WO2023198450A1 - Procédés de récupération élevée d'un produit de clarification de culture cellulaire - Google Patents

Procédés de récupération élevée d'un produit de clarification de culture cellulaire Download PDF

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Publication number
WO2023198450A1
WO2023198450A1 PCT/EP2023/058087 EP2023058087W WO2023198450A1 WO 2023198450 A1 WO2023198450 A1 WO 2023198450A1 EP 2023058087 W EP2023058087 W EP 2023058087W WO 2023198450 A1 WO2023198450 A1 WO 2023198450A1
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cell culture
biomolecule
interest
clarification
arginine
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PCT/EP2023/058087
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English (en)
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Romain METTE
Patrick VETSCH
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Ichnos Sciences SA
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Publication of WO2023198450A1 publication Critical patent/WO2023198450A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types

Definitions

  • the present invention relates to cell culture clarification.
  • the present invention relates to methods for high recovery of a biomolecule of interest, such as an antibody, from a cell culture clarification filter. More in particular, to methods for high recovery of a biomolecule of interest from a cell culture clarification filter using Arginine-HCI buffer.
  • the present invention also discloses methods of cell culture clarification including a filtration step wherein the recovery of the biomolecule of interest from the filter is maximized by a recovery flush step performed with Arginine-HCI buffer.
  • the manufacturing process of a biopharmaceutical molecule is complex and it comprises different steps, each requiring extensive optimizations.
  • the process begins with the selection of a cell line to use for expressing and secreting the biomolecule of interest. Once selected, this cell line is amplified and cultivated at larger scales in bioreactors where the biomolecule of interest is produced under controlled conditions. These steps are referred to as the upstream process (USP) part of the whole biomolecule manufacturing process.
  • USP upstream process
  • a clarification process In order to remove residual biomass and impurities from the culture medium containing the biomolecule of interest, such as an antibody, a clarification process is necessary.
  • Cell culture clarification requires multiple steps directed to the removal of different types of impurities spanning a wide range of size, including residual cells, cell debris and eventually DNA and host cell proteins (HCPs).
  • HCPs DNA and host cell proteins
  • the clarification process must meet the downstream process requirements in terms of product stability and purity and at the same time it needs to be robust enough to permit a high recovery of the obtained biomolecule of interest.
  • a clarification process may comprise one or more steps performed by a depth filter, namely by a filter whose porosity is such that it retains particles of a cell culture throughout the medium, rather than just on the surface.
  • Depth filters can be used for primary or secondary clarification only, or for both primary and secondary clarification, alone or in combination with other clarification units.
  • the cell culture clarification is the step which allows the removal of cell culture material such as cells, cell debris and impurities from the cell culture medium to obtain a clarified cell culture where mainly the biomolecule of interest (herein also called "product") is present in the cell culture medium.
  • the present invention relates to cell culture clarification.
  • the present invention relates to methods for high recovery of a biomolecule of interest, such as an antibody, from a cell culture clarification filter. More in particular, to methods for high recovery of a biomolecule of interest from a cell culture clarification filter using Arginine-HCI buffer.
  • the present invention also discloses methods of cell culture clarification including a filtration step wherein the recovery of the biomolecule of interest from the filter is maximized by a recovery flush step performed with Arginine-HCI buffer.
  • the present invention relates to a method for recovering a biomolecule of interest from a cell culture clarification synthetic depth filter comprising the step of flushing said depth filter with Arginine-HCI buffer.
  • biomolecule of interest is an antibody.
  • biomolecule of interest is a non-naturally occurring antibody.
  • biomolecule is a multispecific and/or multivalent antibody, such as a bispecific antibody.
  • Arginine-HCI buffer is used at a concentration comprised between about 600 and about 1000 mM at a pH comprised between about 5 and about 7.
  • the present invention also relates to a process for clarifying a cell culture including a biomolecule of interest comprising at least a filtration step performed by a synthetic depth filter, the process characterized in that it further comprises recovering said biomolecule of interest from said depth filter according to the method of the present invention for recovering a biomolecule of interest from a cell culture clarification synthetic depth filter comprising the step of flushing said depth filter with the Arginine-HCI buffer disclosed herein.
  • the process disclosed herein comprises the consequent steps of: i. flushing said depth filter with water for injection; ii. flushing said depth filter with 1 X phosphate buffer saline 140 mM; ill. connecting the cell culture to the filter; iv. flushing said depth filter with the cell culture fluid; v. discarding the dead volume; vi. collecting the clarified cell culture fluid; vii. recovering said biomolecule of interest from said depth filter by flushing said depth filter with Arginine-HCI buffer according to the method of the present invention.
  • the process disclosed herein further comprises a step of subjecting said recovered biomolecule of interest to one or more steps of chromatography purification.
  • the present invention also discloses a process of production of a drug substance comprising the steps of: i. seeding cells expressing a biomolecule of interest in a cell culture medium; ii. culturing said cells for a period comprised between 10 and 18 days, preferably for 14 days; ill. subjecting the obtained cell culture to the clarification process according to the present invention; iv. add excipients to the biomolecule of interest purified.
  • biomolecule of interest refers to a product of interest, which is desired to be purified or separated from one or more undesirable entities, e.g., one or more impurities, which may be present in a sample containing the product of interest.
  • the biomolecule of interest is a polypeptide.
  • the biomolecule of interest is a protein.
  • the biomolecule of interest is an antibody or an antibody fragment thereof.
  • the biomolecule of interest is an isolated polypeptide, for instance an antibody such as a monoclonal antibody or a monoclonal antibody fragment thereof.
  • the antibody is a non-naturally occurring or recombinant or engineered molecule, for instance a multispecific antibody.
  • a "recombinant" molecule is one that has been prepared, expressed, created, or isolated by recombinant means.
  • isolated polypeptide is one that: (1) is free of at least some other polypeptides with which it would normally be found, (2) is essentially free of other polypeptides from the same source, e.g., from the same species, (3) is expressed by a cell from a different species, (4) has been separated from at least about 50 percent of polynucleotides, lipids, carbohydrates, or other materials with which it is associated in nature, (5) is not associated (by covalent or noncovalent interaction) with portions of a polypeptide with which the "isolated polypeptide" is associated in nature, (6) is operably associated (by covalent or noncovalent interaction) with a polypeptide with which it is not associated in nature, or (7) does not occur in nature.
  • Such an isolated polypeptide can be encoded by genomic DNA, cDNA, mRNA or other RNA, of synthetic origin, or any combination thereof.
  • the isolated polypeptide is substantially free from polypeptides or other contaminants that are found in its natural environment that would interfere with its use (therapeutic, diagnostic, prophylactic, research or otherwise).
  • antibody and the term “immunoglobulin” are used interchangeably.
  • Antibodies are glycoproteins produced by plasma cells that play a role in the immune response by recognizing and inactivating antigen molecules. In mammals, five classes of immunoglobulins are produced: IgM, IgD, IgG, IgA and IgE. In the native form, immunoglobulins exist as one or more copies of a Y-shaped unit composed of four polypeptide chains: two identical heavy (H) chains and two identical light (L) chains.
  • variable regions are composed of one variable domain (VH), and the constant region is composed of three or four constant domains (CHI, CH2, CH3 and CH4), depending on the antibody class; while the light chain comprises a variable domain (VL) and a single constant domain (CL).
  • the variable regions contain three regions of hypervariability, termed complementarity determining regions (CDRs). These form the antigen binding site and confer specificity to the antibody.
  • CDRs are situated between four more conserved regions, termed framework regions (FRs) that define the position of the CDRs.
  • Antigen binding is facilitated by flexibility of the domains position; for instance, immunoglobulin containing three constant heavy domains present a spacer between CHI and CH2, called “hinge region” that allows movement for the interaction with the target.
  • enzymatic digestion can lead to the generation of antibody fragments.
  • the incubation of an IgG with the endopeptidase papain leads to the disruption of peptide bonds in the hinge region and to the consequent production of three fragments: two antibody binding (Fab) fragments, each capable of antigen binding, and a cristallizable fragment (Fc).
  • Digestion by pepsin instead yields one large fragment, F(ab')2, composed by two Fab units linked by disulfide bonds, and many small fragments resulting from the degradation of the Fc region.
  • antibody as referred to herein includes whole antibodies and any antigen binding fragments or single chains thereof.
  • Naturally occurring antibodies typically comprise a tetramer.
  • Each such tetramer is typically composed of two identical pairs of polypeptide chains, each pair having one full-length "light” chain (typically having a molecular weight of about 25 kDa) and one full-length "heavy” chain (typically having a molecular weight of about 50-70 kDa).
  • the terms “heavy chain” and “light chain” as used herein refer to any immunoglobulin polypeptide having sufficient variable domain sequence to confer specificity for a target antigen.
  • each light and heavy chain typically includes a variable domain of about 100 to 110 or more amino acids that typically is responsible for antigen recognition.
  • the carboxy-terminal portion of each chain typically defines a constant domain responsible for effector function.
  • a full-length heavy chain immunoglobulin polypeptide includes a variable domain (VH) and three constant domains (CHI, CH2, and CH3), wherein the VH domain is at the amino-terminus of the polypeptide and the CH3domain is at the carboxyl-terminus
  • a full-length light chain immunoglobulin polypeptide includes a variable domain (VL) and a constant domain (CL), wherein the VL domain is at the amino-terminus of the polypeptide and the CL domain is at the carboxyl-terminus.
  • Human light chains are typically classified as kappa and lambda light chains, and human heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
  • IgG has several subclasses, including, but not limited to, IgGl, lgG2, lgG3, and lgG4.
  • IgM has subclasses including, but not limited to, IgMl and lgM2.
  • IgA is similarly subdivided into subclasses including, but not limited to, IgAl and lgA2.
  • variable and constant domains typically are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D” region of about 10 more amino acids.
  • the variable regions of each light/heavy chain pair typically form an antigen binding site.
  • the variable domains of naturally occurring antibodies typically exhibit the same general structure of relatively conserved framework regions (FR) joined by three hypervariable regions, also called complementarity determining regions or CDRs.
  • both light and heavy chain variable domains typically comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
  • Fc refers to a molecule comprising the sequence of a non-antigen-binding fragment resulting from digestion of an antibody or produced by other means, whether in monomeric or multimeric form, and can contain the hinge region.
  • the original immunoglobulin source of the native Fc is preferably of human origin and can be any of the immunoglobulins.
  • Fc molecules are made up of monomeric polypeptides that can be linked into dimeric or multimeric forms by covalent ⁇ i.e., disulfide bonds) and non- covalent association.
  • the number of intermolecular disulfide bonds between monomeric subunits of native Fc molecules ranges from 1 to 4 depending on class ⁇ e.g., IgG, IgA, and IgE) or subclass ⁇ e.g., IgGl, lgG2, lgG3, IgAl, lgGA2, and lgG4).
  • a Fc is a disulfide-bonded dimer resulting from papain digestion of an IgG.
  • native Fc as used herein is generic to the monomeric, dimeric, and multimeric forms.
  • a F(ab) fragment typically includes one light chain and the VH and CHI domains of one heavy chain, wherein the VH-CH1 heavy chain portion of the F(ab) fragment cannot form a disulfide bond with another heavy chain polypeptide.
  • a F(ab) fragment can also include one light chain containing two variable domains separated by an amino acid linker and one heavy chain containing two variable domains separated by an amino acid linker and a CHI domain.
  • a F(ab') fragment typically includes one light chain and a portion of one heavy chain that contains more of the constant region (between the CHI and CH2 domains), such that an interchain disulfide bond can be formed between two heavy chains to form a F(ab')2molecule.
  • antibody fragments includes one or more portion(s) of a full-length antibody.
  • Non limiting examples of antibody fragments include: (i) the fragment crystallizable (Fc) composed by two constant heavy chain fragments which consist of CH2 and CH3 domains, in IgA, IgD and IgG, and of CH2, CH3 and CH4 domains, in IgE and IgM, and which are paired by disulfide bonds and non-covalent interactions; (ii) the fragment antigen binding (Fab), consisting of VL, CL and VH, CHI connected by disulfide bonds; (iii) Fab', consisting of VL, CL and VH, CHI connected by disulfide bonds, and of one or more cysteine residues from the hinge region; (iv) Fab'-SH, which is a Fab' fragment in which the cysteine residues contain a free sulfhydryl group; (v) F(ab')2 consisting of two Fab
  • antibodies and antibody fragments can be monomeric or multimeric, monovalent or multivalent, monospecific or multispecific.
  • monospecific antibody refers to any antibody or fragment having one or more binding sites, all binding the same epitope.
  • multispecific antibody refers to any antibody or fragment having more than one binding site that can bind different epitopes of the same antigen, or different antigens.
  • a non-limiting example of multispecific antibodies are bispecific antibody, which have two binding sites that can bind two different epitopes of the same antigen, or two different antigens.
  • MAb refers to a population of antibody molecules that contain only one molecular species of antibody molecule consisting of a unique light chain gene product and a unique heavy chain gene product.
  • CDRs complementarity determining regions
  • the biomolecule of interest such as an antibody
  • host cells refers to all the cells in which the biomolecule of interest codified by the artificially introduced genetic material is expressed, including those cells in which the foreign nucleic acid is directly introduced and their progeny.
  • an expression vectors constructs
  • Such expression vectors normally contain the necessary elements for the transcription and translation of the sequence encoding the biomolecule of interest.
  • Cell lines suitable as host cells include and are not limited to bacteria, mammalian, insect, plant and yeast cells.
  • Cell lines often used for the expression and production of therapeutic antibodies include mammalian cells lines such as Chinese hamster ovary (CHO) cells, NSO mouse myeloma cells, human cervical carcinoma (HeLa) cells and human embryonic kidney (HEK) cells.
  • the cultured cells are mammalian cells, more in particular, they are CHO cells.
  • cell culture and “culture” as used herein are interchangeable and refer to the growth and/or propagation and/or maintenance of cells in controlled artificial conditions, and they indicate a cell culture which comprises a cell culture medium and cell culture material comprising cells, cell debris, for instance generated upon cell death, colloidal particles, such as DNA, RNA and host cell proteins (HCP), and (bio)molecules secreted by the cultured cells, such as the biomolecule of interest.
  • the cells of a cell culture can be cultured in suspension or attached to a solid substrate, in containers comprising a cell culture medium.
  • a cell culture can be grown in tubes, spin tubes, flasks, bags, roller bottles, bioreactors.
  • the obtained titer of the biomolecule of interest is below 10 g/L, in other embodiments the obtained titer of the biomolecule of interest is comprised between about 1 g/L and about 10 g/L.
  • Non limiting examples of the obtained titer of the biomolecule of interest include: about 0.1 g/L, about 0.5 g/L, about 1 g/L, about 1.5 g/L, about 2 g/L, about 2.5 g/L, about 3 g/L, about 3.5 g/L, about 4 g/L, about 4.5 g/L, about 5 g/L, about 5.5 g/L, about 6 g/L, about 6.5 g/L, about 7 g/L, about 7.5 g/L, about 8 g/L, about 8.5 g/L, about 9 g/L, about 9.5 g/L, about 10 g/L.
  • the cell culture to be clarified is also referred herein as cell culture fluid (CCF).
  • CCF cell culture fluid
  • bioreactor refers to any manufactured or engineered device or system that supports a biologically active environment.
  • Optimal culturing conditions are obtained by the control and adjustment of several parameters including: the formulation of the cell culture medium, the bioreactor operating parameters, the nutrient supply modality and the culturing time period.
  • the formulation of the culturing medium has to be optimized to favorite cell vitality and multiplication; examples of constituents of the cell culture medium include but are not limited to essential amino acids, salts, glucose, growth factors and antibiotics.
  • Important bioreactor operating parameters include: initial cell seeding density, temperature, pH, agitation speed, oxygenation and carbon dioxide levels.
  • Nutrients can be supplied in different ways: in the batch mode culture all the necessary nutrients are present in the initial base medium and are used till exhausted while wastes accumulate; in the fed-batch culture additional feed medium is supplied to prevent nutrient depletion and prolong the culture; differently, in the perfusion modality, cells in culture are continuously supplemented with fresh medium containing nutrients that flows in the bioreactor removing cell wastes. The culturing period is important as it needs to be long enough to let the cells produce a consistent amount of product but it cannot be too long to impair their viability.
  • bioreactors are typically cylindrical, ranging in size from liters to cubic meters, and are often made of stainless steel.
  • a bioreactor is made of plastic or of stainless steel. It is contemplated that the total volume of a bioreactor may be any volume ranging from 10 mL to up to 20000 L, e.g., from 100 mL to up to 20000 Liters or more, depending on a particular process.
  • bioreactor volumes include about 100 mL, about 200 mL, about 500 mL, about 800 mL, about 1 L, about 5 L, about 10 L, about 20 L, about 30 L, about 40 L, about 50 L, about 60 L, about 70 L, about 80 L, about 90 L, about 100 L, about 200 L, about 300 L, about 400 L, about 500 L, about 600 L, about 700 L, about 800 L, about 900 L, about 1000 L, about 2000 L, about 3000 L, about 4000 L, about 5000 L, about 6000 L, about 7000 L, about 8000 L, about 9000 L, about 10000 L, about 15000 L, about 20000 L.
  • Bioreactors useful for present inventions include but are not limited to small scale bioreactors, single use bioreactors (SUB), shake flask vessels, large scale bioreactors, batch bioreactors, fed-batch bioreactors.
  • cells are cultured in 3 to 5 L, or 50 L SUBs in fed-batch mode for a number of days comprised between 10 and 20 days, preferably between 12 and 14 days, most preferably for at least 12 days, even more preferably for 12 or 13 or 14 days in a cell culture medium.
  • cell culture medium and “culture medium” and “medium” are used interchangeably herein and they refer to a nutrient solution used for growing cells, such as animal cells, e.g., mammalian cells.
  • a nutrient solution generally includes various factors necessary for cell attachment, growth, and maintenance of the cellular environment.
  • a typical nutrient solution may include a basal media formulation, various supplements depending on the cell type and, occasionally, antibiotics.
  • the cell culture medium may also contain cell culture material such as cell waste products, host cell proteins (HCP) and material from lysed cells.
  • the composition of the culture medium may vary in time during the course of the culturing of cells.
  • clarify refers to one or more steps that aid the removal of a part of the cell culture material from the cell culture (e.g. from the cell culture fluid (CCF)), such as removal of cells, cell debris and colloidal particles, to obtained clarified cell culture, also called clarified cell culture fluid (CCCF) herein, comprising the biomolecule of interest, the biomolecule of interest is also generally referred inhere as the "product”.
  • CCF cell culture fluid
  • the efficiency of the clarification step is crucial to facilitate the subsequent downstream processing steps of purification of the biomolecule of interest.
  • Characteristics of the cell culture that have an impact on the clarification step include the total cell concentration, the cell viability, the initial turbidity of the cell culture to clarify, the concentration of biomolecule produced by the cultured cells.
  • the term “Total cell concentration” (TCC) refers to the number of cells in a given volume of culture.
  • the terms “Viable cell concentration” (VCC) refers to the number of live cells in a given volume of culture, as determined by standard viability assays (such as trypan blue dye exclusion method). The percentage of living cells is called "viability". In general terms, a higher TCC implies higher biomass to be removed from the cell culture and therefore higher impact on the clarification.
  • turbidity refers to the cloudiness or haziness of a liquid caused by large numbers of individual particles.
  • the turbidity indicates the amount of material and small particles inside a liquid capable of light diffusion.
  • the turbidity of a cell culture may be due to the presence of cells, cell debris, colloidal particles, such as DNA, RNA and host cell proteins (HCP), and of the biomolecule of interest.
  • the cell culture clarification can start with a primary clarification step.
  • primary clarification and “primary recovery” as used herein are interchangeable and refer to the removal of large particles such as whole cells and cell debris.
  • the primary clarification can be followed by a secondary clarification step.
  • secondary clarification and “secondary recovery” as used in the present patent application are interchangeable and indicate the removal of smaller particles.
  • Primary and secondary clarification may require one or more clarification operational unit such as filters, centrifuges, acoustic separator etc., and their combinations.
  • clarification operational unit such as filters, centrifuges, acoustic separator etc., and their combinations.
  • the primary clarification step is a depth filtration.
  • depth filtration refers to a technology that exploits filters with a certain porosity to retain particles of a medium throughout the medium, rather than just on the surface.
  • Examples of depth filters used for instance in biopharmaceutical industry include single-use devices in the form of a lenticular disks or cartridges which contain the filter sheets at small scale. Disks or cartridges can be assembled into multilayer housings at larger scales.
  • Depth filters are typically composed of natural fibers such as cellulose fibers and filter aids like diatomaceous earth or perlite bound together into a polymeric resin.
  • cellulose fibers can be replaced by fully synthetic polymeric fibers like polyacrylic or polystyrene.
  • synthetic depth filters are the ones made of synthetic fibers, for instance of polyacrylic or polystyrene.
  • Synthetic filters may further comprise other synthetic or non-synthetic fiber (i.e. natural fibers) and a binder.
  • a synthetic filter may comprise silica fibers and a binder resin. Filters fibers form a three-dimensional network with a certain porosity. The permeability and retention characteristics of the filters are directly correlated with the length and compaction of those fibers. The porosity of the filters matrix can vary along with the filter depth, allowing the coverage a wide range of particle exclusion.
  • Non limiting examples of depth filters useful for the present invention comprise filters made of cellulose and/or resin, and/or synthetic media and/or polypropylene and/or filters aids.
  • a depth filter can be suitable for both primary and/or secondary recovery, or for primary or secondary recovery only.
  • Filters suitable for primary and/or secondary recovery are also known as "single filters”.
  • Single filters can be applied alone to carried out both the primary and the secondary recovery, or they can be used as filters for primary recovery and coupled with the subsequent use of a filter for secondary recovery; or they can be used as filters for secondary recovery for instance after a centrifugation step for primary recovery.
  • single filters are also referred as "SF”
  • filters for primary clarification are referred as "PCF”
  • SCF filters for secondary clarification
  • the present invention relates to methods for high recovery of a biomolecule of interest, such as an antibody, from a cell culture clarification filter, such as a depth filter or a sterile filer for bioburden reduction. More in particular, the present invention relates to methods for high recovery of a biomolecule of interest from a cell culture clarification depth filter, specifically from a cell culture clarification synthetic depth filter, in particular using Arginine-HCI buffer.
  • the present invention also discloses methods of cell culture clarification including a filtration step wherein the recovery of the biomolecule of interest from the filter, in particular from a depth filer, preferably from a synthetic depth filter, is maximized by a recovery flush step performed with Arginine-HCI buffer, wherein the term "flush step” or “flushing” is defined as making the filter in contact with a fluid, such as the water, water for injection, PBS, the cell culture fluid, a buffer, Arginine-HCI buffer, and allows the fluid to go through the filter.
  • Arginine-HCI buffer has a concentration comprised between about 100 mM and 1600 mM, e.g., between about 400 mM and about 1200 mM, preferably comprised between about 600 mM and 1000 mM, more preferably comprised between about 700 mM and 900 mM; particularly Arginine-HCI buffer has a concentration selected from the group comprising about 400 mM, about 200 mM, about 300 mM, about 400 mM, about 500 mM, about 600 mM, about 700 mM, about 800 mM, about 900 mM, about 1000 mM, about 1100 mM, about 1200 mM, about 1300 mM, about 1400 mM, about 1600 mM, about 1600 mM.
  • the concentration of Arginine-HCI buffer is about 800 mM.
  • Arginine-HCI buffer has a pH comprised between about 5 and 7, in particular Arginine-HCI buffer has a pH selected from the group comprising about 5, about 6, about 7.
  • the pH of Arginine-HCI buffer is about 6.
  • the present invention also includes concentration and pH of Arginine-HCI buffer at any intermediate value of the above said ranges. In a particular embodiment Arginine-HCI buffer has a concentration of about 800 mM and a pH of about 6.
  • the present invention also relates to a process for clarifying a cell culture including a biomolecule of interest comprising at least a filtration step performed by synthetic a depth filter, characterized in that said process further comprises recovering said biomolecule of interest from the depth filter by a recovery flush step performed with Arginine-HCI buffer.
  • the process for clarifying a cell culture of the present invention comprising the consequent steps of: i. flushing said depth filter with water for injection; ii. flushing said depth filter with 1 X phosphate buffer saline 140 mM; ill. connecting the cell culture to the filter; iv. flushing said depth filter with the cell culture fluid; v. discarding the dead volume; vi. collecting the clarified cell culture fluid; vii. recovering said biomolecule of interest from said depth filter by flushing said depth filter with Arginine-HCI buffer according to the method of the present invention.
  • process disclosed herein further comprises a step of subjecting said recovered biomolecule of interest to one or more steps of chromatography purification.
  • chromatography refers to the operation of separating compounds of a mixture based on their capability to interact with a stationary phase of a chromatography column, from which they can be retained or eluted.
  • the present invention also discloses a process of production of a drug substance comprising the steps of: i. seeding cells expressing a biomolecule of interest in a cell culture medium; ii. culturing said cells for a period comprised between 10 and 18 days, preferably for 14 days; ill. subjecting the obtained cell culture to the clarification process according to the present invention; iv. add excipients to the biomolecule of interest purified.
  • the primary clarification is performed by a first depth filter selected from the group comprising depth filters for primary clarification and single filters.
  • the first depth filter has an exclusion range equal to or greater than about 0.1 pm and equal to or less than about 50 pm, more particularly the exclusion range is comprised between about 0.25 pm and about 30 pm.
  • the exclusion range is comprised between about 1 pm and about 20 pm, or comprised between about 5 pm and about 30 pm, preferably comprised between about 6 pm and about 30 pm, or comprised between about 0.5 pm and about 10 pm, preferably comprised between about 0.55 pm and about 8 pm, or comprised between about 0.2 pm and about 2 pm, or comprised between about 1.5 pm and about 10 pm, or comprised between about 0.7 pm and about 5 pm, or comprised between about 0.25 pm and about 5 pm.
  • the first depth filter has an exclusion range selected from the group comprising at least about 0.1 pm, at least about 0.2 pm, at least about 0.5 pm, at least about 0.7 pm, at least about 1 pm, at least about 1.5 pm, at least about 2 pm, at least about 5 pm, at least about 7 pm, at least about 10 pm, at least about 20 pm, at least about 30 pm.
  • the present invention also includes first depth filters with an exclusion range at any intermediate value of the above said ranges.
  • the secondary clarification step is carried out by a second depth filtration.
  • the second clarification is performed by a second depth filter selected from the group comprising depth filters for secondary clarification, single filters and postflocculation filters.
  • the second depth filter has an exclusion range comprised between about 0.01 pm and about 10 pm, more in particular comprised between about 0.05 pm and about 5 pm, even more in particular comprised between about 0.1 pm and about 4 pm, in a further particular embodiment the second depth filter exclusion range in comprised between about 0.2 pm and about 3.5 pm.
  • the second depth filter has an exclusion range equal to or less than about 3.5 pm, or equal to or less than about 3 pm, or equal to or less than about 2 pm, or equal to or less than about 1 pm, or equal to or less than about 0.5 pm, or equal to or less than about 0.2 pm, or equal to or less than about 0.1 pm.
  • the present invention also includes second depth filters with an exclusion range at any intermediate value of the above said values.
  • the cell culture when the cell culture has a turbidity comprised between 1000 NTU and 5000 NTU less than about 3000 NTU, e.g. selected from the group comprising about 1000 NTU, about 1500 NTU, about 2000 NTU, about 2500 NTU, about 3000 NTU, about 3500 NTU, about 4000 NTU, about 4500 NTU and about 5000 NTU.
  • the present invention also comprises turbidity at any intermediate values of the ones said above.
  • the surface ratio between the first and the second depth filter is selected from the group comprising 1:1, 2:1, 1:2.
  • the primary clarification step and the secondary clarification step are performed by a single filter with an exclusion range comprised between about 1 pm and about 20 pm.
  • the second clarification is followed by a bioburden reduction.
  • bioburden reduction refers to the reduction of the number of microorganisms in the fluid obtained after the primary and secondary clarification. Normally bioburden reduction is considered the final step of the clarification process and comprises one or more steps of sterile purification.
  • the bioburden reduction is performed by at least one sterile filter having exclusion range equal to or less than about 0.5 pm. In a more specific embodiment, the bioburden reduction is performed by at least one sterile filter having exclusion range from about 0.2 pm to about 0.45 pm.
  • the secondary depth filtration may be followed by a further filtration performed by a membrane absorber with exclusion range equal to less than 0.2 pm.
  • the clarified cell culture may be further subjected to one or more steps of purification to isolate and recover the biomolecule of interest.
  • Standard purification methods include chromatographic techniques, including ion exchange, hydrophobic interaction, affinity, sizing or gel filtration, and reversed-phase, carried out at atmospheric pressure or at high pressure using systems such as FPLC and HPLC. Purification methods also include electrophoretic, immunological, precipitation, dialysis, and chromatofocusing techniques. Ultrafiltration and diafiltration techniques, in conjunction with protein concentration, are also useful.
  • Figure 1 Molecule A (left panel) and B (right panel) cell culture VCC and viability.
  • Figure 2 Molecule A (left panel) and B (right panel) cell culture titer concentration over the fed-batch concentration.
  • FIG. 3 Molecule A (experiments 1 to 8, 12 and 13) and B (experiments 9 to 11) cell culture PCV (Packed cell volume) and turbidity.
  • Figure 8 Screening of equilibration and recovery buffers runs overview.
  • Figure 12 Product yields obtained post screening of arginine-HCI recovery flush.
  • Figure 14 Screening on molecule B CCFs with arginine-HCI flush.
  • Molecule A (bispecific antibody) and B (IgG-like antibody) were produced by two different CHO cell lines derived from the same mother cell line.
  • Table 1 Overview of the cell culture fluid (CCF) used for the optimization of a reference clarification process.
  • CHO cells were thawed and expanded in Power-CHO-2-CD medium (serum free and chemically defined) from Lonza/Sartorius. The duration of a passage was either 2 or 3 days with the aim of reaching 3.0 x 10 s cells/mL at the end of the passage. Cells were diluted to respectively 0.4 x 10 s or 0.9 x 10 s cells/mL for a 2-day or a 3-day passage. Cells were scaled-up in shake flasks before the fed-batch inoculation at 0.8 x 10 s cells/mL.
  • Power-CHO-2-CD medium serum free and chemically defined
  • CHO cells were grown in fed-batch mode either in Thomson 5 L or in single-use BioBlu 3c bioreactors with a final working volume of 2 L or 3 L, respectively.
  • Power-CHO-2-CD medium from Lonza/Sartorius supplemented with 1 g/L of an in-house formulated Kolliphor (shear stress protector) solution was used as a basal medium.
  • the cell culture process was a 14 days fed-batch culture. The feeding started at day 2 consisting in a daily addition of a total of 30%f ee d voiume/working volume of Cell Boost 7a (feed "7a") and 3%feed voiume/working volume of Cell Boost 7b (feed "7b").
  • feeds 7a and 7b are manufactured by Cytiva (previously General Electric) and are chemically defined, animal-derived component-free supplements.
  • Feed 7a supplements cell culture with amino acids, vitamins, salts, trace elements and glucose while feed 7b is a concentrated supplement of amino acids with an alkaline pH. From day 5, glucose threshold and target were respectively set at 5 g/L and 6 g/L meaning that when the glucose concentration went below 5 g/L, in-house formulated glucose was added to reach 6 g/L.
  • CCFs were harvested when the viability dropped below 75 % or at day 14 even if the viability was still above 75 %.
  • molecule A and molecule B CCFs represent two extremes of CCFs for clarification experiments in terms of PCV, viability, turbidity and titer.
  • the cell culture monitoring was performed off-line using specific devices.
  • Viability, Viable Cells Concentration (VCC) and average cell diameter were daily determined using Vi- CellTMXR (Beckman Coulter Ref 38353, USA) cell counter. A sample of 500 pL (diluted if needed) from the cell culture was pumped into the analyser and then automatically mixed with trypan blue to evaluate the amount of dead and viable cells.
  • Metabolite concentrations (glucose, lactate, glutamine, glutamate, ammoniac, lactate dehydrogenase and IgG) were monitored thanks to the CEDEX Bio HT device from Roche which is a high-throughput automated metabolite analyser based on enzymatic assays coupled with spectrophotometry.
  • a 500 pL centrifuged sample was analysed each day of the cell culture process. pH of the cell culture was daily measured off-line for bioreactor culture or at day 0, day 7 and harvest day for Thomson culture using ABL80 Flex analyser from Radiometer. pCO2 and pO2 were also measured using the ABL80.
  • a reference clarification process is composed of 5 steps: a water for injection (WFI) flush, a 1 X phosphate buffer saline (PBS) 140 mM flush, a dead volume (DV) discard step, the filtration of the CCF and a 1 X PBS 140 mM recovery flush.
  • the aim of the WFI flush is to remove all leachables that could have remained in the filters from their manufacturing.
  • the PBS flush aimed to equilibrate the filter media to a suitable pH.
  • the dead volume discard corresponds to the discard of the filters void volume at the beginning of the filtration step to limit the dilution of the product.
  • the recovery flush goal is to recover a maximum of product that is still remaining in the filters.
  • At least one DV of PBS needs to be flushed to recover the harvest volume remaining in the filters and tubing. Another DV of PBS is then flushed to potentially unbind the remaining product bind in the filter media.
  • Table 2 The reference process steps, and flow details for bench scale clarification are shown in Table 2.
  • Bench scale clarification was performed with a primary and a secondary depth filters made of high capacity synthetic media, respectively 270 cm 2 and 140 cm 2 each.
  • the primary clarification depth filter had an exclusion range of 0.55-8 pm; the secondary clarification depth had an exclusion range of «0.1 pm.
  • the clarification runs were conducted based on the reference process limits, meaning a maximum of pressure of respectively 3 bars and 2.4 bars at small and bench scale (filter limit - supplier recommendation) and a turbidity of 20 NTU post clarification for both scales.
  • the classical filtration train used is composed of either a tri-headed FilterTech pump at small scale or peristaltic MasterFlex pumps at bench scale connected to Scilog pressure sensors.
  • the tri-headed pump allows to run three independent CCF clarifications in parallel at the same flow with three different conditions.
  • PCV packed cell volume
  • the PCV corresponds to the proportion of solids (cells, debris) present in the CCF. It is measured by transferring 5 mL of CCF in a 15 mL falcon which is then centrifuged for 5 min at 3000 g. The supernatant is then removed, and the falcon is weighted again to determine the mass of the cell pellet. The PCV is then the calculated with the following equation:
  • the turbidity is measured thanks to a turbidimeter from Hach which measured the diffused light at 90° at a wavelength of 860 nm in NTU.
  • the turbidity indicates the amount of material and small particles inside a liquid capable of light diffusion and is directly correlated with the haziness of a solution.
  • Samples are also taken during the filtration to measure the titer by CEDEX, as well as the glucose and the LDH concentrations.
  • the LDH rate is directly correlated to the cell lysis rate as LDH is an intracellular enzyme which will be detected in the supernatant only when cell lysis increase.
  • Glucose yield was always compared to product yield to ensure no loss of product by dilution effect.
  • Glucose yield was also compared to product yield to detect if any binding of the product was happening. Indeed, glucose is not expected to bind the filters. Therefore, almost 100 % glucose is expected to be recovered.
  • molecule B CCFs reached higher viabilities on the harvest day than molecule A CCFs. Indeed, on average, 54 % viability was obtained with molecule A cell culture while 71 % viability was reached with molecule B cell culture.
  • the Thomson 5L shape could have contributed to minimizing the gas exchange. Due to the specific shape of the Thomson 5L, the gas exchange surface gets smaller with increasing height of the cell culture volume.
  • the final cell culture volume was decreased from 3 L to 2 L to assess the impact of the final cell culture on the viability.
  • the final cell culture volume decreased to 2 L ( Figure 1, Experiment 5, 6 and 7)
  • the cells stayed alive slightly longer than the ones with a final cell culture volume at 3 L ( Figure 1, Experiment 1 to 4). Therefore, the high cell culture volume is the most likely cause explaining an early viability drop probably due to limited gas exchange.
  • the cell density peak is quite similar for both cell lines with around 15 - 20 x 10 s cells/mL at day 8.
  • Figure 2 presents the molecules A and B concentration over the fed-batch duration.
  • fed-batches producing molecule B were also more successful in terms of production compared to cell culture producing molecule A. Indeed, on average fed-batches producing molecule B reached 9 times more titer than fed-batches producing molecule A (0.3 g/L of molecule A and 2.8 g/L of molecule B).
  • the packed cell volume as well as the turbidity of the initial cell broth to be clarified were determined for each experiment.
  • the PCV corresponds to the proportion of solids (cells, debris) present in the CCF. It is measured by transferring 5 mL of CCF in a 15 mL falcon which is then centrifuged for 5 min at 3000 g. The supernatant is then removed, and the falcon is weighted again to determine the mass of the cell pellet. The turbidity is measured by a turbidimeter from Hach which measured the diffused light at 90° at a wavelength of 860 nm in NTU.
  • Example 3 Clarified cell culture fluids (CCCFs) characterization
  • FIG. 4 illustrates the reference clarification process CCCFs results for molecule A CCF clarification at small and bench scale.
  • small and bench scale clarification lead to similar product yield around 76 %.
  • a higher standard deviation is observed at small scale than at bench scale.
  • This variability at small scale can be explained by the low number of small-scale clarifications. Indeed, only 4 small-scale clarifications were performed compared to 9 bench-scale clarifications. Therefore, small-scale clarifications can be considered representative to bench-scale clarifications and CCCFs reference results from both scales can be pooled.
  • the initial difference between molecule A and molecule B CCFs could lead to significant differences in the composition of the CCCFs. Indeed, higher product yields obtained with molecule B CCCFs compared to molecule A CCCFs could be linked to saturation of binding sites on the membranes. If their quantity is constant, it should be expected that with high titer concentration CCFs, like molecule B CCFs, the saturation of the filters could be negligible compared to molecule A where low titer concentration CCFs are clarified.
  • molecule B CCFs contained much more impurities than molecule A CCFs.
  • the first parameter to assess was the clarification flow rate. A higher flow rate was expected to decrease the time of interaction between the product and filter potentially limiting the unwanted binding phenomenon and therefore less product loss.
  • the glucose yield was used to assess whether there was an error in dilution (as glucose is not supposed to bind to the membrane and should be fully recovered). Therefore, glucose yields compared to the product yields obtained with all these reference experiments were plotted to determine if a correlation was observed.
  • Figure 7 presents the high flow impact on the product yield. No significant difference was observed between the reference process and the high flow conditions meaning that the high flow did not impact the product recovery in terms of product yield. The high flow did also not impact the DSP process and the product quality (data not shown).
  • the aim of this experiment was to screen different equilibration and recovery buffers to either prevent the binding of the product on the filters or unbind the maximum of product potentially bound to the membrane filters.
  • recovery buffers were tested to unbind the maximum of product remaining in the filters.
  • Figure 9 depicts the yields obtained with the different salt concentration of PBS equilibration.
  • the salt concentration of the equilibration buffer does not have an impact on the product recovery but negatively impacted the state of the cells in the clarification filters (more lysis).
  • control condition (reference process - PBS recovery 140 mM) was taken from experiment 5 (screening of equilibration buffers) runs as experiments 5 and 6 CCFs were considered similar.
  • FIG. 10 illustrates the yields obtained with the different salt concentration of PBS recovery.
  • CaCL Calcium chloride
  • the CCF used for the experiment 7 had 1000 NTU more (2520 NTU) than the one used for experiment 5 (1532 NTU) and 3 % more PCV meaning that much more impurities (HCP and other molecules) were in the CCF of this experiment 7. It should be expected that when higher impurities are present in the CCF, they also could come in contact with the filter membranes and a competition phenomenon between the antibody and the impurities could appear leading to less binding of our product and a better recovery.
  • the aim of the recovery buffer is to remove the volume of CCF remaining in the filters at the end (filling the void volume of the filters), to ensure a maximum product recovery.
  • PBS is commonly used to flush 1 after clarification to enhance the product recovery.
  • PBS flush was not efficient for recovery of molecule A mainly and molecule B in a less critical manner, as shown above.
  • Other recovery flush, such as WFI were tested, however the use of WFI to make hydrophobic interactions weaker, did not help to recover more product.
  • Arginine-HCI buffer assures maximum product recovery.
  • Figure 12 shows the yields obtained for each experiment for the reference process (after 2 DV of PBS) and after the 2 added DV of arginine-HCI flush.
  • Arginine-HCI flush allowed to recover between 15 to 20 % more product leading to product yields of around 90 %. Arginine-HCI surprisingly enhanced the yield titer, unbinding the product remaining in the filters. Additionally, a second experiment, where PBS flush was directly replaced by arginine-HCI flush, was performed. For this purpose, the PBS flush is directly replaced by an arginine-HCI flush for the trials 2 (both experiment 1 and 2).
  • the first one is that after 2 DVs of arginine-HCI flush, on average 15 % (85.4 - 69.9) more product is recovered than when 2 DVs of PBS are flushed showing that arginine-HCI was responsible for unbinding more product.
  • arginine-HCI flush showed great promise by increasing the product yield by 15 % on average only after a 2 DV flush while a 4 DV flush was needed to reach the same product yield with PBS.

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Abstract

La présente invention concerne la clarification de culture cellulaire. En particulier, la présente invention concerne des procédés de récupération élevée d'une biomolécule d'intérêt, telle qu'un anticorps, à partir d'un filtre de clarification de culture cellulaire. Plus particulièrement, l'invention concerne des procédés de récupération élevée d'une biomolécule d'intérêt à partir d'un filtre de clarification de culture cellulaire à l'aide d'un tampon arginine-HCl. La présente invention concerne également des procédés de clarification de culture cellulaire comprenant une étape de filtration dans laquelle la récupération de la biomolécule d'intérêt à partir du filtre est maximisée par une étape de rinçage de récupération effectuée avec un tampon arginine-HCl.
PCT/EP2023/058087 2022-04-13 2023-03-29 Procédés de récupération élevée d'un produit de clarification de culture cellulaire WO2023198450A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050164929A1 (en) * 2000-11-06 2005-07-28 Lupine Logic, Inc. Methods of preventing and treating inflammatory bowel disease
WO2022008658A1 (fr) * 2020-07-10 2022-01-13 Grifols Worldwide Operations Limited Procédé d'obtention d'une composition comprenant une immunoglobuline m dérivée du plasma humain

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050164929A1 (en) * 2000-11-06 2005-07-28 Lupine Logic, Inc. Methods of preventing and treating inflammatory bowel disease
WO2022008658A1 (fr) * 2020-07-10 2022-01-13 Grifols Worldwide Operations Limited Procédé d'obtention d'une composition comprenant une immunoglobuline m dérivée du plasma humain

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
"FUNDAMENTAL IMMUNOLOGY", 1989, RAVEN PRESS

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