HTST treatment in cell culture, it reduces the precipitation formed in the cell culture medium. In another variation, compared with the scaling of HTST equipment caused by using different mediums for inactivation of viruses during HTST treatment, when the medium provided herein is used for the method for inactivating viruses during HTST treatment, it reduces the scaling of HTST equipment. In yet another variation, when the medium provided herein is used in a method for inactivating viruses in HTST treatment of a culture medium, it reduces the precipitate formed on the HTST-treated equipment compared to the precipitate formed on the HTST-treated equipment using a different culture medium. In another variation, when the medium provided herein is used in a method for inactivating viruses in HTST treatment of a culture medium, it inactivates viruses in a cell culture medium compared to the inactivation of viruses in different culture media. In yet another variation, the medium provided herein reduces the precipitate caused by filter fouling in a HTST treatment method of a culture medium compared to the inactivation of viruses in different culture media.

[0092] One observation is that during HTST treatment, trace metals such as copper and iron that are important for cell culture (including the production of polypeptides / proteins, cell growth, survival and / or proliferation) are reduced. Therefore, the present invention also provides a method for inactivating viruses or exogenous factors in a cell culture medium while maintaining the culture medium for cell culture suitability, the method comprising (a) subjecting the cell culture medium to high temperature transient (HTST) treatment; and (b) adjusting one or more parameters selected from pH, calcium level and phosphate level, wherein the concentration of trace metals can also be adjusted. The trace metal can be iron or copper. The concentration of iron and / or copper can be adjusted independently before HTST treatment. In some instances, if the concentration of iron and / or copper is expected to be reduced and the amount is generally known, iron and / or copper can be added before HTST treatment. In other instances, where the amount of reduction is unknown, iron and / or copper can be reduced (including removal) from the culture medium before HTST treatment. Then after HTST treatment, iron and / or copper are supplemented to the culture medium to a suitable level for cell culture.

[0093] IV. Compositions and Methods of the Invention

[0094] During the HTST treatment, the cell culture medium described in detail herein can be used for methods of inactivating viruses, infectious agents and / or exogenous factors in cell culture medium. The culture medium can be used for methods of culturing cells by batch culture, fed-batch culture or flow culture. The culture medium can be used for methods of culturing cells to produce polypeptides including antibodies. The culture medium can be used for culturing cells to produce cell-based products, including methods for those products used for tissue replacement or gene therapy applications. The culture medium can be used for any cell culture application, wherein preventing possible viral contamination benefits from the use of heat treatment, which inactivates viruses but retains the ability of the culture medium to culture cells. The culture medium can be used for methods of inactivating viruses, infectious agents and / or exogenous factors in cell culture mediums that have undergone any variation or embodiment of the heat treatment described herein.

[0095] Provided is a method for inactivating viruses, infectious agents and / or exogenous factors in a cell culture medium as described in detail herein, wherein the cell culture medium is subjected to HTST treatment. In one variation, the method comprises subjecting the cell culture medium to HTST treatment, wherein the culture medium comprises one or more culture medium components as described in Table 1 (e.g., a culture medium comprising components (a) and (b) in any amount listed in Table 1 or a culture medium comprising components (a) and (c) or a culture medium comprising components (b) and (c) or a culture medium comprising components (a) and (d) or a culture medium comprising components (b) and (d) or a culture medium comprising each of (a)-(c) or (a), (b) and (d)).

[0096] A method for reducing fouling of equipment used for HTST treatment to inactivate viruses, infectious agents and / or exogenous factors, wherein a cell culture medium as described in detail herein is used in the equipment and subjected to HTST treatment. In one variation, the method comprises subjecting the cell culture medium to HTST treatment, wherein the culture medium comprises one or more culture medium components as described in Table 1 (e.g., a culture medium comprising components (a) and (b) or a culture medium comprising components (a) and (c) or a culture medium comprising components (b) and (c) or a culture medium comprising components (a) and (d) or a culture medium comprising components (b) and (d) or a culture medium comprising each of (a)-(c) or (a), (b) and (d) in any amount listed in Table 1). In a specific variation, the fouling is sediment fouling. In one variation, the fouling is filter fouling.

[0097] A. Methods for inactivating viruses and infectious agents and / or exogenous agents in cell culture media

[0098] In some variations, the invention provides methods for inactivating viruses, infectious agents and / or exogenous factors in cell culture media subjected to high temperature treatment, wherein the culture media is compatible with the heat treatment compared to heat-treated incompatible culture media. For example, heat-treated incompatible culture media precipitate or produce precipitation at the temperature required for effective inactivation of viruses, infectious agents and / or exogenous factors. As provided in the culture media disclosed herein, heat-treated compatible culture media do not precipitate or have reduced precipitation at the temperature required for effective inactivation of viruses, infectious agents and / or exogenous factors. In some aspects, heat treatment is HTST treatment. In other aspects, heat treatment is batch heat treatment, including but not limited to sand bath treatment and oil bath method.

[0099] In one variation, the invention provides a method for inactivating viruses in a cell culture medium, comprising subjecting the cell culture medium to HTST treatment, wherein the culture medium has a pH between about pH 5.0 and about pH 6.9 during the HTST treatment. In some aspects, the culture medium has a pH of about pH 5.3 to about pH 6.3 during the HTST treatment. In other aspects, the culture medium has a pH of about pH 6.0 during the HTST treatment. In some aspects, before the polypeptide production stage of the cell culture, during the HTST treatment, the pH of the culture medium is reduced to between about pH 5.0 and about pH 6.9. On the other hand, the pH of the culture medium is then brought to between about pH 6.9 and about pH 7.2 for the polypeptide production stage of the cell culture. In one aspect, after the HTST treatment, the pH of the culture medium is brought to between about pH 6.9 and about pH 7.2 for the polypeptide production stage of the cell culture. In another variation, the invention provides a method for inactivating viruses in a cell culture medium, comprising limiting the total amount of phosphate and calcium in the culture medium to less than about 10 mM during the HTST treatment. On the other hand, during the HTST treatment, the total concentration of phosphate and calcium in the culture medium is limited to less than about 9, 8, 7, 6, 5, 4, 3, 2 or 1 mM. In some aspects, before the polypeptide production stage of the cell culture, during the HTST treatment, the total amount of phosphate and calcium in the culture medium is limited to less than about 10 mM. On the other hand, during the polypeptide production stage of the cell culture, the total amount of phosphate and calcium in the culture medium is then increased to a level sufficient for polypeptide production. In another variation, the present invention provides a method for inactivating viruses in a cell culture medium, comprising subjecting the cell culture medium to HTST treatment, wherein the culture medium has a pH between about pH 5.0 and about pH 6.9, and the total amount of phosphate and calcium contained in the culture medium during the HTST treatment is limited to less than about 10 mM. In some aspects, before the polypeptide production stage of the cell culture, during the HTST treatment, the pH of the culture medium is reduced to between about pH 5.0 and about pH 6.0, and the total amount of phosphate and calcium in the culture medium is limited to less than about 10 mM. On the other hand, during the polypeptide production phase of the cell culture, the pH of the culture medium is brought to between about pH 6.9 and about pH 7.2, and the total amount of phosphate and calcium in the culture medium is increased to a level sufficient for polypeptide production. The virus of any of the methods detailed herein can be any of the viruses detailed herein (e.g., parvovirus), and the culture medium of the present method can be any of the culture medium detailed herein, such as a culture medium comprising the culture medium parameters detailed in Table 1.

[0100] a) Temperature and temperature retention time

[0101] In any of the methods described in detail herein, the heat treatment includes raising the temperature of the culture medium to at least about 85°C for a sufficient time to inactivate the virus in the culture medium. On the other hand, the temperature of the culture medium is raised to at least about 90°C for a sufficient time to inactivate the virus in the culture medium. On the other hand, the temperature of the culture medium is raised to at least about 93°C for a sufficient time to inactivate the virus in the culture medium. On the other hand, the temperature of the culture medium is raised to at least about 95, 97, 99, 101 or 103°C for a sufficient time to inactivate the virus in the culture medium. On the other hand, the temperature of the culture medium is raised to at least about 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115 or 117°C for a sufficient time to inactivate the virus in the culture medium. In yet another aspect, the temperature of the culture medium is raised to at least about 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 100, 111, 112, 113, 114, 115, 116, 117, 118, 119, or 120° C. for a sufficient time to inactivate the viruses in the culture medium. On the other hand, the temperature of the culture medium is raised to at least about 121, 121, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149 or 150 ° C and continued for a sufficient time to inactivate the virus in the culture medium. In some aspects, the temperature of the culture medium is raised to about 95 ° C. In other aspects, the temperature of the culture medium is raised to about 102 ° C. In a variation, the temperature of the culture medium is raised to at least about 85 ° C to about 120 ° C and continued for a sufficient time to inactivate the virus in the culture medium.In another variation, the temperature of the culture medium is raised to about 85°C to about 120°C; about 87°C to about 118°C; about 89°C to about 116°C; about 91°C to about 114°C; about 93°C to about 112°C; about 95°C to about 110°C; about 97°C to about 108°C; about 99°C to about 106°C; about 101°C to about 104°C; about 85°C to about 118°C; about 85°C to about 116°C; about 85°C to about 114°C; about 85°C to about 112°C; about 85°C to about 110 ℃; about 85℃ to about 108℃; about 85℃ to about 106℃; about 85℃ to about 104℃; about 85℃ to about 102℃; about 85℃ to about 100℃; about 85℃ to about 98℃; about 85℃ to about 96℃; about 85℃ to about 94℃; about 85℃ to about 92℃; about 85℃ to about 90℃; about 85℃ to about 88℃; about 87℃ to about 120℃; about 89℃ to about 120℃; about 91℃ to about 120℃; about 93℃ to about 120℃; about 95℃ to about 120 ℃; about 97℃ to about 120℃; about 99℃ to about 120℃; about 101℃ to about 120℃; about 103℃ to about 120℃; about 105℃ to about 120℃; about 107℃ to about 120℃; about 109℃ to about 120℃; about 111℃ to about 120℃; about 113℃ to about 120℃; about 115℃ to about 120℃; about 117℃ to about 120℃; about 85℃ or 86℃ or 88℃ or 90℃ or 92℃ or 94℃ or 96℃ or 98℃ or 100℃ or At least about 85°C or 86°C or 88°C or 90°C or 92°C or 94°C or 96°C or 98°C or 100°C or 102°C or 104°C or 106°C or 108°C or 110°C or 112°C or 114°C or 116°C or 118°C and no more than about 120°C and for a sufficient time to inactivate viruses in the culture medium. In any aspect, during the polypeptide production phase of the cell culture, the culture medium temperature is cooled to between about 15°C and about 40°C for polypeptide production.

[0102] In the temperature holding time (temperature holding time), the heat treatment temperature is maintained to inactivate the virus in the culture medium. The temperature holding time is the amount of time enough to inactivate the virus. In either aspect, the amount of time enough to inactivate the virus in the culture medium is at least about 1 second. On the other hand, the amount of time enough to inactivate the virus in the culture medium is at least about 2 seconds, 3 seconds, 4 seconds. On the other hand, the amount of time enough to inactivate the virus in the culture medium is at least about 5, 8, 10, 12, 14, 16, 18 or 20 seconds. On the other hand, the amount of time enough to inactivate the virus in the culture medium is at least about 5, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58 or 60 seconds. In some aspects, the amount of time enough to inactivate the virus in the culture medium is 10 seconds. In other aspects, the amount of time enough to inactivate the virus in the culture medium is 2 seconds. In another variation, the amount of time sufficient to inactivate the virus in the culture medium is from about 1 to about 60 seconds; about 2 to about 58 seconds; about 6 to about 54 seconds; about 10 to about 50 seconds; about 14 to about 46 seconds; about 18 to about 42 seconds; about 22 to about 38 seconds; about 26 to about 34 seconds; about 30 to about 34 seconds; about 1 to about 56 seconds; about 1 to about 52 seconds; about 1 to about 48 seconds; about 1 to about 44 seconds; about 1 to about 40 seconds; about 1 to about 36 seconds; about 1 to about 32 seconds; about 1 to about 28 seconds; about 1 to about 22 seconds; about 1 to about 18 seconds; about 1 to about 14 seconds; about 1 to about 10 seconds; about 1 to about 6 seconds; about 1 to about 3 seconds; about 4 to about 60 seconds; about 8 to about 60 seconds; about 12 to about 60 seconds; about 16 to about 60 seconds; about 20 to about 60 seconds; about 24 to about 60 seconds; about 28 to about 60 seconds; about 32 to about 60 seconds; about 36 to about 60 seconds; about 40 to about 60 seconds; about 44 to about 60 seconds; about 48 to about 60 seconds; about 52 to about 60 seconds; about 56 to about 60 seconds; about 1 or 2 or 4 or 6 or 8 or 10 or 12 or 14 or 16 or 18 or 20 or 22 or 24 or 26 or 28 or 30 or 32 or 34 or 36 or 38 or 40 or 42 or 44 or 46 or 48 or 50 or 52 or 54 or 56 or 58 or 60 seconds; at least about 1 or 2 or 4 or 6 or 8 or 10 or 12 or 14 or 16 or 18 or 20 or 22 or 24 or 26 or 28 or 30 or 32 or 34 or 36 or 38 or 40 or 42 or 44 or 46 or 48 or 50 or 52 or 54 or 56 or 58 and no more than about 60 seconds. In other aspects, the amount of time sufficient to inactivate the virus in the culture medium is at least about 1 minute. In another aspect, the amount of time sufficient to inactivate the virus in the culture medium is at least about 2.5 minutes.In another aspect, the amount of time sufficient to inactivate the virus in the culture medium is at least about 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 10.5 or 11 minutes. In another variation, the amount of time sufficient to inactivate the virus in the culture medium is about 1 to about 11 minutes; 2 to about 10 minutes; 4 to about 8 minutes; about 1 to about 10 minutes; about 1 to about 8 minutes; about 1 to about 6 minutes; about 1 to about 4 minutes; about 1 to about 2 minutes; about 2 to about 11 minutes; about 4 to about 11 minutes; about 6 to about 11 minutes; about 8 to about 11 minutes; one of about 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 minutes; at least about 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 and no more than about 11 minutes.

[0103] b) Infectious agents

[0104] The present invention provides a method for inactivating viruses and / or exogenous factors in a culture medium, comprising subjecting the culture medium to heat treatment. The virus can be any virus described in detail herein (e.g., parvovirus), and the cell culture medium can be any cell culture medium described in detail herein, such as a culture medium having the components described in detail in Table 1. During the heat treatment, the virus inactivated in the cell culture medium can be any virus industrially associated with product production (e.g., polypeptides, cells, tissues). Industrially relevant viruses are known to those skilled in the art. Non-limiting examples of industrially relevant viruses are: Parvoviridae, paramyoxviradae, orthomyxoviridae, bunyaviridae, rhabdoviridae, reoviridae, togaviridae, caliciviridae, and picornaviridae. In this specification, the formal nomenclature of a virus species (e.g., parvoviridae) is used interchangeably with the less formal nomenclature (e.g., parvovirus). It should be understood that both of the above nomenclatures refer to the same virus species (i.e., parvoviridae includes the same virus species as parvovirus). In one variation, the virus is a non-enveloped virus. In some aspects, non-enveloped viruses include, but are not limited to, single-stranded DNA viruses, double-stranded DNA viruses, double-stranded RNA viruses, and single-stranded RNA viruses. In another aspect, non-enveloped viruses include, but are not limited to, parvoviruses, reoviruses, or picornaviruses. In a variant, viruses are enveloped viruses. In some aspects, enveloped viruses include, but are not limited to, single-stranded DNA viruses, double-stranded DNA viruses, double-stranded RNA viruses, and single-stranded RNA viruses. In another aspect, enveloped viruses include, but are not limited to, retroviruses, herpes viruses, or hepadnaviruses. In any of the variants of the invention, the virus is selected from adenovirus, African swine fever-line virus, arenavirus, arterivirus, astrovirus, baculovirus, badnavirus, barnavirus, birnavirus, bromovirus, bunyavirus, calicivirus, capillovirus, carlavirus, caulimovirus, circovirus, closterovirus, comovirus, coronavirus, cotricovirus, cystovirus, deltavirus,dianthovirus, enamovirus, filovirus, flavivirus, furovirus, fusellovirus, geminivirus, hepadnavirus, herpesvirus, hordeivirus, hypovirus, ideaovirus, inovirus, iridovirus, levivirus, lipothrixvirus, luteovirus, machlomovirus, marafivovirus, microvirus, myovirus, necrovirus, nodavirus, orthomyxovirus, papovavirus, paramyxovirus yxovirus), partitivirus, parvovirus, phycodnavirus, picornavirus, plamavirus, podovirus, polydnavirus, potexvirus, potyvirus, poxvirus, reovirus, retrovirus, rhabdovirus, rhizidiovirus, sequevirus, siphovirus, sobemovirus, tectivirus, tenuivirus, tetravirus, tobaccovirus, tobravirus, togavirus, tombusvirus, totivirus, trichovirus,In some aspects, the virus includes, but is not limited to, epizootic hemorrhagic disease virus (EHDV), mice minute virus (MMV), mouse parvovirus-1 (MPV), cache valley virus, Vesivirus 2117, porcine circovirus (PCV 1), porcine circovirus 2 (PCV 2), canine parvovirus (CPV), bovine parvovirus (BPV) or blue tongue virus (BTV). In specific aspects, the virus is a parvovirus such as murine parvovirus. In a variant, the invention provides a method for inactivating subviral infectious agents in a cell culture medium, comprising heat treating the culture medium. In some aspects, the subviral infectious agent is a viroid or satellite. In another variation, the invention provides a method for inactivating a virus-like agent in a cell culture medium, comprising heat treating the culture medium.

[0105] In another variation, the invention provides a method of inactivating an infectious agent in a cell culture medium, comprising subjecting the culture medium to heat treatment. In some aspects, the infectious agent is a bacterium. In another aspect, the bacterium is a mycoplasma. In another aspect, the infectious agent is a bacterium small enough to pass through a filter, including any of the filters described herein. In another aspect, the infectious agent is a fungus. In yet another aspect, the infectious agent is a parasite. In yet another aspect, the infectious agent is a component of an infectious agent. It will be appreciated by those skilled in the art that a component of an infectious agent can be any component derived from an infectious agent that is not desired in the cell culture medium.

[0106] Still in another variation, the present invention provides a method for inactivating exogenous factors in a cell culture medium, comprising subjecting the culture medium to heat treatment. It will be appreciated by those skilled in the art that any exogenous factor susceptible to inactivation by heat treatment can be inactivated by any of the methods described in detail herein using any of the cell culture media described in detail herein, such as a culture medium having the components described in detail in Table 1. In any variation, during the HTST treatment, at least one or more types of infectious agents and / or exogenous factors are inactivated in the cell culture medium. In some aspects, one or more types of infectious agents and / or exogenous factors are components of viruses, subviral infectious agents, virus-like agents, bacteria, fungi, parasites, or infectious agents and / or exogenous factors. In any variation, the heat treatment is a HTST treatment. It will be appreciated by those skilled in the art that any of the methods for inactivating infectious agents and / or exogenous factors by heat treatment described in detail herein includes subjecting any of the cell culture media, such as a culture medium having the components described in detail in Table 1, to any of the HTST treatments described in detail herein.

[0107] Virus inactivation can be measured by assays known to those skilled in the art. For example, virus inactivation is determined by measuring or evaluating a given log reduction value (LRV) of active virus. In one aspect, a log reduction value of greater than or equal to 3 in a cell culture medium treated with HTST for 2 seconds at a temperature greater than 95°C is determined as inactivating the virus in the culture medium. On the other hand, a log reduction value of about 3 in a cell culture medium treated with HTST for 60 seconds at a temperature of 100°C is determined as inactivating the virus in the culture medium. In either aspect, the viral load is the MVM load.

[0108] c) Heat treatment system

[0109] Methods for heat treating cell culture media for inactivating infectious agents known to those skilled in the art, such as the methods described in Schleh, M. et al. 2009. Biotechnol. Prog. 25(3):854-860 and Kiss, R. 2011. PDA J Pharm Sci and Tech. 65:715-729 (the disclosures of which are incorporated herein by reference in their entirety) can be used to heat treat any of the cell culture media detailed herein, such as a culture medium having the components detailed in Table 1. For example, high temperature transient (HTST) treatment is used in a production process to inactivate viruses. In one variation, the present invention provides a method for inactivating viruses in a cell culture medium, comprising subjecting the cell culture medium to HTST treatment, wherein the culture medium has a pH between about pH 5.0 and about pH 6.9 during the HTST treatment. In another variation, the present invention provides a method for inactivating viruses in a cell culture medium, comprising limiting the total amount of phosphate and calcium in the culture medium to less than about 10 mM during the HTST treatment. In yet another variation, the present invention provides a method for inactivating viruses in a cell culture medium, comprising subjecting the cell culture medium to HTST treatment, wherein the culture medium has a pH between about pH 5.0 and about pH 6.9, and comprising limiting the total amount of phosphate and calcium in the culture medium to less than about 10 mM during the HTST treatment. The HTST treatment may include a HTST cycle, wherein the cell culture medium is heated from ambient temperature or 37°C to about 102°C, wherein the cell culture medium is maintained at 102°C for a temperature holding time of about 10 seconds, and then the cell culture medium is cooled to about ambient temperature or about 37°C. In some aspects, the ambient temperature is between about 15°C and about 30°C. In other aspects, the ambient temperature is between about 18°C ​​and about 25°C. The HTST treatment can be a continuous process using two heat exchangers, one for heating the liquid and one for cooling the liquid, with the piping system between providing the desired temperature holding time for a given flow rate. In one aspect, the inactivation of viruses in the cell culture medium comprises subjecting the cell culture medium to one HTST treatment cycle. In another aspect, the inactivation of viruses in the cell culture medium comprises subjecting the cell culture medium to at least two or more HTST treatment cycles. In one aspect, the flow rate is 100 LPM. In another aspect, the flow rate is 125 LPM. In another aspect, the flow rate can be any flow rate suitable for providing a suitable temperature for inactivation of viruses and / or exogenous factors and retention time at that temperature. In either aspect, the cell culture medium is heated from about 37°C to about 102°C, wherein the cell culture medium is kept at 102°C for an amount of time sufficient to inactivate the virus, and then the cell culture medium is cooled to about 37°C. In another aspect, the amount of time sufficient to inactivate the virus is about 10 seconds. In either aspect, the amount of time sufficient to inactivate the virus is any temperature holding time as described in detail herein.In either aspect, the temperature at which the virus is inactivated during the temperature holding time is any temperature detailed herein. In one aspect, the temperature at which the virus is inactivated is between about 85°C and about 120°C. In one variation, the cell culture medium is heated from about 15°C to 102°C, wherein the cell culture medium is maintained at 102°C during a temperature holding time of about 10 seconds, and then the cell culture medium is cooled to about 37°C. In another variation, the cell culture medium is heated from about 37°C to about 102°C, wherein the cell culture medium is maintained at 102°C during a temperature holding time of about 10 seconds, and then the cell culture medium is cooled to about 37°C.

[0110] For HTST treatment, a cell culture medium with a volume of between about 0.5 and about 20,000 liters (L) can be treated by an apparatus for HTST treatment to inactivate viruses. It will be appreciated by those skilled in the art that any cell culture medium described in detail herein, such as a culture medium with the components detailed in Table 1, can be used to treat a cell culture medium volume of less than about 0.5 L or greater than about 20,000 L by an apparatus for HTST treatment in any of the methods described in detail herein. In one aspect, a cell culture medium volume of between about 0.5 L and about 20,000 L can be treated in one HTST operation. In other aspects, a cell culture medium volume of between about 0.5 L and about 20,000 L can be treated in at least two or more HTST operations. As used herein, the term "HTST operation" may refer to a process in which a specified amount of culture medium is subjected to at least one cycle (e.g., heating, holding, cooling) of HTST treatment in an apparatus for HTST treatment (e.g., HTST skid). It will be appreciated by those skilled in the art that any of the devices for HTST treatment, such as the HTST skid, can be used with any of the cell culture media detailed herein, such as the culture media having the components detailed in Table 1. In one aspect, the HTST operation can include continuously treating at least one or more cell culture media volumes between about 0.5 L and about 20,000 L. In another aspect, the HTST operation includes batch treating at least one or more cell culture media volumes of about 0.5 L to about 20,000 L. In any aspect, at least one or more cell culture media volumes can be cell culture media of the same type (e.g., culture media 1). In any aspect, at least one or more cell culture media volumes can be cell culture media of at least one or more types (e.g., culture media 1 and culture media 2). In any aspect of the methods detailed herein, a cell culture media volume of at least about 0.5 L can be treated by a device for HTST treatment to inactivate viruses. In another aspect, a cell culture media volume of at least about 2 L can be treated by a device for HTST treatment to inactivate viruses. In yet another aspect, the duration is sufficient to inactivate at least about 5, 10, 50, 100, 500, 1000, 5000, 10000, or 15000 liters. In some aspects, a volume of 2 L of cell culture medium is processed through the apparatus for HTST processing to inactivate viruses. In other aspects, a volume of 12000 L of cell culture medium is processed through the apparatus for HTST processing to inactivate viruses.In another variation, about 0.5 to about 20,000; about 2 to about 18,000; about 10 to about 16,000; about 20 to about 14,000; about 40 to about 12,000; about 80 to about 10,000; about 100 to about 8,000; about 200 to about 6,000; about 400 to about 4,000; about 800 to about 2,000; about 0.5 to about 1 8000; about 0.5 to about 16000; about 0.5 to about 14000; about 0.5 to about 12000; about 0.5 to about 10000; about 0.5 to about 8000; about 0.5 to about 6000; about 0.5 to about 4000; about 0.5 to about 2000; about 0.5 to about 800; about 0.5 to about 600; about 0.5 to about 400; about 0.5 to about 200; about 0. 5 to about 100; about 0.5 to about 50; about 0.5 to about 20; about 0.5 to about 10; about 0.5 to about 5; about 0.5 to about 2; about 2 to about 20,000; about 10 to about 20,000; about 100 to about 20,000; about 500 to about 20,000; about 1,000 to about 20,000; about 1,500 to about 20,000; about 2,000 to about 20,000; about 5,000 to about 20,000; about 1,000 to about 20,000; about 15,000 to about 20,000; about 1,750 to about 20,000 liters; about one of 0.5 or 2 or 10 or 100 or 1,000 or 10,000 or 20,000 liters; at least about 0.5 or 2 or 10 or 100 or 1,000 or 10,000 and no more than about 20,000 liters of cell culture medium volume to inactivate viruses.

[0111] Methods for inactivating viruses during cell culture medium treatment are known to those skilled in the art, such as the method described in Kiss, R. 2011. PDA J Pharm Sci and Tech. 65: 715-729, which is incorporated herein by reference in its entirety, and can be used in combination with HTST treatment of any of the cell culture media detailed herein, such as a culture medium having the components detailed in Table 1. For example, a high temperature transient (HTST) treatment can be combined with at least one or more virus barrier treatments in a production process to remove viruses from a cell culture medium. In some aspects, the one or more virus barrier treatments include, but are not limited to, heat sterilization, UV light exposure, gamma irradiation, and filtration. In some aspects, the virus barrier treatment is filtration. The present invention provides methods for removing and / or inactivating viruses in a cell culture medium subjected to HTST treatment.

[0112] or a method for inactivating viruses, wherein after the cell culture medium is subjected to HTST treatment, the virus is removed from the cell culture medium in one step by filtration. In some aspects, the filtration is ultrafiltration. Prior to ultrafiltration, one or more culture medium components, such as those listed in Table 1, are added to the cell culture medium subjected to HTST treatment. In some aspects, prior to ultrafiltration, one or more culture medium components, such as trace metals (e.g., iron or copper), are added to the cell culture medium subjected to HTST treatment.

[0113] Ultrafiltration membranes can be formed from self-regenerating cellulose, polyethersulfone, polyarylsulfone, polysulfone, polyimide, polyamide, polyvinylidene fluoride (PVDF), etc. Representative ultrafiltration membranes include, but are not limited to, membrane, Pro film, 180 film, 70 membranes, NFP membrane, NFR membrane, Retropore TM Film, Virosart CPV Film, Planova 75 Film, Planova 35 Film, Planova 20 Film, Planova15N Film, VAG 300 Film, Ultipor DVD Film, Ultipor DV50 Film, Ultipor DV20 Film, and DVD Zeta Plus VR TM In some aspects, the ultrafiltration membrane can remove cellular virus particles. In some aspects, the ultrafiltration membrane is a parvovirus retention membrane.

[0114] The pore size of the ultrafiltration membrane should be small enough to retain unwanted viral particles while allowing one or more proteins in the aqueous solution to pass through the membrane. In some embodiments of the invention, the pore size of the ultrafiltration membrane is less than 10nm, 10nm, 20nm, 30nm, 40nm, 50nm, 60nm, 70nm, 80nm, 90nm, 100nm, 125nm, 150nm, 175nm or 200nm. In some embodiments, the pore size of the ultrafiltration membrane is 20nm or less.

[0115] Ultrafiltration membrane is characterized by molecular weight cut-off, which represents the average molecular weight of the smallest protein retained by the ultrafiltration membrane. For example, an ultrafiltration membrane with a 1000kD molecular weight cut-off can retain most globular proteins with a molecular weight cut-off of more than 1000kD at a ratio of 80-90%, while most globular proteins with a molecular weight less than 1000kD can pass through the ultrafiltration membrane. In some aspects of the invention, the molecular weight cut-off of the ultrafiltration membrane is between 200kD and 1000kD. In some aspects of the invention, the ultrafiltration membrane has a molecular weight cut-off of 200kD, 300kD, 400kD, 500kD, 600kD, 700kD, 900kD or 1000kD.

[0116] One or more ultrafiltration membranes can be used to effect filtration by dead-end (normal) flow filtration (NFF) or tangential flow filtration (TFF). In NFF, the feed stream passes through the membrane and large molecular weight substances are captured in the filter, while the filtrate is released at the other end. In TFF, the vast majority of the feed stream passes tangentially across the filter surface rather than through the filter. Therefore, during the filtration process, the filter cake is fully flushed, extending the time that the filtration unit can operate. It can be used in cartridge (NFF) such as NFP virus filter or in cassette format (for TFF) The cassette provides an ultrafiltration membrane in either filtration mode. In a preferred embodiment, the filtration is normal flow filtration.

[0117] More than one ultrafiltration membrane may be used in the process of the invention. In some embodiments, more than one ultrafiltration membrane is in parallel contact with the aqueous solution. A combination of more than one viral barrier treatment and HTST treatment may be used to treat any of the cell culture media disclosed herein, such as a cell culture medium having the components detailed in Table 1, for industrial scale production of protein and polypeptide therapeutics.

[0118] The cell culture medium used during the HTST treatment for inactivating infectious agents can be a HTST-compatible medium or a HTST-incompatible medium. As used herein, the term "HTST-compatible medium" can refer to a cell culture medium with reduced or no precipitation during the HTST treatment. As used herein, the term "HTST-incompatible medium" can refer to a cell culture medium with measurable or detectable precipitation during the HTST treatment. In one variation, the present invention provides a method for screening a HTST-compatible cell culture medium for viral inactivation during the HTST treatment, wherein the HTST-compatible medium has reduced precipitation compared to the precipitation in the HTST-incompatible medium. In one aspect, during the HTST treatment, the HTST-compatible cell culture medium has no precipitation compared to the precipitation formed in the HTST-incompatible medium. In either variation, after the HTST treatment, the HTST-compatible medium has a higher level of trace metals compared to the HTST-incompatible medium. In one aspect, the trace metal is at least one or more trace metals selected from iron or copper. In either variation, the HTST-compatible medium has lower levels of trace metals compared to another HTST-compatible medium. In one aspect, the trace metal is at least one or more trace metals selected from iron or copper. The present invention provides a method of converting an HTST-incompatible medium into an HTST-compatible medium. In one variation, the present invention provides a method of converting an HTST-incompatible medium into an HTST-compatible medium, wherein the cell culture medium components are adjusted as detailed in Table 1. In another variation, the present invention provides a method of converting an HTST-incompatible medium into an HTST-compatible medium, wherein the cell culture medium components are adjusted using a response surface as detailed in Example 2. In one variation, the present invention provides a method of converting an HTST-incompatible medium into an HTST-compatible medium, wherein the pH of the culture medium is adjusted to between about pH 5.0 and about pH 6.9. In another variation, the present invention provides a method of converting an HTST-incompatible medium into an HTST-compatible medium, wherein the pH of the culture medium is adjusted to between about pH 5.0 and about pH 7.2. In some aspects, the pH of the HTST-incompatible medium is adjusted to between pH 5.3 and about pH 6.3 to convert the HTST-incompatible medium into a HTST-compatible medium. In another aspect, the pH of the HTST-incompatible medium is adjusted to pH 6.0 to convert the HTST-incompatible medium into a HTST-compatible medium. In some aspects, the pH of the HTST-incompatible medium is lowered to between about pH 5.0 and about pH 6.9 during the HTST treatment prior to the polypeptide production stage of the cell culture.On the other hand, the pH of the HTST incompatible medium is then brought to between about pH 6.9 and about pH 7.2 for use in the polypeptide production phase of cell culture. On the other hand, after HTST treatment, the pH of the HTST incompatible medium is then brought to between about pH 6.9 and about pH 7.2 for use in the polypeptide production phase of cell culture. In some aspects, prior to the polypeptide production phase of cell culture, during HTST treatment, the pH of the HTST incompatible medium is reduced to between about pH 5.0 and about pH 7.2. In a variation, the invention provides a method for converting an HTST incompatible medium into an HTST compatible medium, wherein the total amount of phosphate and calcium in the medium is adjusted to less than about 10 mM. On the other hand, the concentration of total phosphate and calcium in the HTST incompatible medium is adjusted to less than about 9, 8, 7, 6, 5, 4, 3, 2 or 1 mM. In some aspects, the total amount of phosphate and calcium in the HTST-incompatible medium is adjusted to less than about 10 mM prior to the polypeptide production phase of cell culture. In another aspect, during the polypeptide production phase of cell culture, the total amount of phosphate and calcium in the HTST-incompatible medium is then raised to a level sufficient to produce the polypeptide.

[0119] d) Reduction of precipitate formation

[0120] In one variation, the invention provides a method for reducing precipitation in a cell culture medium during a heat treatment for inactivating viruses. The method comprises adjusting one or more levels of calcium, phosphate, and pH in a cell culture medium subjected to heat treatment. In one aspect, the heat treatment is a HTST treatment. In one variation, the invention provides a method for reducing precipitation in a cell culture medium during a heat treatment for inactivating viruses; wherein the culture medium has a pH between about pH 5.0 and about pH 6.9 during the HTST treatment. In another variation, the invention provides a method for reducing precipitation in a cell culture medium during a HTST treatment for inactivating viruses; wherein the culture medium has a pH between about pH 5.0 and about pH 7.2 during the HTST treatment. In some aspects, the culture medium has a pH between about pH 5.3 and about pH 6.3 during the HTST treatment. In other aspects, the culture medium has a pH of about pH 6.0 during the HTST treatment. In some aspects, the pH of the culture medium is reduced to between about pH 5.0 and about pH 6.9 during the HTST treatment, prior to the polypeptide production phase of the cell culture. In some aspects, before the polypeptide production stage of cell culture, during the HTST treatment, the pH of the culture medium is reduced to between about pH 5.0 and about pH 7.2. On the other hand, the pH of the culture medium is then brought to between about pH 6.9 and about pH 7.2 for the polypeptide production stage of cell culture. On the other hand, after the HTST treatment, the pH of the culture medium is then brought to between about pH 6.9 and about pH 7.2 for the polypeptide production stage of cell culture. In one variation, before the polypeptide production stage of cell culture, during the HTST treatment, the pH of the culture medium is between about pH 5.0 and about pH 7.2. In another variation, the invention provides a method for reducing precipitation in a cell culture medium during the HTST treatment for inactivating viruses, including limiting the total amount of phosphate and calcium in the culture medium to less than about 10 mM during the HTST treatment. On the other hand, during the HTST treatment, the concentration of total phosphate and calcium in the culture medium is limited to less than about 9, 8, 7, 6, 5, 4, 3, 2 or 1 mM. In some aspects, prior to the polypeptide production phase of cell culture, during HTST treatment, the total amount of phosphate and calcium in the culture medium is limited to less than about 10 mM. In another aspect, during the polypeptide production phase of cell culture, the total amount of phosphate and calcium in the culture medium is then raised to a level sufficient to produce the polypeptide. In yet another variation, the invention provides a method for reducing precipitation in a cell culture medium during HTST treatment for inactivating viruses, wherein the culture medium has a pH between about pH 5.0 and about pH 6.9, and includes limiting the total amount of phosphate and calcium in the culture medium to less than about 10 mM during HTST treatment.In some aspects, prior to the polypeptide production phase of cell culture, during the HTST treatment, the pH of the culture medium is lowered to between about pH 5.0 and about pH 6.0, and the total amount of phosphate and calcium in the culture medium is limited to less than about 10 mM. In another aspect, during the polypeptide production phase of cell culture, the pH of the culture medium is then brought to between about pH 6.9 and about pH 7.2, and the total amount of phosphate and calcium in the culture medium is raised to a level sufficient for polypeptide production.

[0121] In some aspects of the invention, any of the culture media detailed herein, such as culture media with specific components detailed in Table 1, reduces precipitation during HTST treatment for inactivation of infectious agents. In one aspect, the specific component in the culture medium reduces precipitation by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% when compared to the amount of precipitation present in the same culture medium lacking the specific culture medium component during HTST treatment. Quantitative determination of the amount of precipitation in a culture medium can be performed using techniques well known in the art. In one variation, the invention provides a method of reducing precipitation in a cell culture medium subjected to HTST treatment, wherein the culture medium has a pH of about pH 5.0 to about pH 6.9, and when compared to precipitation in a culture medium that does not have a pH of about pH 5.0 to about pH 6.9 during HTST treatment, the culture medium reduces precipitation by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. In one aspect, the culture medium has a pH of about pH 5.3 to about pH 6.3 during HTST treatment, and when compared to precipitation in a culture medium that does not have a pH of about pH 5.3 to about pH 6.3 during HTST treatment, the culture medium reduces precipitation by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. In another aspect, the culture medium has a pH of about pH 6.0 during HTST treatment, and the culture medium reduces precipitation by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% when compared to precipitation in a culture medium that does not have a pH of about pH 5.3 to about pH 6.0 during HTST treatment. In one variation, the invention provides a method of reducing precipitation in a cell culture medium, comprising limiting the total amount of phosphate and calcium in the culture medium to less than about 10 mM during HTST treatment, and the culture medium reduces precipitation by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% when compared to precipitation in a culture medium that does not have a pH of about pH 5.3 to about pH 6.0 during HTST treatment. In one aspect, the total concentration of phosphate and calcium in the culture medium during the HTST treatment is less than about 9, 8, 7, 6, 5, 4, 3, 2, or 1 mM, and the culture medium reduces precipitation by at least about one of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% when compared to precipitation in a culture medium wherein the total concentration of phosphate and calcium is not less than about 9, 8, 7, 6, 5, 4, 3, 2, or 1 mM during the HTST treatment.In one variation, the invention provides a method of reducing precipitation in a cell culture medium subjected to HTST treatment, wherein the culture medium has a pH of about pH 5.0 to about pH 6.9, and the method comprises limiting the total amount of phosphate and calcium in the culture medium to less than about 10 mM during the HTST treatment, and the culture medium reduces precipitation by at least about one of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% when compared to precipitation in a culture medium that does not have a pH of about pH 5.0 to about pH 6.9 during the HTST treatment and wherein the total amount of phosphate and calcium is greater than about 10 mM.

[0122] The infectious agent of any of the methods detailed herein can be any of the viruses detailed herein (e.g., parvovirus), and the culture medium of the method can be any of the culture media detailed herein, such as a culture medium having the components detailed in Table 1. B. Methods for Reducing Fouling of Equipment Used in Heat Treatment

[0123] The present invention provides methods for reducing fouling of equipment used for HTST treatment to inactivate viruses in cell culture media. The inventors have discovered that adjusting the levels of specific components or parameters in a cell culture medium undergoing HTST treatment can reduce fouling of equipment used for HTST treatment to inactivate viruses. Any of the cell culture media detailed herein, such as a culture medium having the components detailed in Table 1, can be used in any of the methods detailed herein for reducing fouling of equipment used for HTST treatment to inactivate viruses. In any of the methods detailed herein, fouling includes precipitation on equipment used for HTST treatment.

[0124] In one variation, the present invention provides a method for reducing fouling of equipment for HTST treatment, the method comprising subjecting a cell culture medium used in the equipment to HTST treatment, wherein during the HTST treatment, the culture medium has a pH between about pH 5.0 and about pH 6.9. In one aspect, during the HTST treatment, the culture medium has a pH between about pH 5.3 and about pH 6.3. On the other hand, during the HTST treatment, the culture medium has a pH of about pH 6.0. In another aspect, the fouling comprises precipitation on the equipment for HTST treatment. In either aspect, the HTST treatment comprises raising the temperature to at least about 85°C for a sufficient time to inactivate viruses in the culture medium. On the other hand, the temperature of the culture medium is raised to at least about 93°C for a sufficient time to inactivate viruses in the culture medium. In another aspect, the temperature of the culture medium is raised to at least about 95, 97, 99, 101 or 103°C for a sufficient time to inactivate viruses in the culture medium. In one variation, the invention provides a method of reducing fouling of equipment used for HTST treatment, the method comprising limiting the total amount of phosphate and calcium in a cell culture medium used in the equipment to less than about 10 mM during HTST treatment. In one aspect, during HTST treatment, the total concentration of phosphate and calcium in the culture medium is limited to less than about 9, 8, 7, 6, 5, 4, 3, 2, or 1 mM. In another aspect, the fouling comprises precipitation on the equipment used for HTST treatment.

[0125] In either aspect, when a HTST-compatible medium is used, scaling of equipment used for HTST processing is reduced compared to a HTST-incompatible medium. In one aspect, the HTST-compatible medium comprises a pH between about pH 5.0 and about pH 6.9 during HTST processing. In another aspect, the HTST-compatible medium comprises a total amount of less than about 10 mM phosphate and calcium. In another aspect, during HTST processing, the HTST-compatible medium has a pH between about pH 5.0 and about pH 6.9 and a total amount of less than about 10 mM phosphate and calcium. The HTST-compatible medium is any of the cell culture media detailed herein, such as a medium having the components detailed in Table 1.

[0126] Methods for measuring and monitoring fouling of various equipment used in production processes are known to those skilled in the art, such as the methods described in Awad, M. 2011. Heat Transfer: Theoretical Analysis, Experimental Investigations and Industrial Systems. Chapter 20, pages 505-542, which disclosure is incorporated herein by reference in its entirety, and can be used to monitor and measure fouling of equipment used for HTST processing of any of the cell culture media disclosed herein, such as the culture media having the components detailed in Table 1 and the culture media detailed in the Examples. For example, equipment fouling can be measured by measuring the change in heat exchanger steam pressure required to achieve the target culture medium temperature set point or by measuring the change (reduction) in the temperature achieved. In either aspect, fouling includes precipitation on equipment used for HTST processing. In one aspect, due to the use of HTST-incompatible culture media, one or more equipment that is susceptible to fouling includes, but is not limited to, HTST skids, heat exchangers, piping systems, filtration devices, and filter membranes.

[0127] C. Methods for producing polypeptides using heat-treated cell culture medium

[0128] a) Cells

[0129] The methods and compositions provided can be applied to any cell suitable for growing and / or producing polypeptides (e.g., antibodies) in the culture medium described herein, including animal, yeast or insect cells. In one aspect, the cell to which the present methods and compositions can be applied is any mammalian cell or cell type suitable for cell culture and expression of polypeptides. Therefore, the methods (e.g., methods for inactivating viruses in cell culture media) and compositions provided herein can be applied to any suitable type of cell, including animal cells. In one aspect, the present methods and compositions are applied to mammalian cells. The present methods and compositions can also be applied to hybridoma cells. In a variation, the mammalian cell is a non-hybridoma mammalian cell, which has been transformed with exogenous isolated nucleic acids encoding desired polypeptides such as antibodies, antibody fragments (including ligand binding fragments) and chimeric antibodies. In one variation, the mammalian cells used in the methods and compositions are selected from human retinoblasts (PER.C6 (CruCell, Leiden, The Netherlands)); SV40-transformed monkey kidney CV1 cell line (COS-7, ATCC CRL 1651); human embryonic kidney cell line (293 or subcloned 293 cells grown in suspension culture, Graham et al., J. Gen Virol., 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216 (1980)); mouse supporting cells (TM4, Mather, Biol. Reprod., 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1 587); human cervical cancer cells (HeLa, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (HepG2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci., 383:44-68 (1982)); MRC 5 cells; FS4 cells; and human hepatoma cells (Hep G2). In a specific variation, the present methods and compositions are applied to CHO cells. In a specific variation, a CHO cell line is used for culture and the polypeptide (e.g., antibody) is expressed from the CHO cell line.The polypeptide (eg, antibody) can be secreted into the culture medium disclosed herein, where it can be isolated and / or purified, or can be released into the culture medium disclosed herein by lysing cells containing an isolated nucleic acid encoding the polypeptide.

[0130] Methods, vectors and host cells suitable for synthesizing polypeptides of interest in recombinant vertebrate cells are known in the art and are described, for example, in Gething et al., Nature, 293:620-625 (1981); Mantei et al., Nature, 281:40-46 (1979); Levinson et al.; EP 117,060; and EP 117,058. Particularly useful plasmids for expressing polypeptides in mammalian cell culture are pRK5 (P pub. no. 307,247) or pSVI6B (CT pub. no. WO 91 / 08291 published on June 13, 1991).

[0131] Host cells are transformed with expression or cloning vectors and cultured in a nutrient medium modified to be suitable for inducing promoters, selecting transformants or amplifying genes encoding desired sequences. For mammalian cells, preferred are the calcium phosphate precipitation method of Graham and van der Erb, Virology, 52:456-457 (1978) or the cationic liposome method (lipofectamine.TM) (Gibco BRL) of Hawley-Nelson, Focus 15:73 (1193). General aspects of mammalian cell host system transformation are known in the art and are described, for example, in U.S. Patent No. 4,399,216, issued on August 16, 1983, by Axel. For various techniques for transforming mammalian cells, see, e.g., Keown et al., Methods in Enzymology (1989), Keown et al., Methods in Enzymology, 185:527-537 (1990), and Mansour et al., Nature, 336:348-352 (1988).

[0132] The methods and compositions of the present invention also include the use of hybridomas that secrete monoclonal antibodies in cell culture. Monoclonal antibodies are prepared by recovering immune cells (generally spleen cells or lymphocytes from lymph node tissue) from immunized animals, immortalizing cells in a conventional manner (e.g., by fusion with myeloma cells or by transformation with Epstein-Barr virus), and screening clones expressing the desired antibodies. The hybridoma technology originally described by Kohler and Milstein, Eur. J. Immunol., 6: 511 (1976), and also by Hammerling et al., In: Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, NY, pp. 563-681 (1981) is widely used to produce hybrid cell lines that secrete high levels of monoclonal antibodies against many specific antigens.

[0133] b) Peptides

[0134] The polypeptide produced by the compositions (e.g., cells) and methods described in detail herein and present in the compositions provided herein can be homologous to the host cell, or preferably can be exogenous, which means that they are heterologous to the host cell used, i.e., external, such as human proteins produced by Chinese hamster ovary cells or yeast polypeptides produced by mammalian cells. In one variation, the polypeptide is a mammalian polypeptide (e.g., antibody) directly secreted into the culture medium by the host cell. In another variation, the polypeptide is released into the culture medium by cracking the cell of the isolated nucleic acid comprising the encoded polypeptide.

[0135] In one variation, a polypeptide is a sequence of amino acids whose chain length is sufficient to produce higher levels of tertiary and / or quaternary structure. In one aspect, the polypeptide has a molecular weight of at least about 5-20 kD, alternatively at least about 15-20 kD, preferably at least about 20 kD.

[0136] According to the present disclosure, polypeptides that can be expressed in host cells can be produced according to the present disclosure and are present in the provided compositions. The polypeptides can be expressed by genes endogenous to the host cells or introduced into the genes of the host cells by genetic engineering. The polypeptides can be naturally occurring polypeptides, or can alternatively have sequences that are artificially modified or selected. The modified polypeptides can be assembled from other polypeptide segments that exist alone in nature, or can include one or more segments that are not naturally occurring.

[0137] The polypeptides desired to be expressed according to the present invention are generally selected based on the biological or chemical activity of interest. For example, the present invention can be used to express any pharmaceutically or commercially relevant enzyme, receptor, antibody, hormone, regulatory factor, antigen, binding factor, etc.

[0138] Various polypeptides can be produced according to the methods provided herein and are present in the compositions provided herein. Examples of bacterial polypeptides include, for example, alkaline phosphatase and beta-lactamase. Examples of mammalian polypeptides include molecules such as renin, growth hormones, including human growth hormone; bovine growth hormone; growth hormone releasing factor; parathyroid hormone; thyroid stimulating hormone; lipoprotein; alpha-1-antitrypsin; insulin A-chain; insulin B-chain; proinsulin; follicle stimulating hormone; calcitonin; luteinizing hormone; glucagon; coagulation factors such as factor VIIIC, factor IX, tissue factor and von Willebrands. factors; anti-coagulant factors such as protein C; atrial natriuretic peptide; pulmonary surfactant; plasminogen activators such as urokinase or human urinary or tissue-type plasminogen activator (t-PA); bombesin; thrombin; hematopoietic growth factors; tumor necrosis factor alpha and beta; enkephalinase; RANTES (regulatory activation of normal T cells expressed and secreted); human macrophage inflammatory protein-1-alpha (MIP-1-alpha); serum albumins such as human serum albumin; Müllerian inhibitor; relaxin A-chain; relaxin B-chain; prorelaxin n); mouse gonadotropin-related peptide; microbial proteins such as β-lactamase; DNase; inhibin; activin; vascular endothelial growth factor (VEGF); hormone or growth factor receptors; integrin; protein A or D; rheumatoid factor; neurotrophic factors such as bone-derived nerve growth factor (BDNF), neurotrophins-3, -4, -5 or -6 (NT-3, NT-4, NT-5 or NT-6) or nerve growth factors such as NGF-β; platelet-derived growth factor (PDGF); fibroblast growth factor (GFGF); Factors aFGF and bFGF; epidermal growth factor (EGF); transforming growth factors (TGF) such as TGF-α and TGF-β, including TGF-β1, TGF-β2, TGF-β3, TGF-β4 or TGF-β5; insulin-like growth factor-I and -II (IGF-I and IGF-II); des(1-3)-IGF-I (brain IGF-I), insulin-like growth factor binding protein; CD proteins such as CD-3, CD-4, CD-8 and CD-19; erythropoietin ; osteoinductive factors; immunotoxins; bone morphogenetic proteins (BMPs); interferons such as interferon-α, -β and -γ; colony stimulating factors (CSFs), such as M-CSF, GM-CSF and G-CSF; interleukins (ILs), such as IL-1 to IL-10; superoxide dismutase; T-cell receptors; surface membrane proteins; decay accelerating factors; viral antigens, such as proteins of the AIDS envelope; transport proteins; homing receptors; addressing; regulatory proteins; antibodies; and fragments of any of the polypeptides listed above.

[0139] Antibodies are examples of mammalian polypeptides that can be produced according to the methods provided herein, and they can be present in the compositions provided. Antibodies are preferred polypeptides that exhibit binding specificity to a specific antigen. Natural antibodies are typically heterotetrameric glycoproteins of about 150,000 daltons, consisting of two identical light (L) chains and two identical heavy (H) chains. Each light chain is connected to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes is different. Each heavy chain and light chain also have regularly spaced intrachain disulfide bonds. Each heavy chain has a variable domain (V.sub.H) at one end, followed by several constant domains. Each light chain has a variable region (V.sub.L) at one end, and a constant region at the other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain; and the light chain variable domain is aligned with the variable domain of the heavy chain. It is believed that specific amino acid residues form the interface between the light chain and heavy chain variable domains.

[0140] Antibodies are naturally occurring immunoglobulin molecules that have different structures based on the immunoglobulin fold. For example, an IgG antibody has two "heavy" chains and two "light" chains connected by disulfide bonds to form a functional antibody. Each heavy and light chain itself contains a "constant" (C) and a "variable" (V) region. The V region determines the antigen-binding specificity of the antibody, while the C region provides structural support and plays a role in non-antigen-specific interactions with immune effectors. The antigen-binding specificity of an antibody or the antigen-binding fragment of an antibody is the ability of an antibody to specifically bind to a specific antigen.

[0141] The antigen binding specificity of the antibody can be determined by the structural features of the V region. In the variable domain covering a span of 110 amino acids, variability is not evenly distributed. On the contrary, the V region is composed of a relatively unchanged sequence called a framework region (15-30 amino acids), which is separated by a shorter region of high variability called a "hypervariable region" (each region is 9-12 amino acids long). The variable regions of natural heavy and light chains all contain four FRs connected by three hypervariable regions, which form a loop connection, and the FR mainly adopts a β-folded configuration, and in some cases forms a partial β-folded structure. The hypervariable regions in each chain are closely bound together by FRs, and together with the hypervariable regions from other chains, help form the antigen binding site of the antibody (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The constant domain region is not directly involved in the binding of antibodies to antigens, but exhibits various effector functions, such as participation in antibody-dependent cellular cytotoxicity (ADCC) of antibodies.

[0142] Each V region typically contains three complementary determining regions ("CDRs", each CDR containing a "hypervariable loop"), and four framework regions. Thus, an antibody binding site (the minimum structural unit required to bind to a specific desired antigen with substantial affinity) typically includes three CDRs and at least three, preferably four framework regions, which are interspersed between the CDRs to maintain the CDRs in a suitable configuration. Classical four-chain antibodies have an antigen binding site defined by the cooperation of VH and VL. Certain antibodies, such as camel and shark antibodies, lack light chains and rely solely on binding sites formed by heavy chains. Modified single-domain immunoglobulins can be prepared in which the binding site is formed only by heavy chains or light chains in the absence of cooperation between VH and VL.

[0143] The term "variable" refers to the fact that some parts of the variable domain in an antibody are substantially different in sequence, which is used for the binding and specificity of each specific antibody for its specific antigen. However, variability is not evenly distributed throughout the variable domain of an antibody. In both the light and heavy chain variable domains, it is concentrated in three segments called hypervariable regions. The more highly conserved parts of the variable region are called framework regions (FRs). The variable regions of both natural heavy and light chains contain four FRs connected by loops formed by three hypervariable regions, which mainly adopt a β-folded configuration and form a partial β-folded structure in some cases. The hypervariable regions in each chain are bound together in close proximity by FRs, and together with the hypervariable regions from other chains, contribute to the formation of the antigen binding site of the antibody (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The constant region is not directly involved in the binding of an antibody to an antigen, but exhibits various effector functions, such as participation in antibody-dependent cellular cytotoxicity (ADCC) of the antibody.

[0144] The term "hypervariable region" when used herein refers to the amino acid residues of an antibody which are responsible for antigen binding. The hypervariable region comprises amino acid residues from a "complementarity determining region" or "CDR" (e.g., about residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in VL, and about 31-35B (H1), 50-65 (H2), and 95-102 (H3) in VH (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)) and / or those from a "hypervariable loop" (e.g., residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in VL, and 26-32 (H1), 52A-55 (H2), and 96-101 (H3) in VH (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)).

[0145] As defined herein, "framework" or "FR" residues are those variable region residues other than the hypervariable regions.

[0146] Papain digestion of antibodies produces two identical antigen-binding fragments called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, whose name reflects its ability to crystallize readily. Pepsin treatment produces F(ab') fragments with two antigen-binding sites. 2 fragments which are still capable of cross-linking antigens.

[0147] "Fv" is the smallest antibody fragment containing complete antigen recognition and antigen binding sites. This region is composed of a dimer formed by a heavy chain variable domain and a light chain variable domain that are tightly and non-covalently bound. In this configuration, the three hypervariable regions of each variable domain interact with each other to define the antigen binding site on the surface of the VH-VL dimer. Together, the six hypervariable regions confer antigen binding specificity to the antibody. However, despite having a lower affinity than the entire binding site, even a single variable domain (or half of an Fv, i.e., only containing three hypervariable regions specific for an antigen) has the ability to recognize and bind to an antigen.

[0148] The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. The Fab' fragment differs from the Fab fragment by the addition of a few residues at the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH herein refers to Fab' in which the cysteine ​​residues of the constant domains carry at least one free thiol group. F(ab') 2 Antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0149] The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequence of their constant region.

[0150] Depending on the amino acid sequence of the constant region of the heavy chain of the antibody, antibodies can be designated as different species. There are 5 main species of complete antibodies: IgA, IgD, IgE, IgG and IgM, some of which can be further divided into subclasses (isotypes) such as IgG1, IgG2, IgG3, IgG4, IgA and IgA2. The heavy chain constant regions corresponding to different species of antibodies are called α, δ, ε, γ and μ respectively. The subunit structure and three-dimensional configuration of different types of immunoglobulins are well known.

[0151] "Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. In some embodiments, the Fv polypeptide further comprises a polypeptide connector between the VH and VL domains that enables the scFv to form the desired structure for antigen binding. For a review of scFv, see Plückthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0152] The term "diabody" refers to a small antibody fragment with two antigen binding sites, which comprises a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and generate two antigen binding sites. Diabodies are described in more detail, for example, in EP 404,097; WO 93 / 11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993).

[0153] For purposes herein, a "complete antibody" is an antibody comprising a heavy chain variable domain and a light chain variable domain and an Fc region. The constant domain can be a native sequence constant domain (e.g., a human native sequence constant domain) or an amino acid sequence variant thereof. Preferably, the complete antibody has one or more effector functions.

[0154] "Native antibodies" are usually heterotetrameric glycoproteins of about 150,000 daltons, comprising two identical light (L) chains and two identical heavy (H) chains. Each light chain is connected to the heavy chain by a covalent disulfide bond, while the number of disulfide bonds is different in the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable domain (VH) at one end, followed by several constant domains. Each light chain has a variable domain (VL) at one end, and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. It is believed that specific amino acid residues form the interface between the light chain variable domain and the heavy chain variable domain.

[0155] A "naked antibody" is an antibody (as defined herein) that is not conjugated to a heterologous molecule, such as a cytotoxic moiety or a radiolabel.

[0156] The antibody targets an antigen of interest. Preferably, the antigen is a biologically important polypeptide, and administration of the antibody to an individual suffering from a disease or condition can result in a therapeutic benefit in a mammal. However, antibodies targeting non-polypeptide antigens (e.g., tumor-associated glycolipid antibodies; see U.S. Pat. No. 5,091,178) can also be used.

[0157] Where the antigen is a polypeptide, it may be a transmembrane molecule (eg, a receptor) or a ligand such as a growth factor.Exemplary antigens include molecules such as renin, growth hormones, including human growth hormone and bovine growth hormone; growth hormone releasing factor; parathyroid hormone; thyroid stimulating hormone; lipoprotein; alpha-1-antitrypsin; insulin A-chain; insulin B-chain; proinsulin; follicle stimulating hormone; calcitonin; luteinizing hormone; glucagon; coagulation factors such as factor VIIIC, factor IX, tissue factor (TF) and von Willebrands factor; anti-coagulant factors such as protein C; atrial natriuretic peptide; pulmonary surfactant; plasminogen activators such as urokinase or human urinary or tissue-type plasminogen activator (t-PA); bombesin; thrombin; hematopoietic growth factor; tumor necrosis factor alpha and beta; enkephalinase; RANTES (regulatory activation of normal T cells expressed and secreted); human macrophage inflammatory protein (MIP-1-alpha); serum albumin such as human serum albumin; Müllerian inhibitor; relaxin A-chain; relaxin B-chain; pro-relaxin; mouse gonadotropin-related peptide; microbial proteins such as beta-lactamase; DNase; I gE; cytotoxic lymphocyte-associated antigen (CTLA), such as CTLA-4; inhibin; activin; vascular endothelial growth factor (VEGF); hormone or growth factor receptors; integrin; protein A or D; rheumatoid factor; neurotrophic factors such as bone-derived nerve growth factor (BDNF), neurotrophins-3, -4, -5 or -6 (NT-3, NT-4, NT-5 or NT-6) or nerve growth factors such as NGF-β; platelet-derived growth factor (PDGF); fibroblast growth factors aFGF and bFGF; epidermal growth factor (EGF); transferrin Transforming growth factors (TGFs) such as TGF-α and TGF-β, including TGF-β1, TGF-β2, TGF-β3, TGF-β4 or TGF-β5; insulin-like growth factors-I and -II (IGF-I and IGF-II); des(1-3)-IGF-I (brain IGF-I), insulin-like growth factor binding protein; CD proteins such as CD3, CD4, CD8, CD18, CD19, CD20 and CD40; erythropoietin; osteoinductive factors; immunotoxins; bone morphogenetic proteins (BMPs); interferons such as interferon-α, - β and -γ; colony stimulating factors (CSFs), such as M-CSF, GM-CSF and G-CSF; interleukins (ILs), such as IL-1 to IL-10; superoxide dismutase; T-cell receptors; surface membrane proteins; decay accelerating factors; viral antigens, such as proteins of the AIDS envelope; transport proteins; homing receptors; addressins; regulatory proteins; integrins such as CD11a, CD11b, CD11c, CD18, ICAM, VLA-4 and VCAM; tumor associated antigens such as HER2, HER3 or HER4 receptors; and fragments of any of the polypeptides listed above.

[0158] Preferred molecular targets of the antibodies described in detail herein include CD proteins such as CD3, CD4, CD8, CD18, CD19, CD20, CD34 and CD40; members of the ErbB receptor family such as EGF receptor, HER2, HER3 or HER4 receptor; cell adhesion molecules such as LFA-1, Mac1, p150.95, VLA-4, ICAM-1, VCAM, α4 / β7 integrin and αv / β3 integrin including their α or β subunits (such as anti-CD11a, anti-CD18 or anti-CD11b antibodies); growth factors such as VEGF; tissue factor (TF); α interferon (α-IFN); interleukins such as IL-8; IgE; blood group antigens; flk2 / flt3 receptor; obesity (OB) receptor; mpl receptor; CTLA-4; protein C, etc.

[0159] Antibodies (including fragments thereof, including antigen-binding fragments thereof) that can be produced by the methods herein include, but are not limited to, anti-HER2, antibody 2C4, anti-VEGF, antibody C2B8, anti-CD11a, anti-tissue factor, IgG4b, anti-CD40, anti-CD20, anti-IgE, E25 and E26, anti-PCSK9 and anti-β7.

[0160] c) Cell growth and polypeptide production

[0161] Typically, cells are combined (contacted) with any cell culture medium described herein under one or more conditions that promote any cell growth, maintenance and / or polypeptide production. The method for culturing cells and producing polypeptides uses a culture vessel (bioreactor) to contain cells and cell culture medium. The culture vessel can be made of any material suitable for culturing cells, including glass, plastic or metal. Typically, the culture vessel can be at least 1 liter, and can make 10, 100, 250, 500, 1000, 2500, 5000, 8000, 10,000 liters or more. During the culture process, the culture conditions can be adjusted, which include but are not limited to pH and temperature.

[0162] The cell culture is typically maintained in the initial growth phase under conditions conducive to the survival, growth and viability (maintenance) of the cell culture. The exact conditions vary depending on the cell type, the tissue from which the cells are derived, and the nature and characteristics of the expressed polypeptide.

[0163] The temperature of the cell culture during the initial growth phase can be selected primarily based on the temperature range in which the cell culture remains viable. For example, CHO cells grow well at 37° C. during the initial growth phase. In general, most mammalian cells grow well within a range of about 25° C. to 40° C. Depending on the needs of the cells and production requirements, one of ordinary skill in the art can select an appropriate temperature or temperatures for culturing cells.

[0164] In one embodiment of the invention, the temperature of the initial growth stage is maintained at a single constant temperature. In another embodiment, the temperature of the initial growth stage is maintained within a range of temperatures. For example, the temperature can be stably increased or reduced in the initial growth stage. Alternatively, the temperature can be increased or reduced by discontinuous amounts at different times in the initial growth stage. One of ordinary skill in the art can determine whether a single or multiple temperatures can be used, and whether the temperature can be adjusted stably or in discontinuous amounts.

[0165] During the initial growth phase, the cells may be cultured for a longer or shorter amount of time. In one variation, the cells are cultured for a period of time sufficient to achieve a viable cell density that is a given percentage of the maximum viable cell density that the cells would eventually achieve if allowed to grow undisturbed. For example, the cells may be cultured for a period of time sufficient to achieve a desired viable cell density of 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 99% of the maximum viable cell density.

[0166] In another embodiment, the cells are allowed to culture for a defined period of time. For example, depending on the starting concentration of the cell culture, the temperature at which the cells are cultured, and the intrinsic growth rate of the cells, the cells can be cultured for 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 days or more. In some cases, the cells are allowed to culture for a month or longer.

[0167] To increase oxygenation of the cells and dispersion of nutrients, the cell culture may be stirred or shaken during the initial growth phase. According to the present invention, it will be appreciated by those of ordinary skill in the art that during the initial growth phase, it is beneficial to control or adjust certain internal conditions of the bioreactor, including but not limited to pH, temperature, oxygenation, etc. For example, pH may be controlled by supplying an appropriate amount of acid or base, and oxygenation may be controlled using a sparging device well known in the art.

[0168] The initial culture step is a growth phase, in which batch cell culture conditions are modified to promote the growth of recombinant cells, producing a seed train. The growth phase generally refers to the exponential growth phase, in which cells generally divide rapidly, such as growing. During this phase, the cells are cultured for a period of time, generally 1 to 4 days, such as 1, 2, 3 or 4 days, and under these conditions, cell growth is optimal. For a specific host cell, the determination of the growth period of the host cell can be determined by methods known to those skilled in the art.

[0169] In the growth phase, the basal medium and cells can be supplied to the culture container in batches. In one aspect, the culture medium contains less than about 5% or less than 1% or less than 0.1% serum and other animal-derived proteins. However, serum and other animal-derived proteins can be used as needed. In a specific modification, during the HTST treatment of inactivated viruses, the basal medium has a pH of about pH 5.0 to about pH 6.9. In one aspect, before the polypeptide production phase of cell culture, during the HTST treatment of inactivated viruses, the pH of the basal medium is reduced to about pH 5.0 to about pH 6.9. On the other hand, the pH of the culture medium is then brought to about pH 6.9 to about pH 7.2 for the polypeptide production phase of cell culture. On the other hand, after the HTST treatment, the pH of the culture medium is then brought to about pH 6.9 to about pH 7.2 for the polypeptide production phase of cell culture. In another specific modification, during the HTST treatment of inactivated viruses, the total amount of phosphate and calcium contained in the basal medium is limited to less than about 10 mM. In one aspect, before the polypeptide production stage of cell culture, during the HTST treatment of inactivated viruses, the total amount of phosphate and calcium in the culture medium is limited to less than about 10mM. On the other hand, during the polypeptide production stage of cell culture, the total amount of phosphate and calcium in the culture medium is then raised to a level sufficient to produce polypeptide. In another variation, during the HTST treatment of inactivated viruses, the basal medium has a pH of about pH 5.0 to about pH 6.9 and a total amount of less than about 10mM phosphate and calcium. On the one hand, before the polypeptide production stage of cell culture, during the HTST treatment of inactivated viruses, the pH of the basal medium is reduced to about pH 5.0 to about pH 6.9, and the total amount of phosphate and calcium it comprises is less than about 10mM. On the other hand, during the polypeptide production stage of cell culture, the pH of the culture medium is then reached between about pH 6.9 and about pH 7.2, and the total amount of phosphate and calcium in the culture medium is raised to a level sufficient to produce polypeptide. In either aspect, the basal medium is a chemically defined culture medium. In either aspect, the basal medium is a chemically undefined medium. Amino acids, vitamins, trace elements and other medium components may be used at one or two times the ranges specified in European Patent EP 307,247 or US Pat. No. 6,180,401, which are incorporated herein by reference in their entirety.

[0170] Alternatively, commercially available culture media such as Ham's F10 (Sigma), Minimal Essential Medium ([MEM], Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ([DMEM], Sigma) are suitable for culturing animal cells and can be supplemented with the chemically defined culture media components detailed herein (e.g., by using the provided kits). In addition, any of the media described in Ham and Wallace, Meth. Enz., 58:44 (1979), Barnes and Sato, Anal. Biochem., 102:255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762; or 4,560,655; WO 90 / 03430; WO 87 / 00195; U.S. Pat. No. Re. 30,985; or U.S. Pat. No. 5,122,469 (all of which are incorporated herein by reference in their entirety) can be used as culture medium for the host cells, and each of the media can be supplemented with the chemically defined culture media components detailed herein (e.g., by using a provided kit).

[0171] Any of the culture media provided herein may also be supplemented as needed with hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), ions (such as sodium, chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleosides (such as adenosine and thymidine), trace elements (defined as inorganic compounds typically present in the micromolar range at final concentrations), trace metals (such as iron and copper), and glucose or an equivalent energy source. Any other necessary supplements that may also be included at appropriate concentrations are known to those of skill in the art.

[0172] At a specific point in the growth of the cells, the cells can form an inoculum to inoculate the culture medium at the start of the culture in the production phase. Alternatively, the production phase can be continuous with the growth phase. The cell growth phase is usually followed by a polypeptide production phase.

[0173] During the polypeptide production phase, the cell culture is maintained under a second set of culture conditions (compared to the growth phase) that are conducive to cell culture survival and viability and suitable for expressing the desired polypeptide. For example, during the subsequent production phase, CHO cells express recombinant polypeptides and proteins well at 25°C to 35°C. Multiple discontinuous temperature changes can be applied to increase cell density or viability or increase expression of recombinant polypeptides or proteins. As described herein, the term "polypeptide production phase" or "protein expression phase" refers to the cell culture phase in which the cell culture produces a biopharmaceutical (e.g., a polypeptide).

[0174] Cells can be maintained in the subsequent production phase until the desired cell density or product titer is reached. In one embodiment, cells are maintained in the subsequent production phase until the titer of the recombinant polypeptide reaches a maximum. In other embodiments, cultures can be harvested before this point. For example, cells can be maintained for a period of time, and the time is enough to reach 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% of the maximum viable cell density. In some cases, it is desirable to allow viable cell density to reach a maximum, then allow viable cell density to drop to a certain level before harvesting culture.

[0175] In some cases, during the subsequent polypeptide production phase, it is beneficial or necessary to supplement the cell culture with nutrients or other culture medium components that have been exhausted or metabolized by the cell. For example, during monitoring cell culture, it is advantageous to supplement the cell culture with nutrients or other culture medium components that have been exhausted as observed. In some respects, before the subsequent production phase, one or more components that are exhausted include calcium, phosphate, iron and copper. In some respects, the culture medium components supplemented include calcium, phosphate, iron and copper. Alternatively or additionally, it is beneficial or necessary to supplement the cell culture before the subsequent polypeptide production phase. In some respects, before the subsequent production phase, it is beneficial or necessary to supplement the cell culture with one or more culture medium components including calcium, phosphate, iron and copper. As non-limiting examples, it is beneficial or necessary to supplement the cell culture with hormones and / or other growth factors, specific ions (such as sodium, chlorine, calcium, magnesium and phosphate), buffers, vitamins, nucleosides or nucleotides, trace elements (inorganic compounds that are usually present at very low final concentrations), trace metals (such as iron and copper), amino acids, lipids or glucose or other energy sources.

[0176] In a specific modification, the feed medium has a pH between about pH 5.0 and about pH 6.9 during the HTST treatment of inactivated viruses. In some respects, the pH of the feed medium is then brought to a pH between about pH 6.9 and about pH 7.2 for use in the polypeptide production stage of cell culture. In some respects, after the HTST treatment, the pH of the feed medium is then brought to a pH between about pH 6.9 and about pH 7.2 for use in the polypeptide production stage of cell culture. In another specific modification, during the HTST treatment of inactivated viruses, the total amount of phosphate and calcium included in the feed medium is limited to less than about 10mM. In some respects, during the polypeptide production stage of cell culture, the total amount of phosphate and calcium in the feed medium is then raised to a level sufficient to produce polypeptide. In another modification, during the HTST treatment of inactivated viruses, the feed medium has a pH between about pH 5.0 and about pH 6.9, and the total amount of phosphate and calcium is less than about 10mM. In some aspects, during the polypeptide production phase of the cell culture, the pH of the culture medium is then brought to between about pH 6.9 and about pH 7.2, and the total amount of phosphate and calcium in the culture medium is raised to a level sufficient to produce the polypeptide. In either aspect, the feed medium is a chemically defined culture medium. In either aspect, the feed medium is a chemically undefined culture medium. One or two times the amino acids, vitamins, trace elements, and other culture medium components of the specified ranges in European Patent EP 307,247 or U.S. Patent No. 6,180,401 can be used, and the documents are incorporated herein by reference in their entirety.

[0177] D. Test kit

[0178] Kits for supplementing cell culture media with chemically defined components are described. The kit may contain dry components to be reconstituted, and may also contain instructions for use (e.g., for use in supplementing culture media with kit components). The kit may contain culture media components provided herein in amounts suitable for supplementing cell culture media. In one variation, the kit comprises the culture media components of Table 1. In another variation, the kit comprises culture media components for adjusting the pH of the culture media to the pH levels disclosed in Table 1.

[0179] E. Composition

[0180] Also provided are compositions comprising cell culture media and one or more other components, such as cells or desired polypeptides (e.g., antibodies). In one variation, the composition provided comprises: (a) cells comprising isolated nucleic acids encoding polypeptides; and (b) cell culture media as provided herein. In another variation, the composition provided comprises: (a) polypeptides; and (b) cell culture media as provided herein, wherein in one aspect, the polypeptides are secreted into the culture media by cells comprising isolated nucleic acids encoding polypeptides. In another variation, the composition provided comprises: (a) polypeptides; and (b) cell culture media as provided herein, wherein in one aspect, the polypeptides are released into the culture media by lysing cells comprising isolated nucleic acids encoding polypeptides. The cells of the composition can be any of the cells described in detail herein (e.g., CHO cells), and the culture media of the composition can be any of the culture media described in detail herein, such as culture media comprising the culture media components described in detail in Table 1. Similarly, the polypeptide of the composition can be any of the polypeptides described in detail herein, such as antibodies.

[0181] F. Systems for Virus Inactivation

[0182] The present invention contemplates a virus inactivation system and / or process for a cell culture medium. The system may include, but is not limited to, a culture medium, a media containment unit, a pH meter (or another means for measuring pH), a means for measuring and / or quantifying calcium and / or phosphate concentration and / or amount; a means for transferring the culture medium (e.g., a piping system), a heat source for increasing the temperature of the culture medium, a means for adjusting a target set point (e.g., temperature), a holding containment unit (e.g., a holding tube), a cold source for reducing the temperature of the culture medium, an air supply, a means for pressure input and output (e.g., a pressure valve, a peristaltic pump, a centrifugal pump, a positive displacement pump), a means for culture medium input and output, a means for gas input and output, a means for filtering, a means for adjusting flow rate and other aspects related to flow dynamics, and a means optionally connected to a bioreactor for producing a desired polypeptide or cell culture or cell culture medium.

[0183] The virus inactivation system comprising the above elements may also include a system for heat treatment. Figure 1 An exemplary heat treatment system that can use various parameters described herein (e.g., pH, calcium and / or phosphate concentration) is shown in . Such a system described herein can be used to implement a method of inactivating viruses in a cell culture medium so that the culture medium can be used to produce a variety of end products (e.g., polypeptides, antibodies, etc.).

[0184] G. Exemplary Embodiments

[0185] In one aspect, the present invention provides a method for inactivating viruses or exogenous factors in a culture medium while maintaining the culture medium's suitability for cell culture, the method comprising (a) subjecting the cell culture medium to high temperature transient (HTST) treatment; and (b) adjusting one or more parameters selected from the group consisting of pH, calcium level, and phosphate level.

[0186] In any of the above embodiments, the method further comprises adjusting the concentration of the trace metal.

[0187] In any of the above embodiments, the trace metal is selected from iron and copper.

[0188] In any of the above embodiments, the concentration of iron and / or copper in the culture medium is adjusted prior to HTST treatment.

[0189] In any of the above embodiments, iron and / or copper are removed from the culture medium prior to HTST treatment.

[0190] In any of the above embodiments, the method further comprises supplementing iron and / or copper in the culture medium to a level suitable for cell culture after the HTST treatment.

[0191] In any of the above embodiments, when the culture medium comprises calcium and phosphate, the pH is adjusted.

[0192] In any of the above embodiments, the pH is adjusted to a suitable low level when preparing the culture medium prior to HTST treatment.

[0193] In any of the above embodiments, the pH is adjusted by lowering the pH to a suitable level.

[0194] In any of the above embodiments, the pH is adjusted to less than about 7.2.

[0195] In any of the above embodiments, the pH is adjusted to about 5.0-7.2.

[0196] In any of the above embodiments, the method further comprises adjusting the pH to a level suitable for cell culture after the HTST treatment.

[0197] In any of the above embodiments, the pH is adjusted to about 6.9-7.2.

[0198] In any of the above embodiments, when the culture medium comprises phosphate, the calcium level is adjusted.

[0199] In any of the above embodiments, calcium levels are reduced.

[0200] In any of the above embodiments, calcium levels are reduced such that formation of complexes comprising calcium and phosphate is inhibited.

[0201] In any of the above embodiments, calcium is removed from the culture medium prior to HTST treatment.

[0202] In any of the above embodiments, the pH is adjusted such that the formation of complexes comprising calcium and phosphate is inhibited.

[0203] In any of the above embodiments, the method further comprises adjusting the calcium level to a level suitable for cell culture after the HTST treatment.

[0204] In any of the above embodiments, when the culture medium comprises calcium, the phosphate level is adjusted.

[0205] In any of the above embodiments, phosphate levels are reduced.

[0206] In any of the above embodiments, phosphate levels are reduced such that formation of complexes comprising calcium and phosphate is inhibited.

[0207] In any of the above embodiments, phosphate is removed from the culture medium prior to HTST treatment.

[0208] In any of the above embodiments, the pH is adjusted such that the formation of complexes comprising calcium and phosphate is inhibited.

[0209] In any of the above embodiments, the method further comprises adjusting the phosphate level to a level suitable for cell culture after the HTST treatment.

[0210] In any of the above embodiments, during the HTST treatment, the total concentration of phosphate and calcium in the culture medium is less than about 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 mM.

[0211] In any of the above embodiments, calcium and phosphate levels are modulated.

[0212] In any of the above embodiments, pH, calcium and phosphate levels are adjusted.

[0213] On the other hand, the present invention provides a method for inactivating viruses or exogenous factors in a cell culture medium while maintaining the medium for cell culture suitability, the method comprising subjecting the cell culture medium to high temperature transient (HTST) treatment, wherein before and / or during the HTST treatment, the medium has a pH between about pH 5.0 and about pH 7.2. In some embodiments, before and / or during the HTST treatment, the pH is between about pH 5.0 and about pH 6.9, between about pH 5.3 and about pH 6.3, or between about pH 6.9 and about pH 7.2. In some embodiments, the method further comprises adjusting the pH to a level suitable for cell culture after the HTST treatment. In some embodiments, the culture medium comprises calcium and phosphate. In some embodiments, before the HTST treatment, the concentration of trace metals (e.g., iron and / or copper) in the culture medium is adjusted. In some embodiments, before and / or during the HTST treatment, the culture medium does not contain iron and / or copper. In some embodiments, the culture medium further comprises supplementing iron and / or copper to a level suitable for cell culture in the culture medium after the HTST treatment.

[0214] On the other hand, the present invention provides a method for inactivating viruses or exogenous factors in a cell culture medium while maintaining the medium for cell culture suitability, the method comprising subjecting the cell culture medium to high temperature transient (HTST) treatment, wherein the total amount of phosphate and calcium in the culture medium is less than about 10 mM before and / or during the HTST treatment. In some embodiments, the total amount of phosphate and calcium in the culture medium is less than about 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 mM before and / or during the HTST treatment. In some embodiments, the culture medium does not contain phosphate before and / or during the HTST treatment. In some embodiments, the culture medium does not contain calcium before and / or during the HTST treatment. In some embodiments, the method further comprises adjusting the phosphate and / or calcium levels to a level suitable for cell culture after the HTST treatment. In some embodiments, before the HTST treatment, the concentration of trace metals (e.g., iron and / or copper) in the culture medium is adjusted. In some embodiments, before the HTST treatment, the culture medium does not contain iron and / or copper. In some embodiments, the method further comprises supplementing the culture medium with iron and / or copper to a level suitable for cell culture after the HTST treatment.

[0215] In any of the above embodiments, precipitate formation is inhibited.

[0216] In any of the above embodiments, fouling of equipment used for HTST processing is reduced.

[0217] In any of the above embodiments, filter fouling is inhibited.

[0218] In any of the above embodiments, the HTST treatment comprises raising the temperature of the culture medium to at least about 85°C for a time sufficient to inactivate viruses or exogenous factors in the culture medium.

[0219] In any of the above embodiments, the temperature of the culture medium is raised to at least about 93, 95, 97, 99, 101, 102, or 103° C. for a time sufficient to inactivate viruses in the culture medium.

[0220] In any of the above embodiments, the temperature is increased and maintained for at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 seconds.

[0221] In any of the above embodiments, the virus is selected from the group consisting of Parvoviridae, paramyoxviradae, Orthomyxoviridae, Bunyaviridae, Rhabdoviridae, Reoviridae, Togaviridae, Caliciviridae, and Picornaviridae.

[0222] In any of the above embodiments, the virus is an enveloped virus.

[0223] In any of the above embodiments, the virus is a non-enveloped virus.

[0224] In any of the above embodiments, the exogenous agent is a bacterium.

[0225] All features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced with an alternative feature having the same, equivalent or similar purpose. Therefore, unless otherwise expressly indicated, each feature disclosed is only an example of a series of equivalent or similar features.

[0226] The following examples are provided to illustrate but not to limit the present invention.

[0227] All references disclosed herein are hereby incorporated by reference in their entirety for all purposes.

[0228] Example

[0229] Viral infectious agents that contaminate cell culture processes pose a threat to the production of biopharmaceuticals (e.g., recombinant proteins) from cell lines and raise concerns about the potential safety of the ability to remove viral infectious agents during the purification of the product. Several approaches have been taken to minimize the risk of exogenous factors entering the production process, including the use of high temperature transient (HTST) treatment of cell culture media. However, although viruses can be effectively inactivated when properly operated, a given culture medium may be incompatible with the HTST treatment for viral particle inactivation (Schleh, M. et al. 2009. Biotechnol. Prog. 25 (3): 854-860 and Kiss, R. 2011. PDA J Pharm Sci and Tech. 65: 715-729). Culture medium incompatibility can lead to scaling of processing contact surfaces in HTST equipment. One cause of scaling is the precipitation of culture medium caused by the heating and cooling operations of the HTST treatment. Precipitation leads to residual deposition, which scales the heat exchanger and causes the HTST skid to shut down because the system cannot maintain the temperature set point. In addition, precipitation in a culture medium that is incompatible with heat treatment can cause scaling of processing filters that are used to remove bacteria that are not inactivated by HTST treatment in the culture medium. Filter scaling due to precipitation can cause processing failures and significantly increase filtration costs. In addition, precipitation in a culture medium that is incompatible with heat treatment can adversely affect the performance of the cell culture (e.g., product titer, cell growth, cell activity), as well as product quality.

[0230] As described herein, several improvements of culture medium have been confirmed so that the culture medium can be compatible for HTST treatment or other heat treatments for inactivating viruses. Several culture medium preparations have been confirmed, which can reduce or prevent precipitation on equipment for HTST treatment of inactivating viruses. The method for inactivating viruses in culture medium provided herein is described as a method for reducing the precipitate on equipment for HTST treatment of inactivating viruses. In one aspect, during the HTST treatment for virus inactivation, the culture medium can have a pH between about pH 5.0 and about pH 6.9. On the other hand, before the protein expression stage of cell culture, during the HTST treatment for virus inactivation, the culture medium has a pH between about pH 5.0 and about pH 6.9. On the other hand, the culture medium is made to reach a pH between about pH 6.9 and about pH 7.2 for the protein expression stage of cell culture. In one aspect, during the HTST treatment for virus inactivation, the total amount of phosphate and calcium contained in the culture medium is limited to less than about 10mM. On the other hand, before the protein expression stage of cell culture, during the HTST treatment for virus inactivation, the total amount of phosphate and calcium contained in the culture medium is limited to less than about 10mM. On the other hand, during the protein expression stage of cell culture, the total amount of phosphate and calcium that the culture medium has is raised to a level sufficient for protein expression. In either aspect, during the HTST treatment for virus inactivation, the cell culture medium can have a pH between about pH 5.0 and about pH 6.9, and contain a total amount of phosphate and calcium less than about 10mM. It is found that the culture medium can be used for all stages of cell culture, and can be used for basal medium and / or feed medium. It is found that the culture medium can be used to reduce the precipitate on the equipment for HTST treatment for inactivation of viruses. It is also contemplated that a kit for supplementing the cell culture medium with components of known chemical composition is used.

[0231] Example 1: Validation of the sand bath screening method for reproducible observation of HTST skid operation.

[0232] During production-scale HTST processing, a liquid preparation of cell culture medium is heated to 102°C for 10 seconds (100°C) in a continuous flow process using two heat exchangers (one for heating the liquid and one for cooling the liquid with tubing between the two heat exchangers to provide the desired hold time for a given flow rate). Figure 1 The sand bath screening method was used to more rapidly test the performance of media compositions with laboratory-scale (~20 mL) media volumes after heat treatment. The method was based on a "worst-case" heat explosure to screen and identify compatible media for pilot-scale and production-scale HTST skids (Table 2).

[0233] Table 2. Key differences between the sand bath apparatus and the HTST skid at two scales

[0234]

[0235] Materials and methods

[0236] Medium preparation

[0237] The culture medium used in this study includes the basic production culture medium and batch feed culture medium (table 3) with different pH levels. The purified deionized water processed by Millipore SuperQ ultrapure water purification system is used to prepare all culture mediums. Suitable culture medium powder reserves (SAFC and Life Technologies) are used to prepare culture medium. Glass electrode pH probe (Mettler Toledo) and osmometer (Advanced Instruments) are used during liquid preparation to ensure the target pH and permeability of a given preparation. After the component is completely dissolved and the final pH and permeability are adjusted, the culture medium is filtered into a bottle of 250mL to 1L (Corning) for small-scale preparation using a 0.1 μm pore size PES membrane filter.

[0238] Table 3. Medium formulations used to test heat treatment stability

[0239]

[0240] pH Adjustment

[0241] Correction was made for pH deviations due to degassing during the time between completion of the media preparation and application of the heat treatment. Prior to heat treatment, a 30 mL aliquot from each media preparation was transferred to a 50 mL test tube (Falcon) and the original Falcon cap was replaced with a vented cap from a 250 mL Corning Erlenmeyer flask. The test tube was then placed in a humidified atmosphere with CO 2 30 min in an incubator to lower the pH (for pH 6.2 samples, 15% CO 2 , and for all other samples, 12% CO 2 , 200 rpm, 37°C); this step can force the pH below the target. The tube was then manually shaken and the pH was checked using a glass electrode pH probe and meter (Mettler Toledo) until the pH slowly returned to the target pH. The final pH measurement was performed using a NOVAbioprofiler.

[0242] Sand Bath

[0243] For sand bath method, the liquid culture medium prepared by 22mL is transferred in 20mL glass pressure vessel (Aceglassware).Use the threaded cap sealed container with thermocouple sleeve, by filling the container and allowing bottle cap and thermocouple sleeve to discharge too much culture medium, so that head space will not leave air in the solution.Clean the outside of the container, to prevent the fouling of the culture medium outer surface directly exposed to the pyrogen matrix.Teflon tape is used to cover the interface between the bottle mouth and the threaded cap of glass container, to better seal the glass pressure vessel, and prevent the oil of sand or thermocouple hole from entering the sample for heat treatment.Configuration has the fluidized sand bath (Techne SBS-4) (compressed air inlet pressure=5psig, bath temperature=110 ℃) of temperature controller (Techne TC-8D), and given 30 minutes balance.The thermocouple combined with a single VWR digital thermometer is inserted into the sample container thermocouple sleeve at the center of the radial dimension that is geometrically distributed in test tube. Silicone oil was added to the thermocouple hole to provide a heat transfer medium between the thermocouple hole glass wall and the thermocouple. The sample container was placed in a sand bath and the timing was started. The temperature dynamics data was recorded approximately every 30-60 seconds. Once the container reached 102°C on the thermometer reading, it was maintained in the sand bath for 10 seconds. After the heating and holding steps, the container was transferred to a water bath at room temperature until the thermometer temperature reading reached 35°C. After heat treatment, 15 mL of each sample was transferred to a vial for turbidity and visual measurement.

[0244] Sedimentation measurement

[0245] The sediment of the culture medium samples was analyzed before and after heat treatment by the following two methods: 1) turbidity was measured by nephelometer (2100Q Hach); and 2) the samples were centrifuged at 10,000 × g for 10 minutes (Sorvall RC 6plus, SS-34 rotor) in 50-mL Falcon tubes to sediment the sediment, visually identify and qualitatively determine the sediment based on particle size (e.g., invisible, low, medium, high). For visual identification of the sediment, the uncentrifuged samples were also analyzed.

[0246] result

[0247] Two main findings were obtained from the sand bath and HTST heating profiles: 1) the heating profile of the sand bath thermal treatment system was significantly longer than the heating profile of the large-scale HTST skid operation and 2) the sand bath was able to reach the target set point of 102°C in approximately 6.5 minutes ( Figure 2). The sand bath heating profile is typically 5 to 8 minutes with an average time of ~6 to ~6.5 minutes for heating from room temperature (~21-25°C) or 37°C to a target of 102°C and holding for 10 seconds before cooling the sample in a water bath. The areas under the curves for heating, holding, and cooling are significantly larger in the sand bath method relative to a production scale or pilot scale continuous flow HTST system. Media samples measured in the sand bath method are worst-case scenarios for all heating relative to pilot scale and production scale HTST operations. Therefore, any significant changes in the components of a given media formulation that are primarily driven by heat in the sand bath method can provide relevant data for larger scale HTST processes.

[0248] It was observed that lowering the pH of the Medium 1 feed medium formulation from pH 7.0 to pH 6.4 during HTST processing could reduce any HTST operating issues (Table 3). Similarly, for the Medium 2 basal medium formulation, it was found that treating the medium at pH 6.7 rather than at pH 7.0 could also reduce any HTST operating issues (Table 3). In the sand bath method, each of these media was treated at two pH levels to determine whether the sand bath method could accurately reflect the precipitation behavior that occurs in a scaled HTST processing operation. Visibility measurements before and after heat treatment showed that the sand bath system accurately displayed the precipitation behavior ( Figure 3 A and B), this precipitation behavior is caused by heat treatment of a medium known to have HTST-compatibility issues when treated at neutral pH. Consistent with a "worst case" for medium changes based on heat exposure, Medium 2 still showed signs of precipitation in the sand bath, although Medium 2 had significantly reduced precipitation relative to the pH 7.0 treated sample of the same medium. Overall, these results demonstrate that the sand bath method can be used to screen and identify medium formulations that are compatible for use in pilot-scale and production-scale HTST treatments.

[0249] Example 2: pH, calcium and phosphate levels promote precipitation in culture medium during heat treatment for viral inactivation Materials and methods

[0250] Medium preparation

[0251] The culture medium used in this research includes the basic production culture medium and batch feed culture medium with the pH level of about pH 5.9 to about pH 7.5, the calcium concentration of about 0mM to about 3.5mM (or higher in the culture medium of unknown composition), and the phosphate concentration of about 0mM to about 6.5mM (or higher in the culture medium of unknown composition). The deionized water of the purification processed by MilliporeSuperQ ultrapure water purification system is used to prepare all culture mediums. Suitable culture medium powder reserve (SAFC and Life Technologies) is used to prepare culture medium. Glass electrode pH probe (Mettler Toledo) and osmometer (Advanced Instruments) are used during liquid preparation to ensure the target pH and permeability of given preparation. After component is completely dissolved and final pH and permeability are adjusted, 0.1 μm pore size PES membrane filter is used to filter culture medium into the bottle of 250mL to 1L (Corning) for small-scale preparation.

[0252] pH Adjustment

[0253] Correction was made for pH deviations due to degassing during the time between completion of the media preparation and application of the heat treatment. Prior to heat treatment, a 30 mL aliquot from each media preparation was transferred to a 50 mL test tube (Falcon) and the original Falcon cap was replaced with a vented cap from a 250 mL Corning Erlenmeyer flask. The test tube was then placed in a humidified atmosphere with CO 2 30 min in an incubator to lower the pH (for pH 6.2 samples, 15% CO 2 , and for all other samples, 12% CO 2 , 200 rpm, 37°C); this step can force the pH below the target. The tube was then manually shaken and the pH was checked using a glass electrode pH probe and meter (Mettler Toledo) until the pH slowly returned to the target pH. The final pH measurement was performed using a NOVAbioprofiler.

[0254] Sand Bath

[0255] For sand bath method, the liquid culture medium prepared by 22mL is transferred in 20mL glass pressure vessel (Aceglassware).Use the threaded cap sealed container with thermocouple sleeve, by filling the container and allowing bottle cap and thermocouple sleeve to discharge too much culture medium, so that head space will not leave air in the solution.Clean the outside of the container, to prevent the fouling of the culture medium outer surface directly exposed to the pyrogen matrix.Teflon tape is used to cover the interface between the bottle mouth and the threaded cap of glass container, to better seal the glass pressure vessel, and prevent the oil of sand or thermocouple hole from entering the sample for heat treatment.Configuration has the fluidized sand bath (Techne SBS-4) (compressed air inlet pressure=5psig, bath temperature=110 ℃) of temperature controller (Techne TC-8D), and given 30 minutes balance.The thermocouple combined with a single VWR digital thermometer is inserted into the sample container thermocouple sleeve at the center of the radial dimension that is geometrically distributed in test tube. Silicone oil was added to the thermocouple hole to provide a heat transfer medium between the thermocouple hole glass wall and the thermocouple. The sample container was placed in a sand bath and the timing was started. The temperature dynamics data was recorded approximately every 30-60 seconds. Once the container reached 102°C on the thermometer reading, it was maintained in the sand bath for 10 seconds. After the heating and holding steps, the container was transferred to a water bath at room temperature until the thermometer temperature reading reached 35°C. After heat treatment, 15 mL of each sample was transferred to a vial for turbidity and visual measurement.

[0256] Sedimentation measurement

[0257] The sediment of the culture medium samples was analyzed before and after heat treatment by the following two methods: 1) turbidity was measured by nephelometer (2100Q Hach); and 2) the samples were centrifuged at 10,000 × g for 10 minutes (Sorvall RC 6plus, SS-34 rotor) in 50-mL Falcon tubes to sediment the sediment, visually identify and qualitatively determine the sediment based on particle size (e.g., invisible, low, medium, high). For visual identification of the sediment, the uncentrifuged samples were also analyzed.

[0258] result

[0259] Several media formulations were prepared using different pH levels and calcium and phosphate concentrations (Table 4).Precipitation of media prepared before and after heat treatment was assessed by visual inspection of uncentrifuged and centrifuged samples and by turbidity measurements.

[0260] Table 4. Precipitation of test media formulations in the sand bath method

[0261]

[0262]

[0263] The effect of hydrolysate / peptone was not included in the estimation of calcium and phosphate concentrations.

[0264] N / A means not available.

[0265] The measurement results of precipitation in the samples show that the data obtained by the sand bath method reflect the possible situation of overall heating relative to the HTST processing operation, because the medium 4 and medium 5 preparations that did not show operating problems in the pilot or production scale HTST before show measurable turbidity and visible precipitation in the sand bath method. By the sand bath method, it is such components in the medium preparation to measure calcium and phosphate concentrations and pH levels, which contribute to the significant changes in the medium during the heating period of at least 102°C. In several preparations, reducing the pH level, but not modifying the calcium and phosphate concentrations leads to lower turbidity (Table 4). In addition, there is no calcium or the preparation with low calcium concentration will not show precipitation events even at neutral pH, and there is no measurement result of high turbidity (Table 4). Except reducing calcium and phosphate concentrations, there is a correlation between reducing precipitation and reducing the ratio of calcium to phosphate (Table 4). These observations have led to such judgments that the calcium phosphate precipitation formed by heating and cooling during heat treatment is a function of calcium concentration, phosphate concentration and pH level.

[0266] For multivariate analysis of heat treated formulations in a sand bath system, medium 4 was selected for the full factorial design of experiments (DoE) and the turbidity (NTU) response metric was used to generate a precipitation response surface. The variables that could be varied were calcium concentration (0.1 to 2.9 mM), phosphate concentration (0.1, 5.9 mM), and pH (6.0 to 7.2). The pH-dependent calcium phosphate formation that resulted in precipitation in the medium following heat treatment of the calcium and phosphate containing medium formulations was determined by turbidity measurements and confirmed by visual observations. Figure 4 The strongest effect on medium precipitation is the vector product of calcium and phosphate concentrations ( Figure 4 ). This is consistent with the expected reaction kinetics: insoluble calcium phosphate formation depends on calcium and phosphate concentrations. In addition, the turbidity estimated by the product of calcium and phosphate concentrations is also consistent with the turbidity observed when the concentration of calcium or phosphate is zero, with no significant increase in turbidity (as an estimate of HTST compatibility).

[0267] The response surface was then modeled based on the DoE turbidity data and input data (calcium concentration, phosphate concentration, and pH). Relative to the visible precipitation by visual inspection of centrifugal and non-centrifugal heat-treated culture medium samples, the cutoff value for the good running scheme relative to the bad running scheme in the worst-case sand bath heat treatment was selected as a turbidity of 5NTU based on the analysis of all turbidity values. The data show that for ensuring HTST compatibility, multiple options or levels for improving culture medium formulations are possible. In particular, reducing calcium concentration, phosphate concentration, pH, or some combinations of the three parameters can be possible means for preparing culture medium formulations that are compatible with HTST processing or other heat treatment methods (Fig. 5).

[0268] The stability of the media in the sand bath heat treatment study was particularly dependent on calcium and phosphate concentrations and pH levels, according to the modeled response surface generated from the data for heat treated Medium 4. To determine modifications in other media formulations that could transform the media formulation from HTST incompatible to compatible media, other media formulations without hydrolysate (Medium 3, Medium 4, Medium 6, Medium 7, Medium 8, Medium 9, Medium 11, Medium 12, and Medium 13) were plotted on the modeled response surface based on the calcium and phosphate concentrations of the media formulation at pH 7.0. Figure 6 ). The media containing the hydrolyzate added complexity to the analysis due to the different levels of calcium and phosphate. Medium 11 fell within the region of the response surface, indicating that it was outside the precipitation range, thus confirming that medium 11 was likely HTST-compatible ( Figure 6 , point C). Addition of hydrolysate to medium 11 produced medium 5 (Table 4), and based on known estimates of phosphate and calcium levels in the hydrolysate, addition of this hydrolysate caused medium 5 to shift into the precipitation range and thus was likely HTST-incompatible ( Figure 6 , point C arrow). Medium 12 is in the precipitation range, and based on the model, Medium 2 produced by the addition of hydrolysate, which was added (Table 4), is predicted to shift Medium 2 further into the precipitation range ( Figure 6 , dot D arrow). Based on the pattern response surface associated with visible precipitation after sand bath heat treatment, formulations predicted to be within or outside the precipitation range included two formulations that had heat exchanger fouling issues in production scale HTST skid runs. Use of the generated response surface model indicated that precipitation in the cell culture medium formulations after heat treatment was strongly associated with high concentrations of calcium and phosphate near neutral pH (Figures 5 and 6). In addition, a response surface model based on the sand bath data can be generated to provide recommendations for formulation changes for converting HTST-incompatible medium to HTST-compatible medium.

[0269] Example 3: Effect of temperature on the precipitation behavior of culture medium during virus inactivation

[0270] Materials and methods

[0271] Medium preparation

[0272] The culture medium used in this study includes basic production culture medium and batch feed culture medium. All culture mediums are prepared using purified deionized water processed by Millipore SuperQ ultrapure water purification system. Suitable culture medium powder reserves (SAFC and Life Technologies) are used to prepare culture medium. Glass electrode pH probe (Mettler Toledo) and osmometer are used during liquid preparation to ensure target pH and permeability of given preparation. After component is fully dissolved and final pH and permeability are adjusted, 0.1 μm pore size PES membrane filter is used to filter culture medium into 250mL to 1L (Corning) bottle for small-scale preparation.

[0273] pH Adjustment

[0274] Correction was made for pH deviations due to degassing during the time between completion of the media preparation and application of the heat treatment. Prior to heat treatment, a 30 mL aliquot from each media preparation was transferred to a 50 mL test tube (Falcon) and the original Falcon cap was replaced with a vented cap from a 250 mL Corning Erlenmeyer flask. The test tube was then placed in a humidified atmosphere with CO 2 30 min in an incubator to lower the pH (for pH 6.2 samples, 15% CO 2 , and for all other samples, 12% CO 2 , 200 rpm, 37°C); this step can force the pH below the target. The tube was then manually shaken and the pH was checked using a glass electrode pH probe and meter (Mettler Toledo) until the pH slowly returned to the target pH. The final pH measurement was performed using a NOVAbioprofiler.

[0275] Sand Bath

[0276] For sand bath method, the liquid culture medium prepared by 22mL is transferred in 20mL glass pressure vessel (Aceglassware).Use the threaded cap sealed container with thermocouple sleeve, by filling the container and allowing bottle cap and thermocouple sleeve to discharge too much culture medium, so that head space will not leave air in the solution.Clean the outside of the container, to prevent the fouling of the culture medium outer surface directly exposed to the pyrogen matrix.Teflon tape is used to cover the interface between the bottle mouth and the threaded cap of glass container, to better seal the glass pressure vessel, and prevent the oil of sand or thermocouple hole from entering the sample for heat treatment.Configuration has the fluidized sand bath (Techne SBS-4) (compressed air inlet pressure=5psig, bath temperature=110 ℃) of temperature controller (Techne TC-8D), and given 30 minutes balance.The thermocouple combined with a single VWR digital thermometer is inserted into the sample container thermocouple sleeve at the center of the radial dimension that is geometrically distributed in test tube. Silicone oil was added to the thermocouple hole to provide a heat transfer medium between the thermocouple hole glass wall and the thermocouple. The sample container was placed in a sand bath and the timing was started. The temperature dynamics data was recorded approximately every 30-60 seconds. Once the container reached 102°C on the thermometer reading, it was maintained in the sand bath for 10 seconds. After the heating and holding steps, the container was transferred to a water bath at room temperature until the thermometer temperature reading reached 35°C. After heat treatment, 15 mL of each sample was transferred to a vial for turbidity and visual measurement.

[0277] Sedimentation measurement

[0278] The sediment of the culture medium samples was analyzed before and after heat treatment by the following two methods: 1) turbidity was measured by nephelometer (2100Q Hach); and 2) the samples were centrifuged at 10,000 × g for 10 minutes (Sorvall RC 6plus, SS-34 rotor) in 50-mL Falcon tubes to sediment the sediment, visually identify and qualitatively determine the sediment based on particle size (e.g., invisible, low, medium, high). For visual identification of the sediment, the uncentrifuged samples were also analyzed.

[0279] result

[0280] The effect of temperature on media precipitation was also investigated by varying the target set point for heating in the sand bath system. Measurements were made at temperatures of approximately 75, 85, 90, 97, and 102°C for Medium 1 (feed medium, unidentified composition), Medium 2 (basal medium, unidentified composition), Medium 4 (basal medium, identified composition), and Medium 10 (basal medium, unidentified composition), all formulated to a neutral pH of 7.0. Visual inspection of the uncentrifuged and centrifuged samples along with the heating curves showed that all four different media formulations produced precipitation events when the samples reached the 90°C time ( Figure 7 , solid circles). At lower temperatures of 85°C and 75°C, media 4 and 10 continued to show precipitation events ( Figure 7 , solid circles). Medium 2 showed precipitation events during heat treatment at 85°C, but produced no visible precipitate at 75°C, indicating that Medium 2 is compatible with heat treatment at temperatures around or below 75°C ( Figure 7 , open circles). No precipitation was produced in medium 1 at 85°C, indicating that medium 1 is compatible with heat treatment at temperatures around 85°C or below ( Figure 7 , open circles). These observations were confirmed by turbidity measurements showing that all four different media formulations had turbidity measurements of approximately 20 NTU or higher when the samples reached 90°C ( Figure 8 For medium 2, 4 and 10, the turbidity values ​​of the heat-treated medium decreased when the temperature was increased from 97°C to 102°C ( Figure 8 ). In solutions such as culture media, turbidity is a function of particle size distribution, and in particular, a specific range of colloidal particle sizes is more effective in measurement than other parts of the size distribution. Therefore, the reduction in turbidity measured at the highest temperature is due to particle flocculation / aggregation behavior, which results in a change in particle size distribution (e.g., increasing particle size but reducing number), which results in artifacts in the turbidity data. These results indicate that slightly lowering the temperature while still maintaining viral inactivation does not significantly reduce turbidity.

[0281] For large-scale HTST operations, temperatures in excess of 85°C to 90°C for relevant durations are required for an effective method of inactivating viruses, and for industrially relevant parvoviruses, temperatures in excess of 95°C are necessary to achieve the desired log reduction. Although the sand bath method is more stringent for observing heat treatment-derived media precipitation events, data indicate that operations below the current target HTST process set point of 102°C can be used to avoid precipitation events that lead to heat exchanger fouling. Because the purpose of the HTST process is viral inactivation, it is clear that lowering the target temperature set point is not an option, and precipitation of several media formulations is also a potential operational issue for media HTST applications. Therefore, during viral inactivation in media by HTST treatment at temperatures of at least 90°C, calcium and phosphate concentrations and pH levels are components that can be adjusted to reduce or prevent precipitation events.

[0282] Example 4: Contribution of pH, calcium and phosphate levels to precipitation in culture medium during pilot-scale and large-scale HTST medium treatment for viral inactivation

[0283] Materials and methods

[0284] Pilot scale HTST

[0285] For studies using pilot-scale HTST, media formulations were processed from the lowest to the highest concentration of calcium or phosphate to minimize possible carry-over to subsequent runs. Each HTST run required 15L to 20L of media to flush the deionized rinse used between runs and to equilibrate the heating coils to an operating temperature of 102°C (acceptable range: 97°C-110°C) for HTST processing. After equilibration, approximately 20L of media was run through the HTST skid and the outlet flow was collected in a plastic cube container. Samples were collected from media from the media mixing tank before HTST processing and from the media flowing out of the cube container and filtered through a 0.1μm PVDF capsule membrane filter (Millipore) into a media storage bag for use as "before HTST" and "after HTST" samples. All treated and untreated filtered media samples were then stored at 2-8°C prior to cell culture performance assays and analytical testing.

[0286] Production scaleHTST

[0287] For media heat treatment with production-scale HTST skid, 1800 L of Medium 4 or Medium 1 preparation was used for production-scale operational runs. The purpose of this media preparation was to determine: 1) whether the production mix conditions for Medium 4 were adequate to meet specific quality control recipe mix times, and 2) to generate production-scale HTST process performance data using Medium 4. Media samples were collected before and after HTST treatment by aseptically connecting a 6×20-L media bag manifold (Sartorius Stedim Biotech) to the media mix tank and sampling ports on the target bioreactor. For samples taken before HTST treatment, the media was filtered through a 0.1 μm PVDF capsule filter (Millipore) before collection, and for samples taken after HTST treatment, the media was passed through a production filter train consisting of a 0.5 / 0.2 μm double-layer cartridge filter PVDF prefilter (Millipore) and a 0.1 μm Nylon final filter (Pall) before collection. The samples were then used for cell culture performance determinations and analytical testing.

[0288] result

[0289] For pilot-scale and production-scale HTST operations, there were no in-process events that caused the HTST skid to shut down or difficulties in controlling the output temperature when the desired volume of medium 4 was processed. Therefore, there was no indication of significant precipitation events sufficient to cause fouling of heat exchanger surfaces or subsequent filtration. Medium 4 processed at pilot scale was visually free of particles before final filtration and use for cell culture. Due to the arrangement of the system sampling ports, it was not possible to sample the medium 4 processed at production scale before final filtration. Analytical testing and cell culture performance testing were performed on medium 4 with and without HTST treatment. Analytical testing of medium 4 showed that trace metal losses occurred after HTST treatment, indicating that during the HTST treatment, precipitation events occurred that changed the concentration of the medium composition, which was not sufficient precipitation to cause operational difficulties. For production-scale HTST treatment of medium 4, data from inductively coupled plasma mass spectrometry (ICP-MS) and inductively coupled plasma-optical emission spectroscopy (ICP-OES) measurements showed that 24% of Fe (iron) and 16% of Cu (copper) were lost after heat treatment relative to medium 4 that was not heat treated ( Fig. 9 ).

[0290] For pilot-scale and production-scale HTST operations, there were no in-process events that caused the HTST skid to shut down or difficulties in controlling the output temperature when processing the desired volume of medium 1 at pH 7.10. The precipitation events were significant enough to cause scaling of the heat exchanger surfaces. The pilot-scale processed medium 1 had particles before final filtration and use for cell culture. Due to the arrangement of the system sampling holes, the production-scale processed medium 1 could not be sampled before final filtration. Adjusting the pH of medium 1 to approximately pH 6.34 reduced precipitation. For pilot-scale and production-scale HTST operations, there were no in-process events that caused the HTST skid to shut down or difficulties in controlling the output temperature when processing the desired volume of medium 1 at pH 6.34. Therefore, there was no indication of significant precipitation events that were significant enough to cause scaling of the heat exchanger surfaces. The pilot-scale processed medium 1 was visually free of particles before final filtration and use for cell culture. Due to the arrangement of the system sampling holes, the production-scale processed medium 1 could not be sampled before final filtration.

[0291] Example 5: Contribution of pH, calcium and phosphate levels to trace metal losses in culture medium during heat treatment for viral inactivation

[0292] Materials and methods

[0293] Medium preparation

[0294] The culture medium used in this study includes a pH level of about pH 5.9 to about pH 7.5, a calcium concentration of about 0mM to about 3.5mM (or higher in an unidentified culture medium), a phosphate concentration of about 0mM to about 6.5mM (or higher in an unidentified culture medium) and an iron concentration of about 0μM to about 125μM (or higher in an unidentified culture medium). All culture mediums are prepared using purified deionized water processed through a Millipore SuperQ ultrapure water purification system. Culture medium is prepared using a suitable culture medium powder stock (SAFC and Life Technologies). Glass electrode pH probes (Mettler Toledo) and osmometers (AdvancedInstruments) are used during liquid preparation to ensure target pH and permeability of a given preparation. After the components are completely dissolved and the final pH and permeability are adjusted, the culture medium is filtered into a bottle of 250mL to 1L (Corning) for small-scale preparation using a 0.1μm pore size PES membrane filter.

[0295] pH Adjustment

[0296] Corrected for pH deviations due to degassing in the time between completion of culture medium preparation and application of heat treatment.Before heat treatment, 30mL aliquots from each culture medium preparation were transferred to 50mL test tubes (Falcon), and the initial Falcon caps were replaced with ventilating caps from 250mL Corning Erlenmeyer flasks.Then the test tubes were placed in an incubator with CO2 for 30 minutes to reduce pH (15% CO2 for pH 6.2 samples, and 12% CO2 for all other samples, 200 revs / min, 37°C); this step can force pH to be lower than the target.Then manually oscillate the test tubes, and use a glass electrode pH probe and a meter (Mettler Toledo) to detect pH until pH slowly returns to the target pH.Final pH measurements were performed using NOVAbioprofiler.

[0297] Sand Bath

[0298] For sand bath method, the liquid culture medium prepared by 22mL is transferred in 20mL glass pressure vessel (Aceglassware).Use the threaded cap sealed container with thermocouple sleeve, by filling the container and allowing bottle cap and thermocouple sleeve to discharge too much culture medium, so that head space will not leave air in the solution.Clean the outside of the container, to prevent the fouling of the culture medium outer surface directly exposed to the pyrogen matrix.Teflon tape is used to cover the interface between the bottle mouth and the threaded cap of glass container, to better seal the glass pressure vessel, and prevent the oil of sand or thermocouple hole from entering the sample for heat treatment.Configuration has the fluidized sand bath (Techne SBS-4) (compressed air inlet pressure=5psig, bath temperature=110 ℃) of temperature controller (Techne TC-8D), and given 30 minutes balance.The thermocouple combined with a single VWR digital thermometer is inserted into the sample container thermocouple sleeve at the center of the radial dimension that is geometrically distributed in test tube. Silicone oil was added to the thermocouple hole to provide a heat transfer medium between the thermocouple hole glass wall and the thermocouple. The sample container was placed in a sand bath and the timing was started. The temperature dynamics data was recorded approximately every 30-60 seconds. Once the container reached 102°C on the thermometer reading, it was maintained in the sand bath for 10 seconds. After the heating and holding steps, the container was transferred to a water bath at room temperature until the thermometer temperature reading reached 35°C. After heat treatment, 15 mL of each sample was transferred to a vial for turbidity and visual measurement.

[0299] Pilot scale HTST

[0300] For studies using pilot-scale HTST, media formulations were processed from the lowest to the highest concentration of calcium or phosphate to minimize possible carry-over to subsequent runs. Each HTST run required 15L to 20L of media to flush the deionized rinse used between runs and to equilibrate the heating coils to an operating temperature of 102°C (acceptable range: 97°C-110°C) for HTST processing. After equilibration, approximately 20L of media was run through the HTST skid and the outlet flow was collected in a plastic cube container. Samples were collected from media from the media mixing tank before HTST processing and from the media flowing out of the cube container and filtered through a 0.1μm PVDF capsule membrane filter (Millipore) into a media storage bag for use as "before HTST" and "after HTST" samples. All treated and untreated filtered media samples were then stored at 2-8°C prior to cell culture performance assays and analytical testing.

[0301] Production scaleHTST

[0302] The medium 4 preparation from the 1800L operation run was used to support the antibody production program, and the production scale HTSTskid U1281 was used to process the medium. The purpose of this medium preparation was to determine: 1) whether the mixing conditions for medium 4 were sufficient to meet the specific method mixing times (recipe mix time), and 2) to generate production scale HTST processing performance data using medium 4. Medium samples before and after HTST treatment were collected by aseptically connecting a 6×20-L medium bag manifold (Sartorius Stedim Biotech) to the sampling port on the medium mixing tank and the target bioreactor. For samples obtained before HTST treatment, the medium was filtered through a 0.1 μm PVDF capsule filter (Millipore) before collection, and for samples obtained after HTST treatment, the medium was passed through a GMP filter train before collection, which consisted of a 0.5 / 0.2 μm double-layer cartridge filter PVDF prefilter (Millipore) and a 0.1 μm Nylon final filter (Pall). The samples were then used for cell culture performance determination and analytical testing.

[0303] Sedimentation measurement

[0304] The sediment of the culture medium samples was analyzed before and after heat treatment by the following two methods: 1) turbidity was measured by nephelometer (2100Q Hach); and 2) the samples were centrifuged at 10,000 × g for 10 minutes (Sorvall RC 6plus, SS-34 rotor) in 50-mL Falcon tubes to sediment the sediment, visually identify and qualitatively determine the sediment based on particle size (e.g., invisible, low, medium, high). For visual identification of the sediment, the uncentrifuged samples were also analyzed.

[0305] Analysis of changes in concentrations of culture medium components

[0306] The treated supernatant residues before and after HTST were determined to screen for any significant changes in the concentrations of measurable culture medium components. The assays used included: measuring water-soluble vitamins, measuring amino acids, and measuring inorganic phosphates. Trace elements were analyzed using two different inductively coupled plasma mass spectrometry (ICP-MS) assays. In addition, iron, copper, and zinc in the samples were measured by inductively coupled plasma-emission spectroscopy (ICP-OES) for higher throughput quantification of these elements. All statistical analyses and plots were performed using JMP software and Excel when applicable.

[0307] result

[0308] To better understand the trace metal losses that occur at a larger production scale, Medium 4 was used as a model medium formulation for sand bath studies. To determine if trace metal losses were associated with calcium phosphate precipitation events in media containing these components and treated at neutral or higher pH, a half-factorial design was generated to evaluate the effects of different pH, calcium (Ca), inorganic phosphate (PO) and pH 7 after heat treatment. 4 ), iron (Fe) and copper (Cu) on the recovered Fe and Cu levels.

[0309] Table 5. Concentrations of components tested

[0310]

[0311] Note: Except for the above factors, other components in Medium 4 were at normal levels.

[0312] Analysis of trace metal recovery showed that high pH, ​​Ca and PO 4 The level of these three factors was the main factor in Fe loss (Figure 10). Increasing the level of any of these three factors resulted in a decrease in Fe recovery. 4 In comparison, initial Fe concentration showed a similar but smaller main effect, indicating that it had virtually no effect on Fe recovery. pH, Ca, and PO 4The significance of is consistent with the possibility that Fe loss is associated with calcium phosphate precipitation events that are not significant in operational terms (e.g., not significant enough to cause HTST skid operational issues) but are significant in adversely affecting cell culture medium performance (e.g., product titer, cell growth, cell production, and product quality).

[0313] To explain the variance or spread in the main effects plot, we used the four strongest factors (pH, Ca, PO 4 The interaction plot from this analysis shows two interacting effects that affect Fe recovery after thermal treatment: 1) pH*Ca and 2) pH*PO 4 ( Fig.11 ). These results indicate that in order to produce a culture medium formulation that avoids Fe loss after heat treatment, it is necessary to reduce the concentration of calcium and phosphate, reduce the pH level, or reduce some combination of these factors. In addition, these data show that Fe loss still occurs even when no visible precipitation and increased turbidity are observed.

[0314] Mediums with ferrous sulfate levels of 75-150 μM (but otherwise identical to Medium 4) were tested to determine the degree of functional relationship between initial Fe levels and post-HTST Fe levels. The data showed an unexpected nonlinear relationship between initial Fe and post-HTST Fe concentrations ( Fig.12 ). However, it was found that the 125 μM Fe sample experienced a pH shift relative to the other three samples. Of the two groups of samples, the 125 μM Fe sample had the highest pH before the HTST test. For the sample groups of Test 1 and Test 2, the pH ranged from 7.08 to 7.17 and 7.09 to 7.14.

[0315] Results from the DoE experiments showed that in the sand bath thermal treatment system, the medium at pH 7.0 was close to the edge of failure and Fe loss began to occur above pH 6.7. Medium 4 preparations at pH levels of 6.6-7.2 were tested to characterize the protocol. Evidence for significant pH excursions (even as small as fractions of a pH unit) in the sand bath thermal treatment system was shown in the high resolution pH titration data ( Fig.13A ). After pH 6.8, Fe recovery is highly sensitive to pH, decreasing rapidly and reaching a plateau at pH 7.2. This obvious pH dependence indicates that the ferrous sulfate titration results are most likely affected by pH variability. It also shows that very small changes in pH (i.e. 0.2 pH units) may prevent Fe losses in the medium formulation in the scheme.

[0316] The results from the DoE experiments also showed that Ca and PO 4 Relatively small changes in the level can achieve large improvements in Fe recovery ( Fig. 13B ). Ca and PO levels ranging from 0.5 to 1.17× standard medium 4 were tested. 4 Level 4 preparations of medium to better quantify the Ca and PO required to achieve the benefits observed previously 4 For this titration, Ca and PO 4 The ratio of the levels was kept constant at 0.5. Graphical data analysis showed a graph where increasing Fe loss was correlated with increasing Ca and PO 4 Horizontal correlation ( Fig. 13B ). Medium 4 formulation was located on the steepest part of the slope, where Ca and PO 4 Small changes in concentration can greatly improve Fe recovery. For example, making Ca and PO 4 A 30% reduction in concentration (and maintaining the same ratio) can result in a post-sand bath heat treatment that translates to 100% Fe recovery for the HTST process.

[0317] If trace metal losses are associated with calcium phosphate precipitation, then there is a relationship between calcium and phosphate recovery and Fe recovery. Data analysis showed that no clear relationship was established between phosphate recovery and Fe recovery, where Fe recovery was very low and precipitation was confirmed ( Fig.14 , diamonds in circles). It is difficult to detect small concentration differences in samples with relatively high initial phosphate concentrations due to the signal-to-noise ratio. Due to the possibility of interaction between calcium phosphate and iron, iron may be removed in a 1:1 stoichiometry with relatively small amounts of phosphate and phosphate loss is difficult to detect. Another possible reason is related to the fact that the assay detects only specific forms of inorganic phosphate. Calcium recoveries were 60-80% and showed a linear correlation with Fe recoveries of 10-30%, where Fe recoveries were very low and precipitation was confirmed ( Fig.14 , squares in circles). Analysis of this subgroup using least squares regression showed that Fe recovery could explain 69% of the variation in Ca recovery. The data showed that Fe was directly related to Ca recovery.

[0318] Pilot and production scale HTST treatment runs of Medium 4 (and variations of Medium 4) and Medium 9 were also performed. Data were collected from runs for analytical assays and cell culture performance studies to assess the impact of HTST treatment on cell culture performance and product quality. Analytical testing confirmed that there was no significant loss of any component except iron and copper. In addition, no significant changes in key cell culture performance measurements (titer, growth, activity) or product quality (including basic variations thereof) were observed. Copper loss did not result in changes in medium performance or levels (basic variations of medium performance) because the loss was not large enough to illicitly change the host cell line used for testing based on copper titration of the cell line. Since the amount of trace metals such as iron and copper was relatively small (75 μM and 1 μM, respectively) compared to calcium and phosphate in the Medium 4 formulation (1.5 mM and 3 mM, respectively), it is possible that very little calcium phosphate precipitation (CaPO 4) was formed. 4 complex), which does not cause significant scaling of heat exchangers, but can interact with iron and copper present in the liquid culture medium. 4 These complex interactions may somehow chelate the iron and copper to separate them from the liquid medium and separate them from the precipitated CaPO 4 The complex is deposited together somewhere in the process flow. Regarding the mechanism, it is a fact that after HTST treatment of the culture medium, including when the virus in the culture medium has been successfully treated and inactivated, iron and copper losses have been observed. After HTST treatment, if the losses are significant relative to the iron and copper concentrations required in the production phase, the reduction in iron and copper concentrations may adversely affect the successful use of the HTST-treated culture medium in subsequent production phases.

[0319] Pilot and production scale HTST treatments were performed on several different media formulations. Iron levels in the media formulations were measured prior to HTST treatment, and the expected levels of iron after HTST treatment were determined. Analysis of iron levels in the media after HTST treatment (Post-HTST) showed that HTST treatment resulted in a loss of iron levels. The iron loss was 44% in Medium 14, 12% in Medium 15, 20% in Medium 16, 1.3% in Medium 17, and 42% in Medium 18 ( Fig.15 ). Medium 19 and Medium 20 were supplemented with iron after HTST treatment.

[0320] The growth of NS0 cells (murine myeloma cell line) in cell culture medium supplemented with iron and treated with HTST was measured. Analysis of cell growth showed that cells grew over time to a comparable amount in culture medium supplemented with or without iron without HTST treatment ( Fig.16In contrast, when cultured in medium treated with HTST and without iron supplementation, the amount of cell growth was lower. Addition of iron to the HTST-treated cell culture allowed cell growth to recover to higher levels compared to the cell culture medium not treated with HTST.

[0321] In summary, the data show that Fe loss correlates with calcium, phosphate, and pH levels, indicating a close relationship between trace metal loss and calcium phosphate precipitation events during thermal processing, including those that are not necessarily detected, such as visible precipitates, significant turbidity changes, or operational problems. Loss of trace metals such as Fe can adversely affect cell culture performance and product quality in a variety of cell lines.

Claims

1. A method for inactivating viruses in a mammalian cell culture medium for producing biopharmaceuticals, while maintaining the adaptability of the culture medium to mammalian cell culture and reducing iron loss, and reducing the formation of precipitates in the culture medium, the method comprising a) adjusting the pH of the cell culture medium, wherein the cell culture medium has a pH of 5.0 to pH 6.7 prior to the HTST treatment, b) subjecting the cell culture medium to HTST treatment, and c) Raising the pH of the cell culture medium to between 7.0 and 7.2 after HTST treatment for mammalian cell culture.

2. The method according to claim 1, wherein the pH of the cell culture medium is adjusted to pH 5.0 to 6.3 prior to HTST treatment.

3. The method of claim 2, wherein the pH of the cell culture medium is raised to pH 7.0 to pH 7.2 after HTST treatment.

4. The method of claim 1, comprising limiting the total amount of calcium and phosphate in the cell culture medium to less than 9 mM prior to HTST treatment.

5. The method of claim 4, wherein during the HTST treatment, the total phosphate and calcium concentrations in the culture medium are less than 8 mM.

6. The method of claim 2, wherein during the HTST treatment, the total phosphate and calcium concentrations in the culture medium are less than 8 mM.

7. The method of claim 4, wherein during the HTST treatment, the total phosphate and calcium concentrations in the culture medium are less than 4 mM.

8. The method of claim 2, wherein during the HTST treatment, the total phosphate and calcium concentrations in the culture medium are less than 4 mM.

9. The method of claim 4, further comprising adjusting calcium and phosphate levels to levels suitable for cell culture after HTST treatment.

10. The method of claim 7, further comprising adjusting calcium and phosphate levels to levels suitable for cell culture after HTST treatment.

11. The method of claim 1, wherein the HTST treatment comprises raising the temperature of the culture medium from 85 degrees Celsius to 118 degrees Celsius for a sufficient time to inactivate viruses in the culture medium.

12. The method of claim 1, wherein the temperature of the culture medium is raised to 97 degrees Celsius to 120 degrees Celsius for a sufficient time to inactivate viruses in the culture medium.

13. The method of claim 2, wherein the temperature of the culture medium is raised to 97 degrees Celsius to 120 degrees Celsius for a sufficient time to inactivate viruses in the culture medium.

14. The method according to claim 12, in, The temperature was increased for 1 second to 60 seconds.

15. The method according to claim 14, in, The temperature was increased for 1 to 18 seconds.

16. The method of claim 15, wherein the temperature is raised to 102 degrees Celsius for 10 seconds.

17. The method of claim 1, wherein the virus is selected from the group consisting of Parvoviridae, paramyoxviradae, Orthomyxoviridae, Bunyaviridae, Rhabdoviridae, Reoviridae, Togaviridae, calciviridae, and Picornaviridae.

18. The method of claim 1, wherein the virus is an enveloped virus.

19. The method of claim 1, wherein the virus is a non-enveloped virus.

20. The method of claim 1, wherein the mammalian cell culture medium is used to culture CHO cells.

21. The method of claim 1, wherein one or more of iron and copper are absent from the cell culture medium prior to HTST treatment.

22. The method of claim 1, further comprising supplementing one or more of iron and copper to the culture medium after HTST treatment to a level suitable for cell culture.

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