Optimized Transfection Protocol

Abstract

The present invention relates to an optimized method for transfecting cells that requires fewer steps and less DNA than previously disclosed methods. In addition, the method saves scientists days of experimentation.

Description

[Technical field]

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 317,959, filed March 9, 2022, which is incorporated by reference in its entirety.

[0002] Incorporation by Reference The entire Sequence Listing in XML format, identifiable by the following file properties, is incorporated by reference: Filename: 10071-WO01-SEC.XML; File Size: 7,813 bytes; Created: February 17, 2023.

[0003] The present invention relates to an optimized method for transfecting cells that requires fewer steps and less DNA than previously disclosed methods. [Background technology]

[0004] In 2013, Jager et al. (Jager V, Bussow K, Wagner A, Weber S, Hust M, Frenzel A, Schirrmann T. High level transient production of recombinant antibodies and antibody fusion proteins in HEK293 cells. BMC Biotechnol 2013;13:52) reported an optimized HEK293-6E protocol for the expression of monospecific scFv-Fc antibodies. To date, the paper has been cited 163 times in both academic and industrial publications, suggesting that the protocol is widely applied. Briefly, in this protocol, cells are transfected with 0.5mg / L DNA using PEI reagent, and after transfection, they are fed with Sheff-Vax+Tryptone N1 one day later, then glucose is fed a second time on day 3, and 3.75mM sodium valproic (VPA) is added on day 4. Even though Jager's protocol has been shown to maintain recombinant protein expression at high yields and relatively high throughput, the inventors rationalized that this protocol could be further optimized. Furthermore, Jager's protocol only considered scFv-Fc antibodies. In recent years, multispecific antibodies that can simultaneously target multiple targets have shown increasing interest in therapeutic development. Therefore, new HEK293-6E protocols need to be optimized and validated to express good quality multispecific antibodies with high yields and throughput.

[0005] Our goal is to develop a HEK293-6E protocol that can maintain high yields of multispecific antibodies, conserve resources, and increase throughput.

[0006] Transient transfection of human embryonic kidney 293 (HEK293-6E) cells is widely used in the biotechnology industry to rapidly produce recombinant proteins throughout preclinical development. In 2013, Jager et al. reported a HEK293-6E protocol for high-throughput expression of monospecific scFv-Fc antibodies. The present invention is an improvement of Jager's protocol. Compared with Jager's protocol, Grace_v1 requires 50% less DNA and no FBS, and reduces one experimental step, while showing comparable protein yields. With the goal of further optimizing the HEK293-6E protocol, the inventors designed nine new protocols that modified key steps of Grace_v1. When these nine protocols were evaluated together with Grace_v1 in expressing multiple mAbs and multispecific antibodies, two new protocols (Grace_v2 and _v3) showed comparable protein yields and quality to Grace_v1. Surprisingly, these two new protocols do not require the addition of VPA after transfection. Omitting VPA not only saves resources, but also significantly increases experimental throughput and improves flexibility. Furthermore, compared to Jager's protocol, Grace_v2 and _v3 can save resources (50% less DNA, no FBS) and increase throughput (two fewer experimental steps). [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Jager V,Bussow K,Wagner A,Weber S,Hust M,Frenzel A,Schirrmann T.High level transient production of recombinant antibodies and antibody fusion proteins in HEK293 cells.BMC Biotechnol 2013;13:52 Summary of the Invention [Means for solving the problem]

[0008] In one aspect, the invention provides a method for transfecting a population of mammalian cells with DNA encoding a target molecule, comprising the steps of: (a) providing cells in a cell culture medium; (b) carrying out a transfection step by contacting cells with a liposome / DNA complex, the ratio of mg DNA:cell number being about 0.25 mg:1×10 9 Step 1: Cells (c) about 40-56 hours after the transfection step, adding the following to the cell culture medium in any order: Tryptone N1 at a final concentration of approximately 4.5-5.5 g / L; Glucose at a final concentration of about 4.0-5.0 g / L and Approximately 0.8 to 1.2 volumes of fresh cell culture medium, (d) harvesting the target molecule from the cell culture medium about 144 to 192 hours after the transfection step.

[0009] In one embodiment, step (c) is carried out about 48 hours after the transfection step.

[0010] In one aspect, the invention provides a method for transfecting a population of mammalian cells with DNA encoding a target molecule, comprising the steps of: (a) providing cells in a cell culture medium; (b) carrying out a transfection step by contacting cells with a liposome / DNA complex, the ratio of mg DNA:cell number being about 0.25 mg:1×10 9 Step 1: Cells (c) about 2-6 hours after the transfection step, adding the following to the cell culture medium in any order: Tryptone N1 at a final concentration of approximately 4.5-5.5 g / L; Glucose at a final concentration of about 4.0-5.0 g / L and Approximately 0.8 to 1.2 volumes of fresh cell culture medium, (d) harvesting the target molecule from the cell culture medium about 144 to 192 hours after the transfection step.

[0011] In one embodiment, step (c) is carried out about 4 hours after the transfection step.

[0012] In one embodiment, the cells are suspension cells.

[0013] In one embodiment, the cells are adherent cells.

[0014] In one embodiment, the cell is selected from the group consisting of CHO cells, CHOK1 cells, DXB-11 cells, DG-44 cells, COS-7 cells, HEK293-6E cells, BHK cells, TM4 cells, CV1 cells, VERO-76 cells, HELA cells, MDCK cells, BRL 3A cells, W138 cells, Hep G2 cells, MMT cells, TRI cells, MRC 5 cells, and FS4 cells.

[0015] In one embodiment, the cell is a HEK293-6E cell.

[0016] In one embodiment, the cells are about 1×10 5 ~1X10 7 Cells / ml are seeded.

[0017] In one embodiment, the cells are about 1×10 6 / ml.

[0018] In one embodiment, the final concentration of Tryptone N1 is about 5.0 g / L.

[0019] In one embodiment, the final glucose concentration is about 4.5 g / L.

[0020] In one embodiment, about 1 volume of fresh cell culture medium is added.

[0021] In one embodiment, approximately 88-104 hours after the transfection step, valproic acid is added to a final concentration of approximately 3.5-4.0 mM.

[0022] In one embodiment, the final concentration of valproic acid is about 3.75 mM.

[0023] In one embodiment, step (d) is carried out about 168 hours after the transfection step.

[0024] In one embodiment, the valproic acid is added about 96 hours after the transfection step.

[0025] In one embodiment, the targeting molecule is a multispecific antigen-binding protein. [Brief description of the drawings]

[0026] [Figure 1A-1C] Figure 1 shows a comparison of Jager's protocols: Grace_v1 showed higher antibody expression (A), better cell growth (B) and comparable cell viability (C). [Figure 2A] FIG. 2 shows a schematic of the molecule (A) and the experimental design (B). [Figure 2B] FIG. 2 shows a schematic of the molecule (A) and the experimental design (B). [Figure 3A-3C] Figure 3: Comparison of 10 HEK293-6E protocols for expression of 3 mAbs. Grace_v1 (also called protocol_6) is highlighted in red. (A) Viable cell density (VCD) and percentage (%) at harvest. (B) Protein yield after HT kingfisher purification with ProA beads. (C) Analysis of ProA purified proteins by non-reducing MCE and SEC. Intact IgG appears as MP in MCE and SEC. [Figure 4A-4C]Figure 4: Comparison of 10 HEK293-6E protocols for expression of two hetero-IgG bispecific antibodies. Grace_v1 (also called protocol_6) is highlighted in red. (A) Viable cell density (VCD) and percentage (%) at harvest. (B) Protein yield after HT kingfisher purification with ProA beads. (C) Analysis of ProA purified proteins by non-reducing MCE and SEC. Intact IgG appears as MP in MCE and SEC. [Figure 5A] Figure 5 shows a comparison of ten HEK293-6E protocols for the expression of three IgG-scFvs, three IgG-Fabs and four trispecific antibodies. The current 293-6E protocol (#6) is highlighted in red. (A) Protein yields of three IgG-scFvs after HT kingfisher purification with ProA beads. (B) Protein yields of three IgG-Fabs. (C) Protein yields of four trispecific antibodies. [Figure 5B] Figure 5 shows a comparison of ten HEK293-6E protocols for the expression of three IgG-scFvs, three IgG-Fabs and four trispecific antibodies. The current 293-6E protocol (#6) is highlighted in red. (A) Protein yields of three IgG-scFvs after HT kingfisher purification with ProA beads. (B) Protein yields of three IgG-Fabs. (C) Protein yields of four trispecific antibodies. [Figure 5C] Figure 5 shows a comparison of ten HEK293-6E protocols for the expression of three IgG-scFvs, three IgG-Fabs and four trispecific antibodies. The current 293-6E protocol (#6) is highlighted in red. (A) Protein yields of three IgG-scFvs after HT kingfisher purification with ProA beads. (B) Protein yields of three IgG-Fabs. (C) Protein yields of four trispecific antibodies. [Figure 6A]FIG. 6 shows that miniaturized Golden Gate reactions improved ModVec efficiency. A) Schematic of the 11 kb 14-piece (including vector backbone) ModVec assembly. B) Golden Gate overhangs used for this assembly. The underlined overhangs were frequently observed to mispair with non-complementary overhangs. C) Efficiency of ModVec assembly at various Golden Gate reaction volumes. Validation of two improved HEK293-6E protocols (Grace_v2 and Grace_v3) based on experimental simplicity, ProA yield, and product quality. (A) Corrected ProA yield shows that Grace_v2 and _v3 express comparable amounts of protein as Grace_v1. In addition, these two new protocols require one less experimental step than the current protocol. (B) Analysis of ProA purified proteins by non-reducing MCE shows that the two new protocols produce mAbs and bispecific antibodies of comparable quality to the current protocol. [Figure 6B]FIG. 6 shows that miniaturized Golden Gate reactions improved ModVec efficiency. A) Schematic of the 11 kb 14-piece (including vector backbone) ModVec assembly. B) Golden Gate overhangs used for this assembly. The underlined overhangs were frequently observed to mispair with non-complementary overhangs. C) Efficiency of ModVec assembly at various Golden Gate reaction volumes. Validation of two improved HEK293-6E protocols (Grace_v2 and Grace_v3) based on experimental simplicity, ProA yield, and product quality. (A) Corrected ProA yield shows that Grace_v2 and _v3 express comparable amounts of protein as Grace_v1. In addition, these two new protocols require one less experimental step than the current protocol. (B) Analysis of ProA purified proteins by non-reducing MCE shows that the two new protocols produce mAbs and bispecific antibodies of comparable quality to the current protocol. [Figure 7A] Figure 7 shows a summary of HEK293-6E protocols including Jager, Grace_v1, _v2 and _v3. (A) Protocol details. Compared to Jager's protocol, Grace_v1-_v3 improve protein yield, require less DNA for transfection and reduce experimental steps (higher throughput). (B) Grace_v2 and _v3 show higher throughput and better flexibility than Grace_v1. [Figure 7B] Figure 7 shows a summary of HEK293-6E protocols including Jager, Grace_v1, _v2 and _v3. (A) Protocol details. Compared to Jager's protocol, Grace_v1-_v3 improve protein yield, require less DNA for transfection and reduce experimental steps (higher throughput). (B) Grace_v2 and _v3 show higher throughput and better flexibility than Grace_v1. [Figure 8]FIG. 8 shows a table summarizing the various methods of the claimed invention compared to the Jager protocol. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] The present invention relates to a method for transfecting a population of cells with DNA. In one embodiment, the DNA comprises genomic DNA. In one embodiment, the DNA is a vector or plasmid. The present invention utilizes vectors comprising one or more nucleic acids encoding one or more components of a multispecific antigen binding protein (e.g., variable regions, light chains, heavy chains, modified heavy chains, and Fd fragments). The term "vector" refers to any molecule or entity (e.g., nucleic acid, plasmid, bacteriophage, or virus) used to transfer protein coding information into a host cell. Examples of vectors include, but are not limited to, plasmids, viral vectors, non-episomal mammalian vectors, and expression vectors, such as recombinant expression vectors. The term "expression vector" or "expression construct" as used herein refers to a recombinant DNA molecule that contains a desired coding sequence and appropriate nucleic acid control sequences necessary for expression of the operably linked coding sequence in a particular host cell. Expression vectors can include, but are not limited to, sequences that affect or control transcription, translation, and, when introns are present, RNA splicing of the coding region operably linked thereto. Nucleic acid sequences necessary for expression in prokaryotes include a promoter, optionally an operator sequence, a ribosome binding site, and possibly other sequences. Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals. A secretory signal peptide sequence can also be optionally encoded by the expression vector and operably linked to the coding sequence of interest, so that the expressed polypeptide can be secreted by the recombinant host cell, if desired, so that the polypeptide of interest can be more easily isolated from the cell.In certain embodiments, the signal peptide is selected from the group consisting of MDMRVPAQLLGLLLLWLRGARC (SEQ ID NO: 1), MAWALLLLTLLTQGTGSWA (SEQ ID NO: 2), MTCSPLLLTLLIHCTGSWA (SEQ ID NO: 3), MEAPAQLLFLLLLWLPDTTG (SEQ ID NO: 4), MEWTWRVLFLVAAATGAHS (SEQ ID NO: 5), METPAQLLFLLLLWLPDTTG (SEQ ID NO: 6), MKHLWFFLLLVAAPRWVLS (SEQ ID NO: 7), and MEWSWVFLFFLSVTTGVHS (SEQ ID NO: 8).

[0028] Typically, expression vectors used in host cells to produce multispecific antigen binding proteins will contain sequences for plasmid maintenance and sequences for cloning and expression of exogenous nucleotide sequences encoding components of the multispecific antigen binding protein. Such sequences, collectively referred to as "flanking sequences", will typically include, in certain embodiments, one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a complete intron sequence containing donor and acceptor splice sites, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for inserting a nucleic acid encoding a polypeptide to be expressed, and a selectable marker element. Each of these sequences is discussed below.

[0029] Optionally, the vector may contain a "tag" coding sequence, i.e., an oligonucleotide molecule located at the 5' or 3' end of the polypeptide coding sequence, which encodes polyHis (such as hexaHis), FLAG, HA (influenza virus hemagglutinin), myc or another "tag" molecule for which there are commercially available antibodies. This tag is typically fused to the polypeptide upon expression of the polypeptide and can serve as a means for affinity purification or detection of the polypeptide from the host cell. Affinity purification can be achieved, for example, by column chromatography using antibodies against the tag as an affinity matrix. Optionally, the tag can then be removed from the purified polypeptide by various means, such as using certain cleavage peptidases.

[0030] The flanking sequences may be homologous (i.e., derived from the same species and / or strain as the host cell), heterologous (i.e., derived from a species other than the species or strain of the host cell), hybrid (i.e., a combination of flanking sequences derived from more than one source), synthetic or natural. Thus, the source of the flanking sequences may be any prokaryotic or eukaryotic organism, any vertebrate or invertebrate organism, or any plant, provided that the flanking sequences are capable of functioning in and being activated by the host cell machinery.

[0031] The flanking sequences useful in the vector of the present invention may be obtained by any of several methods well known in the art. Typically, the flanking sequences useful herein will have been previously identified by mapping and / or restriction endonuclease digestion and can be isolated from a suitable tissue source using a suitable restriction endonuclease. In some cases, the entire nucleotide sequence of the flanking sequence may be known. In this case, the flanking sequence may be synthesized using conventional methods for nucleic acid synthesis or cloning.

[0032] Whether all or only a portion of the flanking sequences are known, the flanking sequences may be obtained using polymerase chain reaction (PCR) and / or by screening a genomic library with suitable probes, such as oligonucleotides and / or flanking sequence fragments from the same or another species. If the flanking sequences are unknown, fragments of DNA containing the flanking sequences may be isolated from a larger piece of DNA that may contain, for example, a coding sequence or even another gene(s). Isolation may be achieved by producing suitable DNA fragments by restriction endonuclease digestion, followed by isolation using agarose gel purification, Qiagen® column chromatography (Chatsworth, Calif.) or other methods known to those of skill in the art. The selection of suitable enzymes to accomplish this purpose will be readily apparent to those of skill in the art.

[0033] An origin of replication is typically a part of commercially available prokaryotic expression vectors, which aids in the amplification of the vector in a host cell. If the vector of choice does not contain an origin of replication site, one may be chemically synthesized based on a known sequence and ligated into the vector. For example, the origin of replication from the plasmid pBR322 (New England Biolabs, Beverly, Mass.) is suitable for most Gram-negative bacteria, and various viral origins (e.g., SV40, polyoma, adenovirus, vesicular stomatitis virus (VSV) or papillomavirus, such as HPV or BPV) are useful for cloning vectors in mammalian cells. In general, the origin of replication component is not needed for mammalian expression vectors (e.g., the SV40 origin is often used only because it also contains the viral early promoter).

[0034] A transcription termination sequence is typically located at the 3' end of a polypeptide coding region and serves to terminate transcription. Usually, a transcription termination sequence in a prokaryotic cell is a GC-rich fragment followed by a poly-T sequence. This sequence can be easily cloned from a library or even commercially available as part of a vector, but can also be easily synthesized using known nucleic acid synthesis methods.

[0035] A selectable marker gene encodes a protein necessary for the survival and growth of a host cell grown in a selective culture medium. Typical selectable marker genes encode proteins that (a) confer resistance to antibiotics or other toxins, such as ampicillin, tetracycline or kanamycin, to the prokaryotic host cell, (b) complement an auxotrophic deficiency of the cell, or (c) supply vital nutrients not available from complex or defined media. Specific selectable markers are the kanamycin resistance gene, the ampicillin resistance gene and the tetracycline resistance gene. Advantageously, the neomycin resistance gene may also be used for selection in both prokaryotic and eukaryotic host cells.

[0036] Other selection genes may be used to amplify the gene that will be expressed. Amplification is the process by which genes required for the production of a protein critical for growth or cell survival are repeated in tandem in the chromosomes of successive generations of recombinant cells. Examples of suitable selection markers for mammalian cells include the dihydrofolate reductase (DHFR) gene and the promoterless thymidine kinase gene. The mammalian cell transformants are placed under selection pressure in which only the transformants are adapted to survive due to the selection gene present in the vector. Selection pressure is imposed by culturing the transformed cells under conditions in which the concentration of the selection agent in the medium is successively increased, thereby leading to the amplification of both the selection gene and the DNA encoding another gene, such as one or more components of the multispecific antigen binding protein described herein. As a result, large amounts of polypeptides are synthesized from the amplified DNA.

[0037] A ribosome binding site is usually necessary for translation initiation of mRNA and is characterized by a Shine-Dalgarno sequence (prokaryotes) or a Kozak sequence (eukaryotes). This element is typically located 3' to the promoter and 5' to the coding sequence of the polypeptide to be expressed. In certain embodiments, one or more coding regions may be operably linked to an internal ribosome binding site (IRES), allowing translation of two open reading frames from a single RNA transcript.

[0038] In some cases, such as when glycosylation is desired in a eukaryotic host cell expression system, various pre- or pro-sequences may be engineered to improve glycosylation or yield. For example, the peptidase cleavage site of a particular signal peptide may be modified or a pro-sequence may be added, which may also affect glycosylation. The final protein product may have one or more additional amino acids associated with expression at position -1 (relative to the first amino acid of the mature protein), which may not be completely removed. For example, the final protein product may have one or two amino acid residues found within the peptidase cleavage site attached to the amino terminus. Alternatively, the use of some enzyme cleavage sites may result in a slightly truncated form of the desired polypeptide when the enzyme cleaves at such a region within the mature polypeptide.

[0039] Expression and cloning vectors will typically contain a promoter that is recognized by the host organism and operably linked to a molecule encoding a polypeptide. As used herein, the term "operably linked" refers to two or more nucleic acid sequences being linked together to produce a nucleic acid molecule capable of inducing the transcription of a given gene and / or the synthesis of a desired protein molecule. For example, a control sequence in a vector "operably linked" to a protein coding sequence is ligated to the protein coding sequence such that expression of the protein coding sequence is achieved under conditions compatible with the transcriptional activity of the control sequence. More specifically, a promoter and / or enhancer sequence, including any combination of cis-acting transcriptional control elements, is operably linked to a coding sequence if it stimulates or regulates the transcription of the coding sequence in an appropriate host cell or other expression system.

[0040] A promoter is a non-transcribed sequence located upstream (i.e., 5') of the start codon of a structural gene (generally within about 100-1000 bp) and which controls transcription of the structural gene. Promoters are conventionally classified into one of two classes: inducible promoters and constitutive promoters. An inducible promoter initiates increased levels of transcription from the DNA under its control in response to any change in culture conditions, such as the presence or absence of a nutrient or a change in temperature. A constitutive promoter, on the other hand, transcribes the gene to which it is operably linked uniformly, i.e., with little or no control over gene expression. A large number of promoters recognized by a variety of potential host cells are well known. A suitable promoter is operably linked to DNA encoding, for example, the heavy chain, light chain, modified heavy chain or other component of a multispecific antigen-binding protein by removing the promoter from the source DNA by restriction enzyme digestion and inserting the desired promoter sequence into the vector.

[0041] Suitable promoters for use with yeast hosts are also well known in the art. Yeast enhancers are advantageously used with yeast promoters. Suitable promoters for use with mammalian host cells are well known and include, but are not limited to, those obtained from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (such as adenovirus type 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus, and most preferably simian virus 40 (SV40). Other suitable mammalian promoters include heterologous mammalian promoters, such as heat shock promoters and actin promoters.

[0042] Additional promoters that may be of interest include the SV40 early promoter (Benoist and Chambon, 1981, Nature 290:304-310); the CMV promoter (Thornsen et al., 1984, Proc. Natl. Acad. USA 81:659-663); the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto et al., 1980, Cell 22:787-797); the herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. USA 78:1444-1445); promoters and regulatory sequences from metallothionein genes Prinster et al., 1982, Nature 296:39-42) and prokaryotic promoters such as the beta-lactamase promoter (Villa-Kamaroff et al., 1982, Nature 296:39-42). al., 1978, Proc. Natl. Acad. Sci. USA 75:3727-3731) or the tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80:21-25). Also of interest are the following animal transcriptional control regions that exhibit tissue specificity and have been utilized in transgenic animals: the elastase I gene control region, which is active in pancreatic acinar cells (Swift et al., 1984, Cell 38:639-646; Ornitz et al., 1986, Cold Spring Harbor Symp. Quant. Biol. 50:399-409; MacDonald, 1987, Hepatology 7:425-515); the insulin gene control region, which is active in pancreatic beta cells (Hanahan, 1985, Nature 315:115-122); the immunoglobulin gene control region, which is active in lymphoid cells (Grosschedl et al., 1984, Cell 38:647-658; Adames et al., 1985, Nature 318:533-538; Alexander et al., 1987, Mol. Cell. Biol.7:1436-1444); the mouse mammary tumor virus control region, which is active in testis, breast, lymphocytes, and mast cells (Leder et al., 1986, Cell 45:485-495); the albumin gene control region, which is active in the liver (Pinkert et al., 1987, Genes and Devel. 1:268-276); the alpha-fetoprotein gene control region, which is active in the liver (Krumlauf et al., 1985, Mol. Cell. Biol. 5:1639-1648; Hammer et al., 1987, Science 253:53-58); the alpha 1-antitrypsin gene control region, which is active in the liver (Kelsey et al., 1987, Genes and Devel. 1:161-171); the beta-globin gene control region, which is active in bone marrow cells (Mogram et al., 1985, Nature 315:338-340; Kollias et al., 1986, Cell 46:89-94); the myelin basic protein gene control region, which is active in oligodendrocyte cells in the brain (Readhead et al., 1987, Cell 48:703-712); the myosin light chain-2 gene control region, which is active in skeletal muscle (Sani, 1985, Nature 314:283-286), and the gonadotropin releasing hormone gene control region, which is active in the hypothalamus (Mason et al., 1986, Science 234:1372-1378).

[0043] Enhancer sequences may be inserted into vectors to increase transcription of DNA encoding components of a multispecific antigen-binding protein (e.g., light chain, heavy chain, modified heavy chain, Fd fragment) by higher eukaryotes. Enhancers are cis-acting elements of DNA, usually about 10-300 bp in length, that act on a promoter to increase transcription. Enhancers are relatively orientation and position independent and have been found at both 5' and 3' positions relative to the transcription unit. Several enhancer sequences are known that are available from mammalian genes (e.g., globin, elastase, albumin, alpha-fetoprotein, and insulin). However, typically enhancers from viruses are used. SV40 enhancers, cytomegalovirus early promoter enhancers, polyoma enhancers, and adenovirus enhancers known in the art are exemplary enhancing elements for the activation of eukaryotic promoters. Enhancers may be located in the vector either 5' or 3' to the coding sequence, but are typically located at a site 5' from the promoter. A sequence encoding a suitable native or heterologous signal sequence (leader sequence or signal peptide) can be incorporated into the expression vector to facilitate extracellular secretion of the antibody. The choice of signal peptide or leader depends on the type of host cell in which the antibody is to be produced, and the native signal sequence can be replaced with a heterologous signal sequence. Examples of signal peptides are described above. Other signal peptides that function in mammalian host cells include the interleukin-7 (IL-7) signal sequence described in U.S. Pat. No. 4,965,195; the interleukin-2 receptor signal sequence described in Cosman et al., 1984, Nature 312:768; the interleukin-4 receptor signal peptide described in European Patent No. 0 367 566; the type I interleukin-1 receptor signal peptide described in U.S. Pat. No. 4,968,607; and the type II interleukin-1 receptor signal peptide described in European Patent No. 0 460 846.

[0044] The expression vector provided may be constructed from a starting vector, such as a commercially available vector. Such vector may or may not contain all of the desired flanking sequences. If one or more of the flanking sequences described herein are not originally present in the vector, they may be obtained individually and ligated into the vector. The method used to obtain each of the flanking sequences is well known to those skilled in the art. The expression vector can be introduced into a host cell, thereby producing a protein, including a fusion protein, encoded by the nucleic acid described herein.

[0045] In certain embodiments, the nucleic acids encoding the different components of a multispecific antigen-binding protein may be inserted into the same expression vector. In such embodiments, the two nucleic acids may be separated by an internal ribosome entry site (IRES) and under the control of a single promoter, such that the light and heavy chains are expressed from the same mRNA transcript. Alternatively, the two nucleic acids may be under the control of two separate promoters, such that the light and heavy chains are expressed from two separate mRNA transcripts.

[0046] Similarly, for an IgG-scFv multispecific antigen binding protein, the nucleic acid encoding the light chain may be cloned into the same expression vector as the nucleic acid encoding the modified heavy chain (a fusion protein comprising the heavy chain and scFv), where the two nucleic acids are under the control of a single promoter and separated by an IRES, or the two nucleic acids are under the control of two separate promoters. For an IgG-Fab multispecific antigen binding protein, the nucleic acids encoding each of the three components may be cloned into the same expression vector. In some embodiments, the nucleic acid encoding the light chain of the IgG-Fab molecule and the nucleic acid encoding the second polypeptide (comprising the remaining half of the C-terminal Fab domain) are cloned into one expression vector, whereas the nucleic acid encoding the modified heavy chain (a fusion protein comprising the heavy chain and half of the Fab domain) is cloned into a second expression vector. In certain embodiments, all components of the multispecific antigen binding protein described herein are expressed from the same host cell population. For example, even if one or more components are cloned into separate expression vectors, the host cell is transfected simultaneously with both expression vectors, such that one cell produces all components of the multispecific antigen binding protein.

[0047] After a vector has been constructed and one or more nucleic acid molecules encoding the components of a multispecific antigen binding protein described herein have been inserted into the appropriate sites of the vector, the completed vector may be inserted into a suitable host cell for amplification and / or polypeptide expression. Thus, the present invention encompasses an isolated host cell comprising one or more expression vectors encoding the components of a multispecific antigen binding protein. As used herein, the term "host cell" refers to a cell that has been transformed or is capable of being transformed by a nucleic acid, thereby expressing a gene of interest. The term includes the progeny of a parent cell, whether or not the morphology or genetic make-up of the progeny is identical to the original parent cell, so long as the gene of interest is present. A host cell containing an isolated nucleic acid, preferably operably linked to at least one expression control sequence (e.g., a promoter or enhancer), is a "recombinant host cell".

[0048] When cultured under appropriate conditions, the host cells will synthesize the antigen binding protein, which can then be recovered from the culture medium (if the host cells secrete it into the medium) or directly from the host cells that produce it (if it is not secreted). The selection of an appropriate host cell will depend on a variety of factors, such as the desired expression level, modifications of the polypeptide that are desirable or necessary for activity (such as glycosylation or phosphorylation), and the ease of folding into a biologically active molecule.

[0049] Exemplary host cells include prokaryotes, yeast or higher eukaryotic cells. Prokaryotic host cells include eubacteria, such as gram-negative or gram-positive microorganisms, for example Escherichia, Enterobacteriaceae, such as E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, such as Salmonella typhimurium, Serratia, such as Serratia marcescans and Shigella, and Bacillus, such as B. subtilis and B. licheniformis, Pseudomonas and Streptomyces. Eukaryotic microorganisms, such as filamentous fungi or yeast, are suitable cloning or expression hosts for recombinant polypeptides. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms.However, Pichia, e.g. P. pastoris, Schizosaccharomyces pombe; Kluyveromyces, Yarrowia; Candida; Trichoderma reesia; Neurospora crassa; Schwanniomyces, e.g. Schwanniomyces occidentalis; Several other genera, species, and strains are commonly available and useful herein, such as A. occidentalis, and filamentous fungi such as Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts, such as A. nidulans and A. niger.

[0050] Host cells for the expression of glycosylated antigen-binding proteins can be derived from multicellular organisms. Examples of invertebrate cells include plant cells and insect cells. Many strains and mutants of baculoviruses and corresponding insect permissive host cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly) and Bombyx mori have been identified. Various virus strains for transfection of such cells are publicly available, for example the L-1 mutant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV.

[0051] Vertebrate host cells are also suitable hosts, and recombinant production of antigen binding proteins from such cells is routine. Mammalian cell lines available as hosts for expression are well known in the art and include, but are not limited to, immortalized cell lines available from the American Type Culture Collection (ATCC), such as, but not limited to, Chinese hamster ovary (CHO) cells, e.g., CHOK1 cells (ATCC CCL61), DXB-11, DG-44 and Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216, 1980); monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney lines (293 cells or 293 cells subcloned for growth in suspension culture) (Graham et al., J. Gen Virol. 36:59, 1977), such as HEK293-6E cells; baby hamster kidney cells (BHK, ATCC CCL 10); mouse Sertoli 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-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); dog kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocellular carcinoma cells (Hep G2, HB 8065); mouse mammary carcinoma (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals NY Acad. Sci. 383:44-68, 1982); MRC 5 cells or FS4 cells; mammalian myeloma cells and several other cell lines. In another embodiment, a cell line derived from the B cell lineage can be selected that does not produce its own antibodies but has the ability to produce and secrete heterologous antibodies. CHO cells are in some embodiments the preferred host cells for expressing multispecific antigen binding proteins.

[0052] Host cells for the production of multispecific antigen-binding proteins are transformed or transfected with the above-described nucleic acids or vectors and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying genes encoding the desired sequences. In addition, novel vectors and transfected cell lines with multiple copies of transcription units separated by a selectable marker are particularly useful for expressing antigen-binding proteins. Thus, the present invention also provides a method for preparing a multispecific antigen-binding protein as described herein, comprising culturing a host cell comprising one or more expression vectors as described herein in a culture medium under conditions that allow expression of the multispecific antigen-binding protein encoded by the one or more expression vectors, and recovering the multispecific antigen-binding protein from the culture medium.

[0053] The host cells used to produce the antigen binding protein may be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), Minimum Essential Medium ((MEM) (Sigma)), RPMI-1640 (Sigma) and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing the host cells. In addition, any of the media described in Ham et al., Meth. Enz. 58:44, 1979; Barnes et al., Anal. Biochem. 102:255, 1980; U.S. Pat. No. 4,767,704; U.S. Pat. No. 4,657,866; U.S. Pat. No. 4,927,762; U.S. Pat. No. 4,560,655; or U.S. Pat. No. 5,122,469; WO 90103430; WO 87 / 00195; or U.S. Reissue Pat. No. 30,985 may be used as culture media for the host cells. Any of these media may be supplemented as necessary with hormones and / or other growth factors (e.g., insulin, transferrin, or epidermal growth factor), salts (e.g., sodium chloride, calcium, magnesium, and phosphate), buffers (e.g., HEPES), nucleotides (e.g., adenosine and thymidine), antibiotics (e.g., the drug Gentamicin™), trace elements (usually defined as inorganic compounds present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary nutritional supplements may also be included at appropriate concentrations that would be known to one of skill in the art. Culture conditions such as temperature, pH, etc. are those previously used with the host cell selected for expression and will be known to one of skill in the art.

[0054] When the host cells are cultured, the multispecific antigen-binding proteins can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the antigen-binding proteins are produced intracellularly, as a first step, the particulate debris, host cells or lysed fragments are removed, for example, by centrifugation or ultrafiltration. The bispecific antigen-binding proteins can be purified, for example, using hydroxyapatite chromatography, cation or anion exchange chromatography, or preferably affinity chromatography using the antigen of interest or protein A or protein G as affinity ligand. Protein A can be used to purify proteins, including polypeptides based on human gamma 1, gamma 2, or gamma 4 heavy chains (Lindmark et al., J. Immunol. Meth. 62:1-13, 1983). Protein G is recommended for all mouse isotypes and human gamma 3 (Guss et al., EMBO J. 5:15671575, 1986). The matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Physically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. If the protein contains a CH3 domain, Bakerbond ABX™ resin (JT Baker, Phillipsburg, NJ) is useful for purification. Depending on the particular polyspecific antigen-binding protein to be recovered, other techniques for protein purification such as ethanol precipitation, reversed-phase HPLC, chromatofocusing, SDS-PAGE and ammonium sulfate precipitation are also possible.

[0055] As used herein, the term "antibody" refers to a tetrameric immunoglobulin protein comprising two light chain polypeptides (each about 25 kDa) and two heavy chain polypeptides (each about 50-70 kDa). The term "light chain" or "immunoglobulin light chain" refers to a polypeptide comprising, from amino-terminus to carboxyl-terminus, a single immunoglobulin light chain variable region (VL) and a single immunoglobulin light chain constant domain (CL). The immunoglobulin light chain constant domain (CL) can be kappa (κ) or lambda (λ). The term "heavy chain" or "immunoglobulin heavy chain" refers to a polypeptide comprising, from amino-terminus to carboxyl-terminus, a single immunoglobulin heavy chain variable region (VH), immunoglobulin heavy chain constant domain 1 (CH1), an immunoglobulin hinge region, immunoglobulin heavy chain constant domain 2 (CH2), immunoglobulin heavy chain constant domain 3 (CH3), and optionally immunoglobulin heavy chain constant domain 4 (CH4). Heavy chains are classified as mu (μ), delta (Δ), gamma (γ), alpha (α) and epsilon (ε), and define the antibody isotype as IgM, IgD, IgG, IgA and IgE, respectively. IgG and IgA class antibodies are further divided into subclasses, namely IgG1, IgG2, IgG3 and IgG4, and IgA1 and IgA2, respectively. Heavy chains in IgG, IgA and IgD antibodies have three domains (CH1, CH2 and CH3), whereas heavy chains in IgM and IgE antibodies have four domains (CH1, CH2, CH3 and CH4). Immunoglobulin heavy chain constant domains can be from any immunoglobulin isotype, including subtypes. Antibody chains are linked together via interpolypeptide disulfide bonds between the CL and CH1 domains (i.e., between the light and heavy chains) and between the hinge regions of the antibody heavy chains.

[0056] In human antibodies, CH1 refers to the region having the amino acid sequence at positions 118-215 of the EU index. A highly flexible amino acid region called the "hinge region" is located between CH1 and CH2. CH2 represents the region having the amino acid sequence at positions 231-340 of the EU index, and CH3 represents the region having the amino acid sequence at positions 341-446 of the EU index.

[0057] "CL" represents the constant region of the light chain. In the case of the κ chain in a human antibody, CL represents the region having the amino acid sequence at positions 108 to 214 of the EU index. In the case of the λ chain, CL represents the region having the amino acid sequence at positions 108 to 215 of the EU index.

[0058] Both the EU index as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991) and the AHo numbering scheme (Honegger A. and Plueckthun AJ Mol Biol. 2001 Jun 8;309(3):657-70) can be used in the present invention. The amino acid positions of a given antibody as well as the complementarity determining regions (CDRs) and framework regions (FRs) can be identified using either system. For example, EU heavy chain positions 39, 44, 183, 356, 357, 360, 370, 392, 399 and 409 are identical to AHo heavy chain positions 46, 51, 230, 484, 485, 491, 501, 528, 535 and 551, respectively.

[0059] A "binding domain" or "BD" typically comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH), but need not comprise both. An Fd fragment, for example, has two VH regions and often retains some antigen-binding function of an intact antigen-binding domain. Further examples of formats of antibody fragments, antibody variants or binding domains include (1) a Fab fragment, which is a monovalent fragment having the VL, VH, CL and CH1 domains; (2) a F(ab')2 fragment, which is a bivalent fragment having two Fab fragments linked by a disulfide bridge at the hinge region; (3) an Fd fragment having two VH and CH1 domains; (4) an Fv fragment having the VL and VH domains of one arm of an antibody, (5) a dAb fragment having a VH domain (Ward et al., (1989) Nature 341:544-546); (6) isolated complementarity determining regions (CDRs), and (7) single chain Fvs (scFvs), the latter of which are preferred (e.g., from an scFV library).

[0060] The disclosure of each reference mentioned herein is incorporated herein by reference in its entirety.

[0061] The present invention is further illustrated by the following examples. EXAMPLES

[0062] Materials and Methods Cell culture and protein expression Suspension HEK293-6E cell line was licensed from the National Research Council (NRC), Biotechnological Research Institute (BRI), Montreal, Canada. Cells are cultured in FreeStyle F-17 medium (Gibco, Catalog #A1383502) supplemented with 0.1% Kolliphor P188 (Sigma, Catalog #K4894), 25 μg / ml G418 (Gibco, Catalog #10131027) and 6 mM L-glutamine (Gibco, Catalog #25030149). Cell density and viability were determined using a Beckman Coulter Vi-cell Cell Viability Analyzer based on the trypan blue exclusion method. During the retention and proliferation stages, suspension HEK293-6E cells were cultured in 500ml to 2.5L polycarbonate flasks (Corning) with 0.2um vented caps at 37°C in a 5% CO2 atmosphere with gentle shaking at 120 rpm. 6 Once cells / ml were reached, cells were diluted to 3.5X10 for subculture for 2 days. 5 Cells / ml and 2X10 for 3-day subculture 5 Cells / ml were split and these required subculture approximately three times per week.

[0063] 2X10 per ml for optimal transfection 6 To achieve a viable cell density of 1x10 cells with a viability of more than 95% 6Cells were seeded 26 hours prior at a density of 10000 cells / ml. For transfection, 0.5 μg DNA per ml of cells was complexed with 1.5 μl PEImax reagent (Polysciences, Cat#24765-2) in 100 μl FreeStyle F-17 medium for 10 minutes and then added to the cell culture. After transfection, cells were treated separately with different protocols. For Grace_v1, one day after transfection, cells were fed with Tryptone N1 solution (Organotechnie, Cat#19553) and glucose (Thermo Fisher, Cat#A2494001) to a final concentration of 5 g / L and 4.5 g / L, respectively, along with an additional volume of fresh cell culture medium. After 3 days, 3.75 mM VPA (MP Biomedicals, Cat#0215206480) was further added in the hope of enhancing protein expression. Protocols Grace_v2 and _v3 did not require VPA but still required the addition of Trytone N1 and glucose 4 or 24 hours after transfection, respectively. For all protocols, conditioned media was collected 7 days after transfection for complete purification with ProA and CEX columns. For high-throughput kingfisher purification with ProA magnetic beads, 25ul magnetic ProA beads (GE Life Sciences) were added per ml of cell culture 6 days after transfection and then harvested for purification on day 7.

[0064] Plasmid construction The antibody HC and LC genes were synthesized by Twist Bioscience and then individually cloned into mammalian transient expression vectors using the Golden Gate assembly method. 3 To reduce the genetic heterogeneity of the protein, all HCs were constructed from a human IgG1 scaffold (IgG1-SEFL2) with aglycosylation mutations and novel engineered disulfide bonds. 4For hetero-IgGs that require HC heterodimerization, CPM was introduced into the Fc region. After Sanger sequencing, transfection-grade DNA was prepared using Maxi Plasmid Purification Kit (Qiagen, Cat#12165) and then mixed at the following ratios: 1:1 (HC:LC) for monoclonal antibodies and IgG-scFv, 1:1:1:1 (HC1:LC1:HC2:LC2) for hetero-IgG, and 1:1:1 (cLC:HC1:HC2) for IgG-Fab and trispecific antibodies.

[0065] High-throughput protein purification with ProA magnetic beads The KingFisher® Flex system (Thermo Fisher) was used for high-throughput protein purification with magnetic ProA beads (GE Life Sciences). Briefly, 4 ml of 293-6E cells in a 24-well deep block were transfected for protein expression, followed by the addition of 100 μl of magnetic ProA beads and harvesting after 1 day. The beads were then collected and KingFisher purification was performed with a 24-deep-well magnetic head. After washing three times with PBS and two times with Milli-Q water, the protein was eluted with 500 μl of 100 mM sodium acetate at pH 3.6 for 10 min, then immediately neutralized by adding 10 μl of 3 M Tris, pH 11.0. The yield of purified protein was measured by A280.

[0066] Non-reduced microcapillary electrophoresis (MCE) The purity of the purified samples was analyzed by non-reduced MCE and analytical SEC. For non-reduced MCE, 6 μl of protein was mixed with 21 μl of sample buffer (8.4 mM Tris-HCl pH 7.0, 7.98% glycerol, 2.38 mM EDTA, 2.8% SDS and 2.4 mM iodoacetamide), heated at 85° C. for 10 min, and then analyzed using a Caliper LabChip GXII Touch instrument (PerkinElmer).

Claims

1. A method for transfecting a population of mammalian cells with DNA encoding a target molecule, (a) A step of providing cells in a cell culture medium, (b) A step of performing a transfection step by bringing the liposome / DNA complex into contact with the cells, wherein the ratio of mg DNA to the number of cells is approximately 0.25 mg:1 × 10 ９ Step, which is a cell (c) Approximately 40 to 56 hours after the transfection step, add the following to the cell culture medium in any order: Tryptone N1 with a final concentration of approximately 4.5–5.5 g / L. A final concentration of glucose of approximately 4.0–5.0 g / L and Approximately 0.8 to 1.2 volumes of fresh cell culture medium, (d) A method comprising the step of collecting the target molecule from the cell culture medium approximately 144 to 192 hours after the transfection step.

2. The method according to claim 1, wherein the cells are suspension cells.

3. The method according to claim 1, wherein the cells are adherent cells.

4. The method according to claim 1, wherein the cells are selected from the group consisting of CHO cells, CHOK1 cells, DXB-11 cells, DG-44 cells, COS-7 cells, HEK293-6E cells, BHK cells, TM4 cells, CV1 cells, VERO-76 cells, HELA cells, MDCK cells, BRL 3A cells, W138 cells, Hep G2 cells, MMT cells, TRI cells, MRC 5 cells, and FS4 cells.

5. The method according to claim 1, wherein the cells are HEK293-6E cells.

6. The aforementioned cells are approximately 1 x 10 ５ ~1x10 ７ The method according to claim 1, wherein cells are seeded at a rate of cells / ml.

7. The aforementioned cells are approximately 1 x 10 ６ The method according to claim 6, wherein the seeds are sown at a rate of / ml.

8. The method according to claim 1, wherein the final concentration of Tryptone N1 is approximately 5.0 g / L.

9. The method according to claim 1, wherein the final concentration of glucose is approximately 4.5 g / L.

10. The method according to claim 1, wherein approximately 1 volume of fresh cell culture medium is added.

11. The method according to claim 1, wherein valproic acid is added approximately 88 to 104 hours after the transfection step to a final concentration of approximately 3.5 to 4.0 mM.

12. The method according to claim 11, wherein the final concentration of valproic acid is approximately 3.75 mM.

13. The method according to claim 1, wherein step (c) is performed about 48 hours after the transfection step.

14. The method according to claim 1, wherein step (d) is performed approximately 168 hours after the transfection step.

15. The method according to claim 11 or 12, wherein the valproic acid is added approximately 96 hours after the transfection step.

16. A method for transfecting a population of mammalian cells with DNA encoding a target molecule, (a) A step of providing cells in a cell culture medium, (b) A step of performing a transfection step by bringing the liposome / DNA complex into contact with the cells, wherein the ratio of mg DNA to the number of cells is approximately 0.25 mg:1 × 10 ９ Step, which is a cell (c) Approximately 2 to 6 hours after the transfection step, add the following to the cell culture medium in any order: Tryptone N1 with a final concentration of approximately 4.5–5.5 g / L. A final concentration of glucose of approximately 4.0–5.0 g / L and Approximately 0.8 to 1.2 volumes of fresh cell culture medium, (d) A method comprising the step of collecting the target molecule from the cell culture medium approximately 144 to 192 hours after the transfection step.

17. The method according to claim 1, wherein the cells are suspension cells.

18. The method according to claim 1, wherein the cells are adherent cells.

19. The method according to claim 1, wherein the cells are selected from the group consisting of CHO cells, CHOK1 cells, DXB-11 cells, DG-44 cells, COS-7 cells, HEK293-6E cells, BHK cells, TM4 cells, CV1 cells, VERO-76 cells, HELA cells, MDCK cells, BRL 3A cells, W138 cells, Hep G2 cells, MMT cells, TRI cells, MRC 5 cells, and FS4 cells.

20. The method according to claim 1, wherein the cells are HEK293-6E cells.

21. The aforementioned cells are approximately 1 x 10 ５ ~1x10 ７ The method according to claim 1 or 16, wherein cells are seeded at a rate of cells / ml.

22. The aforementioned cells are approximately 1 x 10 ６ The method according to claim 6, wherein the seeds are sown at a rate of / ml.

23. The method according to claim 1 or 16, wherein the final concentration of Tryptone N1 is approximately 5.0 g / L.

24. The method according to claim 1 or 16, wherein the final concentration of glucose is approximately 4.5 g / L.

25. The method according to claim 1 or 16, wherein approximately 1 volume of fresh cell culture medium is added.

26. The method according to claim 1 or 16, wherein valproic acid is added approximately 88 to 104 hours after the transfection step to a final concentration of approximately 3.5 to 4.0 mM.

27. The method according to claim 26, wherein the final concentration of valproic acid is approximately 3.75 mM.

28. Step (c) is performed approximately four hours after the transfection step, according to the method of claim 1 or 16.

29. Step (d) is performed approximately 168 hours after the transfection step, according to the method of claim 1 or 16.

30. The method according to claim 26, wherein the valproic acid is added approximately 96 hours after the transfection step.

31. The method according to claim 1 or 16, wherein the target molecule is a multispecific antigen-binding protein.

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