Methods for improving protein expression

Abstract

The present disclosure relates to nucleic acids comprising a nucleotide sequence encoding an immunoglobulin heavy chain, wherein the nucleotide sequence at one or two intron positions in the immunoglobulin heavy chain is deleted, such nucleic acids being useful for increasing immunoglobulin expression.

Description

[Technical Field]

[0001] The present disclosure relates to improved methods of expressing a polypeptide of interest. Nucleic acids comprising a nucleotide sequence encoding an immunoglobulin heavy chain and an intron deletion are useful for increasing the cell-specific productivity of immunoglobulins. [Background technology]

[0002] DNA is composed of sequences of introns and exons, and introns are removed during mRNA processing by splicing. This process is closely linked to mRNA export through the nuclear pore complex. This export is fundamental for expression and has been extensively documented in the literature. See Non-Patent Document 1, Non-Patent Document 2, and Non-Patent Document 3.

[0003] The presence of an intron within a codon-optimized heavy chain constant region encoded in an expression vector has been shown to improve harvest titers compared to the same nucleotide sequence without the intron in this region. However, a risk associated with using an intron-containing expression vector is that intron retention and mis-splicing events can occur during expression of a protein of interest, such as an antibody or immunoglobulin, resulting in unwanted aberrant protein species that must be removed during purification. This adds extra complexity to the purification process and can result in batch-to-batch variability if these additional species cannot be removed during downstream processing.

[0004] It has been shown that the use of a non-codon-optimized sequence containing the heavy chain constant region introns can eliminate the occurrence of splice variants in the heavy chain constant region. The nucleotide sequence changes resulting from codon optimization are thought to induce cryptic splicing to produce these alternative species. It has also been shown that removing each of the introns individually (thereby leaving two of the three introns) results in a surprising improvement in the recovered titer.

[0005] Removal of one or two introns from the immunoglobulin heavy chain constant region can reduce the risk of intron retention and mis-splicing events while increasing immunoglobulin expression, resulting in the de novo generation of expression vectors and a lower risk of generating aberrant protein species during the production of antibody- or immunoglobulin-based recombinant proteins. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Kohler A. et al., Nature Review Molecular Cell Biology 8:761-773(2007) [Non-patent document 2] Bjork, P. et al., Seminars in Cell & Developmental Biology 32:47-54(2014) [Non-patent document 3] Reed, R.Current Opinion in Cell Biology,15:326-331(2003) Summary of the Invention [Means for solving the problem]

[0007] The present disclosure is generally directed to an isolated nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain, wherein the nucleotide sequences of intron 2 and intron 3 of the immunoglobulin heavy chain constant region are deleted.

[0008] The present disclosure is also directed to an isolated nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain, wherein the nucleotide sequence of one intron of the immunoglobulin heavy chain constant region is deleted. In one embodiment, the nucleotide sequence of intron 1 of the immunoglobulin heavy chain constant region is deleted. In another embodiment, the nucleotide sequence of intron 2 of the immunoglobulin heavy chain constant region is deleted. In another embodiment, the nucleotide sequence of intron 3 of the immunoglobulin heavy chain constant region is deleted.

[0009] The present disclosure is also directed to an isolated nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain, wherein the nucleotide sequences of the two introns of the immunoglobulin heavy chain constant region are deleted. In one embodiment, the nucleotide sequences of intron 1 and intron 2 of the immunoglobulin heavy chain constant region are deleted. In another embodiment, the nucleotide sequences of intron 1 and intron 3 of the immunoglobulin heavy chain constant region are deleted.

[0010] The present disclosure is also directed to an isolated nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain, wherein the nucleotide sequence of intron 1 of the immunoglobulin heavy chain constant region has been deleted, and the nucleotide sequence of intron 2 and / or intron 3 of the immunoglobulin heavy chain constant region has been deleted, and the nucleotide sequence of intron 2 and / or intron 3 has been replaced with the nucleotide sequence of intron 1. In one embodiment, the nucleotide sequence of intron 2 of the immunoglobulin heavy chain constant region has been replaced with the nucleotide sequence of intron 1. In one embodiment, the nucleotide sequence of intron 3 of the immunoglobulin heavy chain constant region has been replaced with the nucleotide sequence of intron 1.

[0011] The present disclosure is also directed to an isolated nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain, wherein the nucleotide sequence of intron 2 and / or intron 3 of the immunoglobulin heavy chain constant region is replaced with the nucleotide sequence of intron 1. In one embodiment, the nucleotide sequence of intron 2 of the immunoglobulin heavy chain constant region is replaced with the nucleotide sequence of intron 1. In one embodiment, the nucleotide sequence of intron 3 of the immunoglobulin heavy chain constant region is replaced with the nucleotide sequence of intron 1.

[0012] The present disclosure is also directed to an isolated nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain, in which the nucleotide sequence of intron 3 of the immunoglobulin heavy chain constant region is replaced with a nucleotide sequence of an intron comprising approximately the same number of nucleotides as the nucleotide sequence of intron 1 of the immunoglobulin heavy chain constant region.

[0013] The present disclosure is also directed to an isolated nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain, in which the nucleotide sequence of intron 2 of the immunoglobulin heavy chain constant region is replaced with a nucleotide sequence of an intron comprising approximately the same number of nucleotides as the nucleotide sequence of intron 1 of the immunoglobulin heavy chain constant region. In one embodiment, the nucleotide sequence of intron 1 of the immunoglobulin heavy chain constant region is deleted.

[0014] In one embodiment, when the nucleic acid is expressed together with a nucleic acid encoding an immunoglobulin light chain, it expresses immunoglobulins at higher titers than a nucleic acid containing all the intron sequences of the immunoglobulin heavy chain constant region. In another embodiment, when the nucleic acid is expressed together with a nucleic acid encoding an immunoglobulin light chain, it expresses immunoglobulins at higher titers than a nucleic acid that does not contain the intron sequences of the immunoglobulin heavy chain constant region. In another embodiment, the immunoglobulin light chain is a kappa light chain or a lambda light chain. In another embodiment, the nucleic acid is codon-optimized. In another embodiment, the expressed immunoglobulin has an IgG1, IgG2, IgG3, or IgG4 isotype. In another embodiment, the expressed immunoglobulin is a human, humanized, chimeric, or resurfaced immunoglobulin. In another embodiment, the nucleic acid encoding an immunoglobulin heavy chain is deoxyribonucleic acid (DNA).

[0015] In another aspect, the present disclosure is directed to a vector or expression vector comprising a nucleic acid of the present disclosure. In another aspect, the present disclosure is directed to a host cell comprising a vector or expression vector of the present disclosure. In one aspect, the host cell is a eukaryotic cell, such as a Chinese hamster ovary (CHO) cell.

[0016] The present disclosure also generally relates to methods for producing immunoglobulins, the method comprising culturing host cells in a culture medium under conditions in which the cells express the immunoglobulin, the host cells comprising a nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain lacking the nucleotide sequences of intron 2 and intron 3 of the immunoglobulin heavy chain constant region, and a nucleic acid encoding an immunoglobulin light chain, wherein the host cells express the immunoglobulin at a higher titer than host cells comprising a nucleic acid encoding an immunoglobulin heavy chain in which either all or none of introns 1-3 of the immunoglobulin heavy chain constant region are present, and a nucleic acid encoding an immunoglobulin light chain.

[0017] The present disclosure also generally relates to a method for producing immunoglobulins, the method comprising culturing host cells in a medium under conditions in which the cells express the immunoglobulins, the host cells comprising a nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain in which the nucleotide sequence of one intron of the immunoglobulin heavy chain constant region is deleted, and a nucleic acid encoding an immunoglobulin light chain, the host cells expressing the immunoglobulins at a higher titer than host cells comprising a nucleic acid encoding an immunoglobulin heavy chain in which all or none of introns 1 to 3 of the immunoglobulin heavy chain constant region are present, and a nucleic acid encoding an immunoglobulin light chain. In one embodiment, the nucleotide sequence of intron 1 of the immunoglobulin heavy chain constant region is deleted. In another embodiment, the nucleotide sequence of intron 2 of the immunoglobulin heavy chain constant region is deleted. In another embodiment, the nucleotide sequence of intron 3 of the immunoglobulin heavy chain constant region is deleted.

[0018] The present disclosure is also generally directed to a method of producing immunoglobulins, the method comprising culturing host cells in a medium under conditions in which the cells express the immunoglobulins, the host cells comprising a nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain in which the nucleotide sequences of two introns of the immunoglobulin heavy chain constant region are deleted, and a nucleic acid encoding an immunoglobulin light chain, the host cells expressing immunoglobulins at higher titers than host cells comprising a nucleic acid encoding an immunoglobulin heavy chain in which all or none of introns 1-3 of the immunoglobulin heavy chain constant region are present, and a nucleic acid encoding an immunoglobulin light chain. In one embodiment, introns 1 and 2 of the immunoglobulin heavy chain constant region are deleted. In another embodiment, introns 1 and 3 of the immunoglobulin heavy chain constant region are deleted.

[0019] The present disclosure is also generally directed to a method of producing immunoglobulins, the method comprising culturing host cells in a culture medium under conditions in which the cells express the immunoglobulins, the host cells comprising a nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain in which the nucleotide sequence of intron 1 of the immunoglobulin heavy chain constant region has been deleted, the nucleotide sequence of intron 2 and / or intron 3 of the immunoglobulin heavy chain has been deleted, and the nucleotide sequence of intron 2 and / or intron 3 has been replaced with the nucleotide sequence of intron 1, and a nucleic acid encoding an immunoglobulin light chain, the host cells expressing immunoglobulins at a higher titer than host cells comprising a nucleic acid encoding an immunoglobulin heavy chain in which all or none of introns 1-3 of the immunoglobulin heavy chain constant region are present and a nucleic acid encoding an immunoglobulin light chain. In one aspect, the nucleotide sequence of intron 2 of the immunoglobulin heavy chain constant region has been replaced with the nucleotide sequence of intron 1. In another embodiment, the nucleotide sequence of intron 3 of the immunoglobulin heavy chain constant region is replaced with the nucleotide sequence of intron 1.

[0020] The present disclosure is also generally directed to a method of producing an immunoglobulin, the method comprising culturing host cells in a culture medium under conditions in which the cells express the immunoglobulin, the host cells comprising a nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain in which the nucleotide sequence of intron 2 and / or intron 3 of the immunoglobulin heavy chain constant region is replaced with the nucleotide sequence of intron 1, and a nucleic acid encoding an immunoglobulin light chain, the host cells expressing the immunoglobulin at a higher titer than host cells comprising a nucleic acid encoding an immunoglobulin heavy chain in which all or none of introns 1-3 of the immunoglobulin heavy chain constant region are present and a nucleic acid encoding an immunoglobulin light chain. In one embodiment, the nucleotide sequence of intron 2 of the immunoglobulin heavy chain constant region is replaced with the nucleotide sequence of intron 1. In another embodiment, the nucleotide sequence of intron 3 of the immunoglobulin heavy chain constant region is replaced with the nucleotide sequence of intron 1.

[0021] The present disclosure is also generally directed to a method of producing immunoglobulins, the method comprising culturing host cells in a culture medium under conditions in which the cells express the immunoglobulins, the host cells comprising a nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain in which the nucleotide sequence of intron 3 of the immunoglobulin heavy chain constant region is replaced with a nucleotide sequence of an intron comprising approximately the same number of nucleotides as the nucleotide sequence of intron 1 of the immunoglobulin heavy chain constant region, and a nucleic acid encoding an immunoglobulin light chain, the host cells expressing immunoglobulins at higher titers than host cells comprising a nucleic acid encoding an immunoglobulin heavy chain and a nucleic acid encoding an immunoglobulin light chain in which all or none of introns 1-3 of the immunoglobulin heavy chain constant region are present. In one embodiment, the nucleotide sequence of intron 1 of the immunoglobulin heavy chain constant region is deleted.

[0022] The present disclosure is also generally directed to a method of producing immunoglobulins, the method comprising culturing host cells in a medium under conditions in which the cells express the immunoglobulin, the host cells comprising a nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain in which the nucleotide sequence of intron 2 of the immunoglobulin heavy chain constant region is replaced with a nucleotide sequence of an intron containing approximately the same number of nucleotides as the nucleotide sequence of intron 1 of the immunoglobulin heavy chain constant region, and a nucleic acid encoding an immunoglobulin light chain, the host cells expressing immunoglobulins at higher titers than host cells comprising a nucleic acid encoding an immunoglobulin heavy chain and a nucleic acid encoding an immunoglobulin light chain in which all or none of introns 1-3 of the immunoglobulin heavy chain constant region are present. In one embodiment, the nucleotide sequence of intron 1 of the immunoglobulin heavy chain constant region is deleted.

[0023] In one embodiment, the expressed immunoglobulin has an isotype of IgG1, IgG2, IgG3, or IgG4, hi another embodiment, the expressed immunoglobulin is human, humanized, chimeric, or resurfaced.

[0024] In one embodiment, the expressed immunoglobulins produced from the pool of clones have a recovered titer of at least 1,000 mg / L, at least 1,500 mg / L, at least 2,000 mg / L, at least 2,500 mg / L, or at least 3,000 mg / L. In another embodiment, the expressed immunoglobulins produced from the highest expressing clones have a recovered titer of at least 1,000 mg / L, at least 1,500 mg / L, at least 2,000 mg / L, at least 3,000 mg / L, at least 4,000 mg / L, at least 5,000 mg / L, at least 6,000 mg / L, at least 7,000 mg / L, at least 8,000 mg / L, at least 9,000 mg / L, at least 10,000 mg / L, at least 11,000 mg / L, or at least 12,000 mg / L.

[0025] In one embodiment, the host cell is a eukaryotic cell, hi another embodiment, the eukaryotic cell is a CHO cell. [Brief explanation of the drawings]

[0026] [Figure 1] 1 shows a high-performance size-exclusion chromatograph of MAb1 and a depiction of the immunoglobulin variants produced. [Figure 2] Figure 2A shows a schematic representation of the non-codon-optimized genomic DNA (gDNA) and codon-optimized complementary DNA (cDNA) of the immunoglobulin heavy chain constant region of MAb2. Figure 2B shows the immunoglobulin titer (mg / L) on day 14 of MAb2 production using either the gDNA or cDNA nucleotide sequence. [Figure 3] Figure 3A shows a schematic diagram of non-codon-optimized genomic DNA (gDNA) of the immunoglobulin heavy chain constant region, codon-optimized complementary DNA (cDNA) of the immunoglobulin heavy chain constant region of MAb2, gDNA without intron 1 of the immunoglobulin heavy chain constant region of MAb2, gDNA without intron 2 of the immunoglobulin heavy chain constant region of MAb2, gDNA without intron 3 of the immunoglobulin heavy chain constant region of MAb2, and gDNA without any introns of the immunoglobulin heavy chain constant region of MAb2. Figure 3B shows the immunoglobulin titer (mg / L) on day 13 of MAb2 production using each of the following constructs: (1) non-codon-optimized genomic DNA (gDNA) of the immunoglobulin heavy chain constant region, (2) codon-optimized complementary DNA (cDNA) of the immunoglobulin heavy chain constant region of MAb2, (3) gDNA without intron 1 of the immunoglobulin heavy chain constant region of MAb2, (4) gDNA without intron 2 of the immunoglobulin heavy chain constant region of MAb2, (5) gDNA without intron 3 of the immunoglobulin heavy chain constant region of MAb2, and (6) gDNA without any introns of the immunoglobulin heavy chain constant region of MAb2. [Figure 4]FIG. 4A shows a graph depicting the mean viable cell count (VCN) (×106 / mL) for each of the following constructs: (1) non-codon-optimized genomic DNA (gDNA) of the immunoglobulin heavy chain constant region, (2) codon-optimized complementary DNA (cDNA) of the immunoglobulin heavy chain constant region of MAb2, (3) gDNA without intron 1 of the immunoglobulin heavy chain constant region of MAb2, (4) gDNA without intron 2 of the immunoglobulin heavy chain constant region of MAb2, (5) gDNA without intron 3 of the immunoglobulin heavy chain constant region of MAb2, and (6) gDNA without any introns of the immunoglobulin heavy chain constant region of MAb2. Figure 4B shows a graph depicting the percent cell viability for each of the following constructs: (1) non-codon-optimized genomic DNA (gDNA) of the immunoglobulin heavy chain constant region, (2) codon-optimized complementary DNA (cDNA) of the immunoglobulin heavy chain constant region of MAb2, (3) gDNA without intron 1 of the immunoglobulin heavy chain constant region of MAb2, (4) gDNA without intron 2 of the immunoglobulin heavy chain constant region of MAb2, (5) gDNA without intron 3 of the immunoglobulin heavy chain constant region of MAb2, and (6) gDNA without any introns of the immunoglobulin heavy chain constant region of MAb2. FIG. 4C shows a graph depicting the integrated viable cell concentration (IVC) (10 cells-days / L) for each of the following constructs: (1) non-codon-optimized genomic DNA (gDNA) of the immunoglobulin heavy chain constant region, (2) codon-optimized complementary DNA (cDNA) of the immunoglobulin heavy chain constant region of MAb2, (3) gDNA without intron 1 of the immunoglobulin heavy chain constant region of MAb2, (4) gDNA without intron 2 of the immunoglobulin heavy chain constant region of MAb2, (5) gDNA without intron 3 of the immunoglobulin heavy chain constant region of MAb2, and (6) gDNA without any introns of the immunoglobulin heavy chain constant region of MAb2.FIG. 4D shows a graph depicting the cellular productivity (qP) (pg / (cells per day)) for each of the following constructs: (1) non-codon-optimized genomic DNA (gDNA) of the immunoglobulin heavy chain constant region, (2) codon-optimized complementary DNA (cDNA) of the immunoglobulin heavy chain constant region of MAb2, (3) gDNA without intron 1 of the immunoglobulin heavy chain constant region of MAb2, (4) gDNA without intron 2 of the immunoglobulin heavy chain constant region of MAb2, (5) gDNA without intron 3 of the immunoglobulin heavy chain constant region of MAb2, and (6) gDNA without any introns of the immunoglobulin heavy chain constant region of MAb2. [Figure 5]Figure 5A shows the following constructs: (1) gDNA with intron 2 removed from the immunoglobulin heavy chain constant region of MAb2 (non-codon optimized), (2) gDNA with introns 1 and 2 removed from the immunoglobulin heavy chain constant region of MAb2 (non-codon optimized), (3) gDNA with introns 2 and 3 removed from the immunoglobulin heavy chain constant region of MAb2 (non-codon optimized), (4) gDNA with no introns in the immunoglobulin heavy chain constant region of MAb2 (non-codon optimized), (5) gDNA with no introns in the immunoglobulin heavy chain constant region of MAb2 (non-codon optimized), and (6) gDNA with no introns in the immunoglobulin heavy chain constant region of MAb2 (non-codon optimized). Figure 1 shows the immunoglobulin titers (mg / L) on day 11 of MAb2 production using gDNA with intron 2 removed from the immunoglobulin heavy chain constant region (codon optimized), (5) gDNA with introns 1 and 2 removed from the immunoglobulin heavy chain constant region of MAb2 (codon optimized), (6) gDNA with introns 2 removed from the immunoglobulin heavy chain constant region of MAb2 (codon optimized), (7) gDNA with introns 2 and 3 removed from the immunoglobulin heavy chain constant region of MAb2 (codon optimized), and (8) gDNA with no introns in the immunoglobulin heavy chain constant region of MAb2 (codon optimized). Figure 5B shows the following constructs: (1) gDNA with intron 2 removed from the immunoglobulin heavy chain constant region of MAb2 (non-codon optimized), (2) gDNA with introns 1 and 2 removed from the immunoglobulin heavy chain constant region of MAb2 (non-codon optimized), (3) gDNA with introns 2 and 3 removed from the immunoglobulin heavy chain constant region of MAb2 (non-codon optimized), (4) gDNA with no introns in the immunoglobulin heavy chain constant region of MAb2 (non-codon optimized), (5) gDNA with no introns in the immunoglobulin heavy chain constant region of MAb2 (non-codon optimized), (6) gDNA with no introns in the immunoglobulin heavy chain constant region of MAb2 (non-codon optimized), (7) gDNA with no introns in the immunoglobulin heavy chain constant region of MAb2 (non-codon optimized), (8) gDNA with no introns in the immunoglobulin heavy chain constant region of MAb2 (non-codon optimized), (9) gDNA with no introns in the immunoglobulin heavy chain constant region of MAb2 (non-codon optimized), (10) gDNA with no introns in the immunoglobulin heavy chain constant region of MAb2 (non-codon optimized), (11) gDNA with no introns in the immunoglobulin heavy chain constant region of MAb2 (non-codon optimized), (12) gDNA with no introns in the immunoglobulin heavy chain constant region of MAb2 (non-codon optimized), (13) gDNA with no introns in the immunoglobulin heavy chain constant region of MAb2 (non-codon optimized), (14) gDNA with no introns in the immunoglobulin heavy chain constant region of MAb2 (non-codon optimized), (15) gDNA with no introns in the immunoglobulin heavy chain constant region of MAb2 (non-codon optimized), (16) gDNA with no introns in the immunoglobulin heavy chain constant region of MAb2 (non-codon optimized), (17 Graphs showing the mean viable cell count (VCN) (×106 / ml) for each of (5) gDNA with intron 2 removed from the immunoglobulin heavy chain constant region of MAb2 (codon optimized), (6) gDNA with introns 1 and 2 removed from the immunoglobulin heavy chain constant region of MAb2 (codon optimized), (7) gDNA with introns 2 and 3 removed from the immunoglobulin heavy chain constant region of MAb2 (codon optimized), (8) gDNA with no introns in the immunoglobulin heavy chain constant region of MAb2 (codon optimized), and (9) MAb3 (positive control).Figure 5C shows the following constructs: (1) gDNA with intron 2 removed from the immunoglobulin heavy chain constant region of MAb2 (non-codon optimized), (2) gDNA with introns 1 and 2 removed from the immunoglobulin heavy chain constant region of MAb2 (non-codon optimized), (3) gDNA with introns 2 and 3 removed from the immunoglobulin heavy chain constant region of MAb2 (non-codon optimized), (4) gDNA with no introns in the immunoglobulin heavy chain constant region of MAb2 (non-codon optimized), (5) gDNA with no introns in the immunoglobulin heavy chain constant region of MAb2 (non-codon optimized), (6) gDNA with no introns in the immunoglobulin heavy chain constant region of MAb2 (non-codon optimized), (7) gDNA with no introns in the immunoglobulin heavy chain constant region of MAb2 (non-codon optimized), (8) gDNA with no introns in the immunoglobulin heavy chain constant region of MAb2 (non-codon optimized), (9) gDNA with no introns in the immunoglobulin heavy chain constant region of MAb2 (non-codon optimized), (10) gDNA with no introns in the immunoglobulin heavy chain constant region of MAb2 (non-codon optimized), (11) gDNA with no introns in the immunoglobulin heavy chain constant region of MAb2 (non-codon optimized), (12) gDNA with no introns in the immunoglobulin heavy chain constant region of MAb2 (non-codon optimized), (13) gDNA with no introns in the immunoglobulin heavy chain constant region of MAb2 (non-codon optimized), (14) gDNA with no introns in the immunoglobulin heavy chain constant region of MAb2 (non-codon optimized), (15) gDNA with no introns in the immunoglobulin heavy chain constant region of MAb2 (non-codon optimized), (16) gDNA with no introns in the immunoglobulin heavy chain constant region of MAb2 (non-codon optimized), (17 1 shows a graph depicting the integrated viable cell concentration (IVC) (10 cell-h / L) for each of (5) gDNA from which intron 2 was removed from the immunoglobulin heavy chain constant region of MAb2 (codon optimized), (6) gDNA from which introns 1 and 2 were removed from the immunoglobulin heavy chain constant region of MAb2 (codon optimized), (7) gDNA from which introns 2 and 3 were removed from the immunoglobulin heavy chain constant region of MAb2 (codon optimized), (8) gDNA from which no introns are present in the immunoglobulin heavy chain constant region of MAb2 (codon optimized), and (9) MAb3 (positive control). Figure 5D shows the following constructs: (1) gDNA with intron 2 removed from the immunoglobulin heavy chain constant region of MAb2 (non-codon optimized), (2) gDNA with introns 1 and 2 removed from the immunoglobulin heavy chain constant region of MAb2 (non-codon optimized), (3) gDNA with introns 2 and 3 removed from the immunoglobulin heavy chain constant region of MAb2 (non-codon optimized), (4) gDNA with no introns in the immunoglobulin heavy chain constant region of MAb2 (non-codon optimized), (5 Graphs showing cellular productivity (qP) (pg / (cells per day)) are shown for (1) gDNA in which intron 2 has been removed from the immunoglobulin heavy chain constant region of MAb2 (codon optimized), (2) gDNA in which introns 1 and 2 have been removed from the immunoglobulin heavy chain constant region of MAb2 (codon optimized), (3) gDNA in which introns 2 and 3 have been removed from the immunoglobulin heavy chain constant region of MAb2 (codon optimized), and (4) gDNA in which no introns are present in the immunoglobulin heavy chain constant region of MAb2 (codon optimized). [Figure 6]Figure 6A shows the following constructs: (1) non-codon-optimized genomic DNA (gDNA) of the immunoglobulin heavy chain constant region of MAb2, (2) non-codon-optimized gDNA lacking intron 2 of the immunoglobulin heavy chain constant region of MAb2, (3) non-codon-optimized gDNA lacking introns 2 and 3 of the immunoglobulin heavy chain constant region of MAb2, (4) non-codon-optimized gDNA lacking introns 1 and 2 of the immunoglobulin heavy chain constant region of MAb2, (5) non-codon-optimized gDNA with intron 3 in place of intron 1 of the immunoglobulin heavy chain constant region of MAb2, (6) non-codon-optimized gDNA with a modified intron 3 nucleotide sequence in place of intron 1 of the immunoglobulin heavy chain constant region of MAb2, and (7) non-codon-optimized gDNA with a modified intron 3 nucleotide sequence in place of intron 1 of the immunoglobulin heavy chain constant region of MAb2. Schematic diagrams of (1) non-codon-optimized gDNA having intron 1 in place of intron 3 of the immunoglobulin heavy chain constant region of MAb2, (2) non-codon-optimized gDNA having no introns at all in the immunoglobulin heavy chain constant region of MAb2, (3) non-codon-optimized gDNA having all introns of the immunoglobulin heavy chain constant region of MAb2 and wild-type IgG1, (4) non-codon-optimized gDNA having intron 1 in place of intron 3 of the immunoglobulin heavy chain constant region of MAb2, (5) non-codon-optimized gDNA having all introns of the immunoglobulin heavy chain constant region of MAb2 and wild-type IgG1, (6) non-codon-optimized gDNA having intron 1 in place of intron 3 of the immunoglobulin heavy chain constant region of MAb2, (7) non-codon-optimized gDNA having intron 1 in place of intron 3 of the immunoglobulin heavy chain constant region of MAb2, (8) non-codon-optimized gDNA having no introns at all in the immunoglobulin heavy chain constant region of MAb2, (9) non-codon-optimized genomic DNA (gDNA) having all introns of the immunoglobulin heavy chain constant region of MAb2 and wild-type IgG1, (10) non-codon-optimized gDNA having introns 2 and 3 of the immunoglobulin heavy chain constant region of MAb2 but wild-type IgG1, (11) non-codon-optimized gDNA having introns 1 and 2 of the immunoglobulin heavy chain constant region of MAb2 but wild-type IgG1, and (12) non-codon-optimized gDNA having the immunoglobulin heavy chain constant region of MAb2 and wild-type IgG1.Figure 6B shows the following constructs: (1) non-codon-optimized genomic DNA (gDNA) of the immunoglobulin heavy chain constant region of MAb2, (2) non-codon-optimized gDNA lacking intron 2 of the immunoglobulin heavy chain constant region of MAb2, (3) non-codon-optimized gDNA lacking introns 2 and 3 of the immunoglobulin heavy chain constant region of MAb2, (4) non-codon-optimized gDNA lacking introns 1 and 2 of the immunoglobulin heavy chain constant region of MAb2, (5) non-codon-optimized gDNA with intron 3 in place of intron 1 of the immunoglobulin heavy chain constant region of MAb2, (6) non-codon-optimized gDNA with a modified intron 3 nucleotide sequence in place of intron 1 of the immunoglobulin heavy chain constant region of MAb2, and (7) non-codon-optimized gDNA with a modified intron 3 nucleotide sequence in place of intron 1 of the immunoglobulin heavy chain constant region of MAb2. (8) non-codon-optimized gDNA with intron 1 at the position of intron 3 in the immunoglobulin heavy chain constant region of MAb2; (9) non-codon-optimized genomic DNA (gDNA) with all introns in the immunoglobulin heavy chain constant region of MAb2 and wild-type IgG1; (10) non-codon-optimized gDNA without introns 2 and 3 in the immunoglobulin heavy chain constant region of MAb2 and wild-type IgG1; (11) non-codon-optimized gDNA without introns 1 and 2 in the immunoglobulin heavy chain constant region of MAb2 and wild-type IgG1; and (12) non-codon-optimized gDNA of the immunoglobulin heavy chain constant region of MAb2 and wild-type IgG1. [Figure 7] Schematic diagrams of non-codon-optimized genomic DNA (gDNA) of the immunoglobulin heavy chain constant region of MAb2, MAb1, MAb3, and MAb4, non-codon-optimized gDNA without introns 2 and 3 of the immunoglobulin heavy chain constant region of MAb2, MAb1, MAb3, and MAb4, and non-codon-optimized gDNA without any introns of the immunoglobulin heavy chain constant region of MAb2, MAb1, MAb3, and MAb4 are shown. [Figure 8]Shown are immunoglobulin titers (mg / L) on day 11 for non-codon-optimized genomic DNA (gDNA) of the immunoglobulin heavy chain constant regions of MAb2, MAb1, MAb3, and MAb4, non-codon-optimized gDNA lacking introns 2 and 3 of the immunoglobulin heavy chain constant regions of MAb2, MAb1, MAb3, and MAb4, and non-codon-optimized gDNA lacking any introns of the immunoglobulin heavy chain constant regions of MAb2, MAb1, MAb3, and MAb4. [Figure 9] Graphs showing the time course of titer levels of non-codon-optimized genomic DNA (gDNA) of the immunoglobulin heavy chain constant regions of MAb2, MAb1, MAb3, and MAb4, non-codon-optimized gDNA lacking introns 2 and 3 of the immunoglobulin heavy chain constant regions of MAb2, MAb1, MAb3, and MAb4, and non-codon-optimized gDNA lacking any introns of the immunoglobulin heavy chain constant regions of MAb2, MAb1, MAb3, and MAb4. [Figure 10] 1 shows a schematic representation of the genomic DNA sequence of the immunoglobulin heavy chain constant region. DETAILED DESCRIPTION OF THE INVENTION

[0027] To facilitate understanding of this disclosure, several terms and phrases are defined below.

[0028] I. Definition As used herein, the terms "immunoglobulin," "antibody," and "antibodies" are terms of art and may be used interchangeably herein to refer to a molecule or complex of molecules that have at least one antigen-binding site that specifically binds to an antigen.

[0029] Antibodies can include, for example, monoclonal antibodies, recombinantly produced antibodies, human antibodies, humanized antibodies, resurfaced antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies comprising two heavy chain molecules and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain-antibody heavy chain pairs, intrabodies, heteroconjugate antibodies, single domain antibodies, monovalent antibodies, single chain antibodies or single chain Fvs (scFvs), camelid antibodies, affibodies, Fab fragments, F(ab')2 fragments, disulfide-linked Fvs (sdFvs), anti-idiotypic (anti-Id) antibodies (including, for example, anti-anti-Id antibodies), bispecific antibodies, and multispecific antibodies. In certain embodiments, the antibody described herein refers to a polyclonal antibody population. An antibody can be any type (e.g., IgG, IgE, IgM, IgD, IgA, or IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2), or any subclass (e.g., IgG 2a or IgG 2b ). In certain embodiments, the antibodies described herein are IgG antibodies, or classes thereof (e.g., human IgG1, IgG2, or IgG4) or subclasses thereof. In certain embodiments, the antibodies are humanized monoclonal antibodies. In other particular embodiments, the antibodies are human monoclonal antibodies, e.g., they are immunoglobulins. In certain embodiments, the antibodies described herein are IgG1, IgG2, or IgG4 antibodies.

[0030] As used herein, the terms "antigen-binding domain," "antigen-binding region," "antigen-binding site," and similar terms refer to the portion of an antibody molecule (e.g., the complementarity-determining region (CDR)) that contains the amino acid residues that confer specificity to the antibody molecule for an antigen. The antigen-binding region can be derived from any animal species, including rodents (e.g., mice, rats, or hamsters) and humans.

[0031] A "monoclonal" antibody refers to a homogeneous antibody population responsible for highly specific recognition and binding of a single antigenic determinant or epitope. This is in contrast to polyclonal antibodies, which typically include different antibodies directed against different antigenic determinants. The term "monoclonal" antibody encompasses both intact and full-length immunoglobulin molecules, as well as Fab, Fab', F(ab')2, Fv), single chain (scFv), fusion proteins containing an antibody portion, and any other modified immunoglobulin molecule containing an antigen recognition site. Furthermore, "monoclonal" antibody refers to antibodies produced in a number of ways, including, but not limited to, by hybridoma, phage selection, recombinant expression, and transgenic animals.

[0032] The term "chimeric" antibody refers to an antibody whose amino acid sequences are derived from two or more species. Typically, the variable regions of both the light and heavy chains correspond to the variable regions of antibodies derived from one species of mammal (e.g., mouse, rat, rabbit, etc.) having the desired specificity, affinity, and capacity, while the constant regions are homologous to sequences in antibodies derived from another species (usually human) to avoid eliciting an immune response in that species.

[0033] The term "humanized" antibody refers to forms of non-human (e.g., murine) antibodies that contain minimal non-human (e.g., murine) sequences. Typically, humanized antibodies are human immunoglobulins in which complementarity-determining region (CDR) residues are replaced by CDR residues from a non-human species (e.g., mouse, rat, rabbit, hamster) having the desired specificity, affinity, and capacity ("CDR-grafted") (Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science 239:1534-1536 (1988)). In some cases, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding residues in an antibody from a non-human species having the desired specificity, affinity, and capacity. The humanized antibody can be further modified by substitution of additional residues within the Fv framework regions and / or within the replaced non-human residues to refine and optimize the specificity, affinity, and / or capacity of the antibody. Generally, a humanized antibody will comprise substantially all of at least one, and typically two or three, variable domains, containing all or substantially all of the CDR regions corresponding to a non-human immunoglobulin, while all or substantially all of the FR regions are of human immunoglobulin consensus sequences. The humanized antibody may also comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. Examples of methods used to generate humanized antibodies are described in U.S. Pat. No. 5,225,539, Roguska et al., Proc. Natl. Acad. Sci., USA, 91(3):969-973 (1994), and Roguska et al., Protein Eng. 9(10):895-904 (1996).

[0034] The term "resurfaced antibody" or "multiple resurfaced antibodies" refers to a murine antibody that has been redesigned to resemble a human antibody by humanizing only the amino acids available on the surface of the variable region of the recombinant FV. Resurfacing a murine monoclonal antibody to reduce its immunogenicity can be beneficial in maintaining the binding ability of the original monoclonal antibody in the resurfaced form, since the native framework-CDR interactions are preserved.

[0035] The term "human antibody" means an antibody having an amino acid sequence derived from the human immunoglobulin locus, such an antibody being made using any technique known in the art.

[0036] The variable region typically refers to a portion of an antibody, generally a portion of either the light or heavy chain, typically the amino-terminal 110-125 amino acids in the mature heavy chain and approximately 90-115 amino acids in the mature light chain, which vary extensively in sequence among antibodies and are used in the binding and specificity of a particular antibody to its specific antigen. Sequence variability is concentrated in regions called complementarity-determining regions (CDRs), while the more highly conserved regions in the variable domain are called framework regions (FRs). While not wishing to be bound by any particular mechanism or theory, it is believed that the CDRs of the light and heavy chains are primarily responsible for the interaction and specificity of the antibody with the antigen. In certain embodiments, the variable region is a human variable region. In certain embodiments, the variable region comprises rodent or murine CDRs and human framework regions (FRs). In certain embodiments, the variable region is a primate (e.g., non-human primate) variable region. In certain embodiments, the variable region comprises rodent or murine CDRs and primate (e.g., non-human primate) framework regions (FRs).

[0037] As used herein, the terms "constant region" and "constant domain" are used interchangeably and have their common meanings in the art. The constant region is the portion of an antibody (e.g., the carboxyl-terminal portion of the light and / or heavy chain) that is not directly involved in binding the antibody to an antigen but may exert various effector functions, such as interacting with Fc receptors. The constant region of an immunoglobulin molecule generally has a more conserved amino acid sequence compared to the immunoglobulin variable domain. An immunoglobulin "constant region" or "constant domain" can contain a CH1 domain, a hinge, a CH2 domain, and a CH3 domain, or a subset of these domains, e.g., a CH2 domain and a CH3 domain. In certain embodiments provided herein, the immunoglobulin constant region does not contain a CH1 domain. In certain embodiments provided herein, the immunoglobulin constant region does not contain a hinge. In certain embodiments provided herein, the immunoglobulin constant region does not contain a CH2 domain or a CH3 domain.

[0038] "Fc region" or "Fc domain" refers to a polypeptide sequence corresponding to or derived from the portion of a source antibody responsible for binding to cellular antibody receptors and the C1q component of complement. Fc means "fragment crystallizable" and refers to a fragment of an antibody that readily forms protein crystals. The different protein fragments originally described by proteolytic digestion can define the overall general structure of an immunoglobulin protein. An "Fc region" or "Fc domain" contains the CH2 domain, the CH3 domain, and optionally all or part of the hinge. An "Fc region" or "Fc domain" can refer to a single polypeptide or two disulfide-bonded polypeptides. For reviews of immunoglobulin structure and function, see Putnam, The Plasma Proteins, Vol. V (Academic Press, Inc., 1987), pp. 49-140; and Padlan, Mol. Immunol. 31:169-217, 1994. As used herein, the term Fc includes native sequence variants.

[0039] A "wild-type immunoglobulin hinge region" refers to the naturally occurring upper and middle hinge amino acid sequences found in naturally occurring antibody heavy chains that intersect and connect the CH1 and CH2 domains (in the case of IgG, IgA, and IgD) or the CH1 and CH3 domains (in the case of IgE and IgM). In certain embodiments, the wild-type immunoglobulin hinge region sequence is human and can include a human IgG hinge region. A "modified wild-type immunoglobulin hinge region" or "modified immunoglobulin hinge region" is a wild-type immunoglobulin hinge region that has (a) up to 30% amino acid changes (e.g., up to 25%, 20%, 15%, 10%, or 5% amino acid substitutions or deletions), or (b) from about 5 amino acids in length (e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids) to about 120 amino acids in length (e.g., about 10 to about 40 amino acids in length or about 15 to 20 amino acids in length). "Immunoglobulin hinge region" refers to a portion of a wild-type immunoglobulin hinge region (having a length of about 30 amino acids, or about 15 to about 20 amino acids, or about 20 to about 25 amino acids) having up to about 30% amino acid changes (e.g., up to about 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% amino acid substitutions or deletions, or a combination thereof), and having an IgG core hinge region as disclosed in U.S. Patent Application Publication No. 2013 / 0129723 and U.S. Patent Application Publication No. 2013 / 0095097.

[0040] As used herein, the term "heavy chain" when used in reference to an antibody can refer to any of the distinct types, e.g., alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), based on the amino acid sequence of the constant region, which give rise to the IgA, IgD, IgE, IgG, and IgM classes of antibodies, respectively, and include the subclasses of IgG, e.g., IgG1, IgG2, IgG3, and IgG4.

[0041] As used herein, the term "light chain" when used in reference to an antibody can refer to any distinct type, e.g., kappa (κ) or lambda (λ), based on the amino acid sequence of the constant region. Light chain amino acid sequences are well known in the art. In certain embodiments, the light chain is a human light chain.

[0042] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymers may be linear or branched, may comprise modified amino acids, and may be interrupted by non-amino acids. The term also includes amino acid polymers that are modified, naturally or by intervening processes, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are polypeptides containing, for example, one or more analogs of an amino acid (including, for example, unnatural amino acids), as well as other modifications known in the art. It will be understood that because the polypeptides of the invention are based on antibodies, in certain embodiments the polypeptides can occur as single chains or associated chains.

[0043] As used herein, the terms "nucleic acid," "nucleic acid molecule," or "polynucleotide" refer to deoxyribonucleotides or ribonucleotides, or polymers thereof, in either single- or double-stranded form. Unless otherwise limited, these terms encompass nucleic acids containing analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized similarly to natural nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the explicitly specified sequence. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al. (1991) Nucleic Acid Res. 19:5081; Ohtsuka et al. (1985) J. Biol. Chem. 260:2605-2608; Cassol et al. (1992); Rossolini et al. (1994) Mol. Cell. Probes 8:91-98). The term nucleic acid is used interchangeably with gene, cDNA, and mRNA encoded by a gene. As used herein, the terms "nucleic acid," "nucleic acid molecule," or "polynucleotide" are intended to include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of DNA or RNA generated using nucleotide analogs, and derivatives, fragments, and homologs thereof.

[0044] The term "intron," as used herein, refers to a sequence of nucleotides that is transcribed into RNA and then removed from the RNA, typically by splicing, to create a mature RNA, e.g., mRNA. Typically, an intron nucleotide sequence is not incorporated into the mature RNA, and intron sequences, or portions thereof, are typically not translated or incorporated into polypeptides. Splice signal sequences, such as splice donors and splice acceptors, are used by the cellular splicing machinery to remove introns from RNA.

[0045] The term "vector," as used herein, refers to a linear or circular nucleic acid that contains a segment of nucleic acid of interest.

[0046] The term "expression vector," as used herein, refers to a linear or circular nucleic acid molecule that contains one or more expression units. In addition to one or more expression units, an expression vector can also contain additional nucleic acid segments, such as... Expression vectors are generally derived from plasmid or viral DNA, or can contain elements of both.

[0047] As used herein, the term "host cell" can be any type of cell, e.g., a primary cell, a cultured cell, or a cell from a cell line. In certain embodiments, the term "host cell" refers to a cell transfected with a nucleic acid molecule and the progeny or potential progeny of such a cell. The progeny of such a cell may not be identical to the parent cell transfected with the nucleic acid molecule due, for example, to mutations or environmental influences that may occur in subsequent generations or in the integration of the nucleic acid molecule into the host cell genome.

[0048] The term "viable cell count," as used herein, refers to the number of living (surviving) cells present in a culture.

[0049] The term "cell viability," as used herein, refers to the ability of cultured cells to survive under a given set of culture conditions or experimental variations. This term, as used herein, also refers to the fraction of cells that are alive at a particular time in culture relative to the total number of live and dead cells at that time.

[0050] The term "recovery titer" or "titer," as used herein, refers to the total amount of expressed polypeptide or immunoglobulin produced in a cell culture divided by a given amount of medium volume.

[0051] The term "qP," as used herein, refers to cell-specific productivity, determined from total immunoglobulin produced divided by the integral viable cell concentration.

[0052] The term "IVC" as used herein refers to integral viable cell concentration;

number

[0053] An "isolated" polypeptide, antibody, nucleic acid, vector, cell, or composition is a polypeptide, antibody, nucleic acid, vector, cell, or composition in a form not found in nature. Isolated polypeptides, antibodies, nucleic acids, vectors, cells, or compositions include those that have been purified to the extent that they are no longer in the form found in nature. In some embodiments, an isolated antibody, nucleic acid, vector, cell, or composition is substantially pure. As used herein, "substantially pure" refers to a material that is at least 50% pure (i.e., free from contaminants). In some examples, the material is at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.

[0054] It will be understood that the terms "a" and "an," as used herein, refer to "one or more" of the listed components, unless otherwise indicated.

[0055] Unless otherwise specified or clear from the context, as used herein, the term "or" is understood to be inclusive. When used herein in phrases such as "A and / or B," the term "and / or" is intended to include both "A and B," "A or B," "A" and "B." Similarly, when used in phrases such as "A, B and / or C," the term "and / or" is intended to encompass each of the following aspects: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0056] It should be understood that whenever an embodiment is described herein with the word "comprising," otherwise similar embodiments described with the terms "consisting of" and / or "essentially consisting of" are also provided and are part of this disclosure. In this disclosure, "comprises," "comprising," "containing," and "having" and the like can have the meanings they have in U.S. and European patent law and can mean "includes," "including," and the like. "Consisting essentially of" or "consisting essentially of" likewise have the meanings under U.S. and European patent law. It should be understood that, as far as U.S. patent law is concerned, the term is open-ended, allowing for the presence of more than what is recited, as long as the basic or novel characteristics of what is recited are not altered by the presence of more than what is recited, but excluding prior art aspects. It should also be understood that, as far as European patent law is concerned, the use of "essentially consisting of" or "essentially comprising" means that certain additional components, i.e., those that do not materially affect the essential characteristics of the compound or composition, can be present.

[0057] As used herein, the terms "about" and "approximately," when used to modify a numerical value or numerical range, indicate a deviation of up to 5% above or below that value or range while remaining within the intended meaning of the stated value or range.

[0058] II. Nucleic Acids Encoding Immunoglobulins Human immunoglobulin G (IgG) contains heavy and light chain polypeptides, which together form immunoglobulins. Immunoglobulin light chains have variable light chains, which contain complementarity-determining regions (CDRs) that help bind to epitopes. Immunoglobulin light chains also contain light chain constant regions. IgG light chains can be either kappa or lambda light chains.

[0059] Immunoglobulin heavy chains have variable heavy chains, which contain complementarity-determining regions (CDRs) that help bind to epitopes. Immunoglobulin heavy chains also contain heavy chain constant regions. In the IgG heavy chain constant region, there are three constant domains (CH1, CH2, and CH3) and a hinge region.

[0060] The nucleotide sequence encoding the human IgG heavy chain constant region contains three introns (see Figure 10). Intron 1 in the human IgG heavy chain constant region is located between the exon encoding the CH1 region and the exon encoding the hinge region. Intron 2 in the human IgG heavy chain constant region is located between the exon encoding the hinge region and the exon encoding the CH2 region. Intron 3 in the human IgG heavy chain constant region is located between the exon encoding the CH2 region and the exon encoding the CH3 region.

[0061] Introns are known to be involved in mRNA nuclear export through the nuclear pore complex. For general information, see Kohler A. et al., Nature Review Molecular Cell Biology 8:761-773 (2007); Bjork, P. et al., Seminars in Cell & Developmental Biology 32:47-54 (2014); Reed, R. Current Opinion in Cell Biology, 15:326-331 (2003). Therefore, when preparing nucleic acids for recombinant immunoglobulin production, endogenous introns are typically not excised, because nucleic acids with introns typically increase immunoglobulin production titers compared to the corresponding cDNA versions. See, for example, Figure 2.

[0062] Although immunoglobulin production using nucleic acids containing endogenous introns in human IgG heavy chains increases immunoglobulin titers, the introns can introduce incorrect splice sites that can result in immunoglobulin fragments or variants, thereby reducing product purity. Introduction of immunoglobulin fragments or variants into the immunoglobulin pool can increase the difficulty in purifying these fragments and variants, thereby unduly burdening the purification process and contributing to reduced product purity.

[0063] In one aspect, deletion of one or two intronic nucleotide sequences in the IgG heavy chain constant region reduces production of immunoglobulin fragments or variants that, when expressed together with appropriate nucleic acid encoding an IgG light chain, result in increased immunoglobulin titers.

[0064] In certain embodiments, the present disclosure encompasses nucleic acids comprising a nucleotide sequence encoding an immunoglobulin heavy chain, wherein the nucleotide sequences of the second and third introns of the immunoglobulin heavy chain constant region are deleted. In certain embodiments, the present disclosure encompasses nucleic acids comprising a nucleotide sequence encoding an immunoglobulin heavy chain, wherein the nucleotide sequence comprises the sequence of the first intron and the sequences of the second and third introns of the immunoglobulin heavy chain constant region are deleted. In certain embodiments, the present disclosure encompasses nucleic acids comprising a nucleotide sequence encoding an immunoglobulin heavy chain, wherein the nucleotide sequence comprises only the first intron of the immunoglobulin heavy chain constant region. In certain embodiments, the present disclosure encompasses nucleic acids comprising a nucleotide sequence encoding an immunoglobulin heavy chain, wherein the nucleotide sequence comprises only the second intron of the immunoglobulin heavy chain constant region. In certain embodiments, the present disclosure encompasses nucleic acids comprising a nucleotide sequence encoding an immunoglobulin heavy chain, wherein the nucleotide sequence comprises only the third intron of the immunoglobulin heavy chain constant region.

[0065] In certain embodiments, the present disclosure encompasses nucleic acids comprising a nucleotide sequence encoding an immunoglobulin heavy chain, wherein the nucleotide sequence of one of the three endogenous introns of the immunoglobulin heavy chain constant region is deleted. In some examples, the first intron is deleted. In some examples, the second intron is deleted. In some examples, the third intron is deleted. In certain embodiments, the present disclosure encompasses nucleic acids comprising a nucleotide sequence encoding an immunoglobulin heavy chain, wherein the nucleotide sequence comprises the sequences of the second and third introns, but the sequence of the first intron of the immunoglobulin heavy chain constant region is deleted. In certain embodiments, the present disclosure encompasses nucleic acids comprising a nucleotide sequence encoding an immunoglobulin heavy chain, wherein the nucleotide sequence comprises the sequences of the first and third introns, but the sequence of the second intron of the immunoglobulin heavy chain constant region is deleted. In certain aspects, the disclosure encompasses a nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain, wherein the nucleotide sequence comprises the sequence of the first and second introns, but lacks the sequence of the third intron of an immunoglobulin heavy chain constant region.

[0066] In certain embodiments, the present disclosure encompasses nucleic acids comprising a nucleotide sequence encoding an immunoglobulin heavy chain in which the nucleotide sequences of two of the three endogenous introns in the immunoglobulin heavy chain constant region are deleted. In some examples, the first and second introns are deleted. In some examples, the first and third introns are deleted. In some examples, the second and third introns are deleted. In certain embodiments, the present disclosure encompasses nucleic acids comprising a nucleotide sequence encoding an immunoglobulin heavy chain in which the nucleotide sequence comprises the sequence of the first intron but the sequences of the second and third introns of the immunoglobulin heavy chain constant region are deleted. In certain embodiments, the present disclosure encompasses nucleic acids comprising a nucleotide sequence encoding an immunoglobulin heavy chain in which the nucleotide sequence comprises the sequence of the second intron but the sequences of the first and third introns of the immunoglobulin heavy chain constant region are deleted. In certain aspects, the disclosure encompasses a nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain, wherein the nucleotide sequence includes the sequence of the third intron but lacks the sequences of the first and second introns of an immunoglobulin heavy chain constant region.

[0067] In certain embodiments, the present disclosure encompasses nucleic acids comprising a nucleotide sequence encoding an immunoglobulin heavy chain, in which the nucleotide sequence of the first intron has been deleted, the nucleotide sequence of the second and / or third intron of an immunoglobulin heavy chain constant region has been deleted, and the nucleotide sequence of the second and / or third intron has been replaced with the nucleotide sequence of the first intron. In some examples, the nucleotide sequence of the second intron has been replaced with the nucleotide sequence of the first intron. In some examples, the nucleotide sequence of the third intron has been replaced with the nucleotide sequence of the first intron. In certain embodiments, the present disclosure encompasses nucleic acids comprising a nucleotide sequence encoding an immunoglobulin heavy chain, in which the nucleotide sequences of the first and second introns have been deleted from an immunoglobulin heavy chain constant region, and the nucleotide sequence of the second intron has been replaced with the nucleotide sequence of the first intron. In certain aspects, the disclosure encompasses nucleic acids comprising a nucleotide sequence encoding an immunoglobulin heavy chain, in which the nucleotide sequences of the first and third introns have been deleted from the immunoglobulin heavy chain constant region and the nucleotide sequence of the third intron has been replaced with the nucleotide sequence of the first intron. In certain aspects, the disclosure encompasses nucleic acids comprising a nucleotide sequence encoding an immunoglobulin heavy chain, in which the nucleotide sequence of the first intron has been deleted, the nucleotide sequences of the second and third introns of the immunoglobulin heavy chain constant region have been deleted, and the nucleotide sequences of the second and third introns have each been replaced with the nucleotide sequence of the first intron.

[0068] In certain embodiments, the present disclosure encompasses nucleic acids comprising a nucleotide sequence encoding an immunoglobulin heavy chain, in which the nucleotide sequence of the second and / or third intron in the immunoglobulin heavy chain constant region is replaced with the nucleotide sequence of the first intron. In some examples, the nucleotide sequence of the second intron is replaced with the nucleotide sequence of the first intron. In some examples, the nucleotide sequence of the third intron is replaced with the nucleotide sequence of the first intron. In certain embodiments, the present disclosure encompasses nucleic acids comprising a nucleotide sequence encoding an immunoglobulin heavy chain, in which the nucleotide sequence of the second intron in the immunoglobulin heavy chain constant region is replaced with the nucleotide sequence of the first intron, and the sequences of the first and third introns are not deleted. In certain embodiments, the present disclosure encompasses nucleic acids comprising a nucleotide sequence encoding an immunoglobulin heavy chain, in which the nucleotide sequence of the third intron in the immunoglobulin heavy chain constant region is replaced with the nucleotide sequence of the first intron, and the sequences of the first and second introns are not deleted. In certain aspects, the present disclosure encompasses a nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain, in which the nucleotide sequences of the second and third introns in an immunoglobulin heavy chain constant region are replaced with the nucleotide sequence of the first intron, and the sequence of the first intron is not deleted.

[0069] In certain embodiments, the present disclosure encompasses nucleic acids comprising a nucleotide sequence encoding an immunoglobulin heavy chain in which the nucleotide sequence of the third intron in the immunoglobulin heavy chain constant region has been replaced with the nucleotide sequence of an intron comprising approximately the same number of nucleotides as the nucleotide sequence of the first intron of the immunoglobulin heavy chain constant region. In some examples, the nucleotide sequence of the first intron in the immunoglobulin heavy chain constant region has been deleted. In certain embodiments, the present disclosure encompasses nucleic acids comprising a nucleotide sequence encoding an immunoglobulin heavy chain in which the nucleotide sequence of the third intron in the immunoglobulin heavy chain constant region has been replaced with the nucleotide sequence of an intron comprising approximately the same number of nucleotides as the nucleotide sequence of the first intron of the immunoglobulin heavy chain constant region, and the sequences of the first and / or second introns have been deleted.

[0070] In certain embodiments, the present disclosure encompasses nucleic acids comprising a nucleotide sequence encoding an immunoglobulin heavy chain in which the nucleotide sequence of a second intron in the immunoglobulin heavy chain constant region has been replaced with a nucleotide sequence of an intron comprising approximately the same number of nucleotides as the nucleotide sequence of the first intron of the immunoglobulin heavy chain constant region. In some examples, the nucleotide sequence of the first intron in the immunoglobulin heavy chain constant region has been deleted. In certain embodiments, the present disclosure encompasses nucleic acids comprising a nucleotide sequence encoding an immunoglobulin heavy chain in which the nucleotide sequence of a second intron in the immunoglobulin heavy chain constant region has been replaced with a nucleotide sequence of an intron comprising approximately the same number of nucleotides as the nucleotide sequence of the first intron of the immunoglobulin heavy chain constant region, and the sequences of the first and / or third introns have been deleted.

[0071] In any of the above aspects, a nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain can also be expressed together with a nucleic acid encoding an immunoglobulin light chain. In some examples, the immunoglobulin light chain is a kappa light chain. In some examples, the immunoglobulin light chain is a lambda light chain.

[0072] In any of the above aspects, the nucleic acid expresses immunoglobulins at higher titers than a nucleic acid containing all intron sequences in the heavy chain constant region when the nucleic acid is expressed together with a nucleic acid encoding an immunoglobulin light chain.

[0073] In any of the above aspects, the nucleic acid expresses immunoglobulins at higher titers than a nucleic acid that does not contain an intron sequence in the heavy chain constant region when the nucleic acid is expressed together with a nucleic acid encoding an immunoglobulin light chain.

[0074] In any of the above embodiments, the nucleic acid is not codon optimized. In any of the above embodiments, the nucleic acid is codon optimized.

[0075] In any of the above embodiments, the expressed immunoglobulin has an isotype of IgG1, IgG2, IgG3, or IgG4.

[0076] In any of the above aspects, the expressed immunoglobulin is a human, humanized, chimeric, or resurfaced immunoglobulin.

[0077] The nucleic acid of the present disclosure can be in the form of RNA or DNA. The DNA includes genomic DNA or synthetic DNA, and can be double-stranded or single-stranded, and if single-stranded, can be the coding strand or non-coding (antisense) strand. In some embodiments, the nucleic acid is DNA that lacks another endogenous intron.

[0078] In some embodiments, the nucleic acid comprises non-naturally occurring nucleotides. In some embodiments, the nucleic acid is recombinantly produced.

[0079] In certain embodiments, the nucleic acid is isolated.

[0080] III. Cells and Vectors Also provided are vectors and cells containing the nucleic acids described herein.

[0081] In certain aspects, provided herein are cells (e.g., host cells) comprising expression vectors that express (e.g., recombinantly) a nucleic acid described herein encoding an immunoglobulin. Provided herein are vectors (e.g., expression vectors) comprising nucleotide sequences encoding immunoglobulin heavy and light chains for recombinant expression in a host cell, e.g., a mammalian host cell. Also provided herein are host cells comprising such vectors for recombinantly expressing a nucleic acid comprising a nucleotide sequence encoding an immunoglobulin.

[0082] Recombinant expression of immunoglobulins requires an expression vector containing a nucleic acid comprising a nucleotide sequence encoding an immunoglobulin (e.g., IgG1, IgG2, IgG3, or IgG4) described herein. In some embodiments, the vector comprises a nucleic acid described herein containing a nucleotide sequence encoding an immunoglobulin. In some embodiments, a host cell comprises the vector. The disclosure also provides constructs in the form of plasmids, vectors, transcription cassettes, or expression cassettes comprising a nucleic acid described herein. In some embodiments, the vector is an expression vector, additional nucleotide sequences (e.g., a promoter) to facilitate recombinant immunoglobulin production in the host cell.

[0083] The expression vector can be introduced into cells (e.g., host cells) by conventional techniques, and the resulting cells can then be cultured by conventional techniques to produce the immunoglobulins described herein. Thus, provided herein are host cells containing a polynucleotide encoding an immunoglobulin or polypeptide thereof described herein operably linked to a promoter for expression of such sequence in the host cell.

[0084] In certain embodiments, the host cell contains vectors comprising nucleic acids encoding the immunoglobulin heavy and light chain polypeptides of the immunoglobulins described herein, hi certain embodiments, the host cell contains multiple different vectors comprising nucleic acids encoding all of the immunoglobulin polypeptides described herein.

[0085] A vector or combination of vectors can comprise nucleic acids encoding two or more polypeptides that interact to form an immunoglobulin as described herein: for example, a first nucleic acid encoding a heavy chain and a second nucleic acid encoding a light chain. When the two polypeptides are encoded by nucleic acids in two separate vectors, the vectors can be transfected into the same host cell.

[0086] A variety of host-expression vector systems can be utilized to express the immunoglobulins or polypeptides thereof (e.g., immunoglobulin constant regions; heavy or light chains) described herein. Such host-expression systems are either vehicles in which a coding sequence of interest can be produced and subsequently purified, or cells that, when transformed or transfected with appropriate nucleotide coding sequences, are capable of expressing the immunoglobulins or polypeptides thereof described herein in situ. These include microorganisms such as bacteria (e.g., E. coli and B. subtilis) transfected with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing the antibody coding sequences; yeast (e.g., Saccharomyces Pichia) transfected with recombinant yeast expression vectors containing the antibody coding sequences; insect cell systems infected with recombinant viral expression vectors (e.g., baculovirus) containing the antibody coding sequences; and plant cell systems (e.g., Chlamydomonas reinhardtii) infected with recombinant viral expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) containing the antibody coding sequences or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing the antibody coding sequences. reinhardtii); or mammalian cell lines (e.g., COS (e.g., COS1 or COS), CHO, BHK, MDCK, HEK293, NS0, PER.C6, VERO, CRL7030, HsS78Bst, HeLa, and NIH3T3, HEK-293T, HepG2, SP210, R1.1, BW, LM, BSC1, BSC40, YB / 20, and BMT10 cells) harboring a recombinant expression construct containing a promoter derived from the genome of a mammalian cell (e.g., a metallothionein promoter) or a promoter derived from a mammalian virus (e.g., an adenovirus late promoter, a vaccinia virus 7.5K promoter).

[0087] Once an immunoglobulin or polypeptide thereof (e.g., immunoglobulin constant region; heavy or light chain) described herein is produced by recombinant expression, it can be purified by any antibody purification method known in the art, such as chromatography (e.g., ion exchange chromatography, particularly affinity chromatography for a particular antigen followed by protein A, and sizing column chromatography), centrifugation, differential solubility, or any other standard protein purification technique. Additionally, the antibodies described herein can be fused to heterologous polypeptide sequences (e.g., FLAG tags, his tags, or avidin) described herein or otherwise known in the art to facilitate purification.

[0088] IV. Immunoglobulin production Immunoglobulins can be produced by any method for synthesizing immunoglobulins known in the art, for example, by recombinant expression techniques. The methods described herein employ, unless otherwise indicated, conventional techniques in molecular biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, and related fields that are within the skill of the art. These techniques are described and explained in detail, for example, in the references cited herein. For example, Maniatis T et al., (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; Sambrook J et al., (1989), Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press; Sambrook J et al., (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel FM et al. al.,Current Protocols in Molecular Biology,John Wiley & Sons(1987 and annual revision);Current Protocols in Immunology,John Wiley & Sons(1987 and annual revision)Gait(ed.)(1984)Oligonucleotide Synthesis:A Practical Approach,IRL Press;Eckstein(ed.)(1991)Oligonucleotides and Analogues:A Practical Approach,IRL Press;Birren B et See, e.g., (eds.) (1999) Genome Analysis: A Laboratory Manual, Cold Spring Harbor Laboratory Press.

[0089] In some aspects, isolated nucleic acids are provided having a nucleotide sequence encoding any of the immunoglobulin heavy chain constant regions, and optionally, the immunoglobulin heavy chain variable region and / or the immunoglobulin light chain of the present disclosure. L and / or C H Such nucleic acids may encode an amino acid sequence (e.g., an immunoglobulin light constant chain and / or heavy constant chain) comprising an immunoglobulin V L and / or V H In some embodiments, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided. In some embodiments, host cells comprising such nucleic acids are also provided. In some embodiments, the host cell comprises (e.g., is transfected with) (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising an immunoglobulin light chain and an amino acid sequence comprising an immunoglobulin heavy chain, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising an immunoglobulin light chain and a second vector comprising a nucleic acid encoding an amino acid sequence comprising an immunoglobulin heavy chain.

[0090] In some embodiments, host cells containing nucleic acids of the present disclosure encoding immunoglobulins are cultured under conditions suitable for expression of the antibody. For recombinant production of immunoglobulins using nucleic acids of the present disclosure, one or more nucleic acids encoding the heavy and / or light chains are isolated and inserted into one or more vectors for further cloning and / or expression in host cells as described herein.

[0091] In certain embodiments, a host cell comprises a nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain from which the nucleotide sequences of the second and third introns of the immunoglobulin heavy chain constant region are deleted, and a nucleic acid encoding an immunoglobulin light chain, and the host cell expresses immunoglobulins at higher titers than a host cell comprising a nucleic acid encoding an immunoglobulin heavy chain constant region containing all or no intron sequences. In certain embodiments, the cell is an isolated cell. In some embodiments, the core sequence of the first intron is deleted. In some embodiments, the core sequence of the second intron is deleted. In some embodiments, the core sequence of the third intron is deleted. In some embodiments, the nucleotide sequences of the first and second introns are deleted. In some embodiments, the nucleotide sequences of the first and third introns are deleted. In some embodiments, the nucleotide sequence of the second intron is replaced with the nucleotide sequence of the first intron. In some embodiments, the nucleotide sequence of the third intron is replaced with the nucleotide sequence of the first intron. In some embodiments, the nucleotide sequence of the second intron is replaced with the nucleotide sequence of the first intron. In some embodiments, the nucleotide sequence of the third intron is replaced with the nucleotide sequence of the first intron. In certain embodiments, the nucleotide sequence of the third intron in the immunoglobulin heavy chain constant region is replaced with the nucleotide sequence of an intron that comprises approximately the same number of nucleotides as the nucleotide sequence of the first intron of the immunoglobulin heavy chain constant region. In certain embodiments, the nucleotide sequence of the second intron in the immunoglobulin heavy chain constant region is replaced with the nucleotide sequence of an intron that comprises approximately the same number of nucleotides as the nucleotide sequence of the first intron of the immunoglobulin heavy chain constant region. In some embodiments, the nucleotide sequence of the first intron in the immunoglobulin heavy chain constant region is also deleted.

[0092] In any of the above aspects, the immunoglobulin light chain is a kappa light chain or a lambda light chain.

[0093] In any of the above embodiments, the nucleic acid is codon optimized. In any of the above embodiments, the nucleic acid is not codon optimized.

[0094] In any of the above embodiments, the immunoglobulin has an isotype of IgG1, IgG2, IgG3, or IgG4.

[0095] In any of the above aspects, the immunoglobulin is a human, humanized, chimeric, or resurfaced immunoglobulin.

[0096] In any of the above embodiments, the immunoglobulins produced from the pool of clones have a recovered titer of at least 1,000 mg / L. In any of the above embodiments, the immunoglobulins produced from the pool of clones have a recovered titer of at least 1,500 mg / L. In any of the above embodiments, the immunoglobulins produced from the pool of clones have a recovered titer of at least 2,000 mg / L. In any of the above embodiments, the immunoglobulins produced from the pool of clones have a recovered titer of at least 2,500 mg / L. In any of the above embodiments, the immunoglobulins produced from the pool of clones have a recovered titer of at least 3,000 mg / L.

[0097] In any of the above embodiments, the immunoglobulin produced from the highest expressing clone has a recovered titer of at least 1,000 mg / L. In any of the above embodiments, the immunoglobulin produced from the highest expressing clone has a recovered titer of at least 1,500 mg / L. In any of the above embodiments, the immunoglobulin produced from the highest expressing clone has a recovered titer of at least 2,000 mg / L. In any of the above embodiments, the immunoglobulin produced from the highest expressing clone has a recovered titer of at least 3,000 mg / L. In any of the above embodiments, the immunoglobulin produced from the highest expressing clone has a recovered titer of at least 4,000 mg / L. In any of the above embodiments, the immunoglobulin produced from the highest expressing clone has a recovered titer of at least 5,000 mg / L. In any of the above embodiments, the immunoglobulin produced from the highest expressing clone has a recovered titer of at least 6,000 mg / L. In any of the above embodiments, the immunoglobulin produced from the highest expressing clone has a recovered titer of at least 7,000 mg / L. In any of the above embodiments, the immunoglobulin produced from the highest expressing clone has a recovered titer of at least 8,000 mg / L. In any of the above embodiments, the immunoglobulin produced from the highest expressing clone has a recovered titer of at least 9,000 mg / L. In any of the above embodiments, the immunoglobulin produced from the highest expressing clone has a recovered titer of at least 10,000 mg / L. In any of the above embodiments, the immunoglobulin produced from the highest expressing clone has a recovered titer of at least 11,000 mg / L. In any of the above embodiments, the immunoglobulin produced from the highest expressing clone has a recovered titer of at least 12,000 mg / L.

[0098] In any of the above embodiments, the host cell is a eukaryotic cell. In any of the above embodiments, the eukaryotic cell is a CHO cell.

[0099] In some embodiments, an exogenous nucleic acid has been introduced into the cell.

[0100] In some aspects, the method further comprises purifying the immunoglobulin from the cell or host cell.

[0101] Aspects of the present disclosure can be further defined by reference to the following non-limiting examples, which describe in detail the preparation of certain antibodies of the present disclosure and methods of using the antibodies of the present disclosure. It will be apparent to those skilled in the art that numerous modifications, both to materials and methods, can be made without departing from the scope of the present disclosure. [Example]

[0102] It should be understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes in light thereof will be suggested to those skilled in the art, which are to be included within the spirit and scope of the present application.

[0103] Example 1. Characterization of mis-spliced ​​immunoglobulin variants Introns are important in several areas, including regulating alternative splicing, enhancing gene expression, and controlling the transport of mRNA from the nucleus. For this reason, nucleic acids used in expression vectors for producing immunoglobulins contain endogenous introns. However, this can result in mis-spliced ​​immunoglobulin variants that can be difficult to purify and / or reduce the recovered titer of the immunoglobulin product.

[0104] Cell culture production of MAb1 resulted in three immunoglobulin variants due to intron splice variants. Expression vectors containing the light and heavy chain sequences of each immunoglobulin, including endogenous intron sequences in the heavy chain constant region, were linearized and transfected into Chinese hamster ovary (CHO) cells. Transfection of the linearized expression vector was performed by nucleofection to generate pools of cells. The linearized expression vector was integrated into the CHO genome by random integration. The pool of cells expressing MAb1 was cloned to generate cell lines that produced the clones using a cell line development process appropriate at that time.

[0105] CHO cells were grown and induced to initiate MAb1 production. Immunoglobulins were collected and purified using high-performance size-exclusion chromatography (HPSEC). For HPSEC fractionation, MAb1 was injected onto a TSK-gel G3000SW×L column (7.8 mm × 30 cm; Tosoh Bioscience, King of Prussia, PA, USA) at ambient column temperature. The sample was eluted isocratically with a mobile phase composed of 0.1 M sodium phosphate, 0.1 M sodium sulfate, pH 6.8, at a flow rate of 1.0 mL / min. Fractions collected from multiple injections were pooled and concentrated prior to characterization and analysis. See Harris, C. et al., MABS, 11:1452-1463 (2019).

[0106] As shown in Figure 1, HPSEC revealed three splice variants: a monomeric immunoglobulin with an extension, and two fragments. The extended immunoglobulin variant was confirmed to have an additional lambda light chain constant domain at the C-terminus of the immunoglobulin. This was due to an alternative heavy chain transcript with an additional lambda light chain constant domain at the C-terminus of the heavy chain. Two fragments were determined to be splice variants associated with intron 2 between the hinge and CH2 regions. Specifically, one fragment resulted in a truncated heavy chain due to an in-frame stop codon, while the other fragment resulted in a frameshift resulting in a stop codon and a truncated heavy chain due to a missplicing event. Thus, immunoglobulin fragment variants are produced by introns in the immunoglobulin heavy chain constant region.

[0107] Example 2. Manipulation of nucleic acids to reduce immunoglobulin splice variants To eliminate the immunoglobulin fragments produced in Example 1, a codon-optimized cDNA nucleotide sequence was generated without any introns in the immunoglobulin heavy chain constant region of MAb2. The cDNA version removed intron 2 in the immunoglobulin heavy chain constant region, preventing the production of two fragments.

[0108] However, it is well known that introns are required to enhance protein expression in CHO cells. Therefore, CHO cells were transfected by nucleofection with non-codon-optimized gDNA or codon-optimized cDNA to generate pools, which were grown and induced to produce immunoglobulins in shake flasks as in a pooled fed-batch process. See Figure 2A. Figure 2B shows that the cDNA version of MAb2 produced significantly less immunoglobulin compared to the genomic DNA (gDNA) version, as indicated by the recovery titer.

[0109] Because the two immunoglobulin fragment variants result from missplicing in intron 2, individual introns were removed from the immunoglobulin heavy chain constant region and compared with cDNA or gDNA versions lacking any introns in the immunoglobulin heavy chain constant region. The following constructs were generated and tested: (1) gDNA containing all three introns in the heavy chain constant region (non-codon optimized), (2) cDNA (codon optimized), (3) gDNA with intron 1 removed (non-codon optimized), (4) gDNA with intron 2 removed (non-codon optimized), (5) gDNA with intron 3 removed (non-codon optimized), and (6) gDNA with all introns removed (non-codon optimized). See Figure 3A.

[0110] Nucleic acids were prepared, transfected into CHO cells, and pooled as described in Example 1. Pools were screened for single-cell clones using a Single Cell Printer™ (Cytena), which deposits droplets containing single cells into wells of a 384-well plate. Single-cell deposition was confirmed using a Cellavista® plate reader (Synentec). The highest-expressing clones were selected for further characterization. The highest clones were expanded and induced to produce MAb2 for each construct. MAb2 was recovered on day 13. Figure 3B shows that the cDNA and gDNA (intron-free) yielded the lowest recovered titers. In contrast, gDNA constructs lacking either intron 1, intron 2, or intron 3 yielded increased recovered titers compared to gDNA, gDNA (intron-free), and cDNA constructs containing all three introns.

[0111] Figure 4A-C shows that the mean viable cell count (VCN), cell viability, and integral viable cell concentration (IVC) were similar across all constructs, while Figure 4D reveals that the increased titer resulted from increased cellular productivity (qP). It was surprising that removing one intron increased the recovered titer, as introns are known to increase immunoglobulin expression.

[0112] Example 3. Manipulation of nucleic acids to increase recovery titers To further determine the importance of each intron to the ability of CHO cells to produce immunoglobulins, the following MAb2 constructs were generated: (1) gDNA with intron 2 removed (non-codon optimized), (2) gDNA with introns 1 and 2 removed (non-codon optimized), (3) gDNA with introns 2 and 3 removed (non-codon optimized), and (4) gDNA without any introns (non-codon optimized). To determine the importance of using codon-optimized nucleotide sequences, a set of identical constructs was generated except that they used codon-optimized sequences.

[0113] The constructs were transfected into CHO cells as described in Example 2. Similarly, CHO cells were grown and then induced for immunoglobulin production as described in Example 2. Immunoglobulin products were harvested on day 11, and the recovery titer, qP, VCN, and IVC were all determined. Figure 5A reveals that the highest recovery titers were obtained for immunoglobulins produced from gDNA lacking intron 2 and gDNA lacking introns 2 and 3, regardless of whether the nucleotide sequence was codon-optimized or not. However, gDNA lacking introns 1 and 2 had similar titer levels to gDNA lacking any introns. Figures 5B and 5C show that the VCN and IVC were nearly identical for each. However, cellular productivity (qP) revealed that gDNA lacking intron 2 and gDNA lacking introns 2 and 3 were the most productive. See Figure 5D. This suggests that intron 1 is important for increased immunoglobulin production.

[0114] To further understand the importance of intron 1 for increased immunoglobulin production, several novel MAb2 constructs were generated. In addition to the non-codon-optimized constructs generated above (i.e., gDNA with all introns (non-codon-optimized), gDNA without intron 2 (non-codon-optimized), gDNA without introns 2 and 3 (non-codon-optimized), and gDNA without introns 1 and 2 (non-codon-optimized)), the following constructs were generated: (1) gDNA with intron 3 moved to the position of intron 1 and introns 1 and 2 deleted (non-codon-optimized), (2) gDNA with intron 3 moved to the position of intron 1, where the nucleotide sequence of intron 3 was modified to enhance the 5' splice donor site by making a single nucleotide change in the intron sequence. (3) gDNA (non-codon optimized) with intron 1 moved to the position of intron 3 and introns 2 and 3 deleted, (4) gDNA without any introns (non-codon optimized), (5) gDNA with wild-type IgG1 and all introns (non-codon optimized), (6) gDNA with wild-type IgG1 and without introns 2 and 3 (non-codon optimized), (7) gDNA with wild-type IgG1 and without introns 1 and 2 (non-codon optimized), and (8) gDNA with wild-type IgG1 and without any introns (non-codon optimized). See Figure 6A.

[0115] These constructs were transfected into CHO cells as described in Example 1. The CHO cells were grown and induced to produce immunoglobulins as described in Example 1. Immunoglobulins were harvested on day 11, and the recovery titers were determined. Figure 6B shows that moving intron 1 to the position of intron 3 results in similar recovery titers as gDNA with all introns and gDNA with only intron 1. Figure 6B also confirms that the effect of intron 1 in maintaining similar titers to constructs containing all three introns is independent of whether the construct is wild-type or half-life-extended IgG1.

[0116] Example 4. Manipulation of nucleic acids in additional immunoglobulins to determine if there is an increase in recovery titer To determine whether the results seen with MAb2 are specific to that immunoglobulin, additional immunoglobulin molecules will be tested. Figure 7 shows the different constructs made for MAb2, MAb1, MAb3, and MAb4. For each immunoglobulin, the following constructs were made: (1) gDNA with all introns (non-codon optimized), (2) gDNA with only intron 1 (non-codon optimized), and (3) gDNA without any introns (non-codon optimized). MAb2 and MAb3 contain kappa light chains, while MAb1 and MAb4 contain lambda light chains. The lambda light chains have different introns between the variable and constant light chains and different polyA tails. These constructs were made as previously described in Example 1. These constructs were transfected into CHO cells to generate pools as described in Example 2. The CHO cells were grown and induced to produce immunoglobulins as described in Example 2. Immunoglobulins were harvested on day 11 and recovery titers determined.

[0117] Figure 8 shows that the presence of only intron 1 in the heavy chain constant region of MAb2, MAb3, MAb1, and MAb4 resulted in similar immunoglobulin titers compared to constructs with all introns in the heavy chain constant region. In addition, constructs with only intron 1 increased titers compared to gDNA lacking any introns in the immunoglobulin heavy chain constant region. Furthermore, Figure 9 shows that for MAb2, MAb3, MAb1, and MAb4, constructs with all introns in the heavy chain constant region had similar immunoglobulin titers from days 7 to 11 compared to constructs with only intron 1 in the heavy chain constant region. For all molecules tested, gDNA lacking any introns resulted in significantly lower immunoglobulin titers than constructs with all introns in the heavy chain constant region and constructs with only intron 1.

[0118] These data provide further evidence that intron 1 of the heavy chain constant region is important in maintaining higher immunoglobulin titer levels. Furthermore, the data show that the increased titer levels are not limited to MAb2. Furthermore, the presence of kappa or lambda light chains on the immunoglobulin does not affect titer levels.

[0119] Example 5. Manipulation of nucleic acid to replace intron 3 with an intron of the same size as intron 1 To determine why intron 1 can increase the recovered titer, this example will demonstrate that the reason is the size of intron 1 rather than the nucleotide sequence itself. Intron 1 in MAb2 is 391 nucleotides, while intron 3 is 97 nucleotides. The following constructs will be made: (1) MAb2 gDNA (non-codon optimized) with all introns, (2) MAb2 (non-codon optimized) without introns 2 and 3, (3) MAb2 (non-codon optimized) without introns 1 and 2 but with the number of nucleotides in intron 3 increased to approximately the same size as intron 1, (4) MAb2 (non-codon optimized) but with the size of intron 1 reduced to approximately the size of intron 3 and introns 2 and 3 deleted, and (5) MAb2 gDNA (non-codon optimized) without any introns.

[0120] These constructs will be made as previously described in Example 2. These constructs will be transfected into CHO cells as described in Example 2. The CHO cells will be grown and induced to produce immunoglobulins as described in Example 1. The immunoglobulins will be harvested on day 11, and the recovery titer will be determined. The results will show that increasing the size of intron 3 will result in an increased recovery titer, comparable to MAb2 lacking introns 2 and 3. However, reducing the size of intron 1 will result in comparable results to MAb2 lacking introns 1 and 2. This example will demonstrate that the size of intron 1 is more important than its nucleotide sequence itself.

[0121] Aspects of the present invention are further described in the following sections: [Section 1] An isolated nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain, wherein the nucleotide sequences of intron 2 and intron 3 of the immunoglobulin heavy chain constant region are deleted. [Section 2] An isolated nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain constant region from which the nucleotide sequence of one intron of said immunoglobulin heavy chain is deleted. [Section 3] Item 3. The nucleic acid according to Item 2, wherein the nucleotide sequence of intron 1 of the immunoglobulin heavy chain constant region is deleted. [Section 4] Item 3. The nucleic acid according to Item 2, wherein the nucleotide sequence of intron 2 of the immunoglobulin heavy chain constant region is deleted. [Section 5] Item 3. The nucleic acid according to Item 2, wherein the nucleotide sequence of intron 3 of the immunoglobulin heavy chain constant region is deleted. [Section 6] An isolated nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain constant region from which the nucleotide sequences of two introns of said immunoglobulin heavy chain are deleted. [Section 7] Item 7. The nucleic acid according to Item 6, wherein the nucleotide sequences of intron 1 and intron 2 of the immunoglobulin heavy chain constant region are deleted. [Section 8] Item 7. The nucleic acid according to Item 6, wherein the nucleotide sequences of intron 1 and intron 3 of the immunoglobulin heavy chain constant region are deleted. [Section 9] 1. An isolated nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain constant region, wherein the nucleotide sequence of intron 1 of the immunoglobulin heavy chain constant region is deleted, and the nucleotide sequence of intron 2 and / or intron 3 of the immunoglobulin heavy chain constant region is deleted, and the nucleotide sequence of intron 2 and / or intron 3 is replaced with the nucleotide sequence of intron 1. [Section 10] Item 10. The nucleic acid according to Item 9, wherein the nucleotide sequence of intron 2 of the immunoglobulin heavy chain constant region is replaced with the nucleotide sequence of intron 1. [Section 11] Item 10. The nucleic acid according to Item 9, wherein the nucleotide sequence of intron 3 of the immunoglobulin heavy chain constant region is replaced with the nucleotide sequence of intron 1. [Section 12] An isolated nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain constant region, wherein the nucleotide sequence of intron 2 and / or intron 3 of said immunoglobulin heavy chain constant region is replaced with the nucleotide sequence of intron 1. [Section 13] Item 13. The nucleic acid according to Item 12, wherein the nucleotide sequence of intron 2 of the immunoglobulin heavy chain constant region is replaced with the nucleotide sequence of intron 1. [Section 14] Item 13. The nucleic acid according to Item 12, wherein the nucleotide sequence of intron 3 of the immunoglobulin heavy chain constant region is replaced with the nucleotide sequence of intron 1. [Section 15] An isolated nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain, in which the nucleotide sequence of intron 3 of the immunoglobulin heavy chain constant region is replaced with a nucleotide sequence of an intron containing approximately the same number of nucleotides as the nucleotide sequence of intron 1 of the immunoglobulin heavy chain constant region. [Section 16] An isolated nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain, in which the nucleotide sequence of intron 2 of the immunoglobulin heavy chain constant region is replaced with a nucleotide sequence of an intron comprising approximately the same number of nucleotides as the nucleotide sequence of intron 1 of the immunoglobulin heavy chain constant region. [Section 17] 17. The nucleic acid according to item 15 or 16, wherein the nucleotide sequence of intron 1 of the immunoglobulin heavy chain constant region is deleted. [Section 18] 18. The nucleic acid according to any one of items 1 to 17, wherein when the nucleic acid is expressed together with a nucleic acid encoding an immunoglobulin light chain, the nucleic acid expresses an immunoglobulin with a higher titer than a nucleic acid containing all intron sequences of the immunoglobulin heavy chain constant region. [Section 19] 18. The nucleic acid according to any one of items 1 to 17, wherein when the nucleic acid is expressed together with a nucleic acid encoding an immunoglobulin light chain, the nucleic acid expresses an immunoglobulin with a higher titer than a nucleic acid that does not contain an intron sequence of the immunoglobulin heavy chain constant region. [Section 20] 20. The nucleic acid according to item 18 or 19, wherein the immunoglobulin light chain is a kappa light chain or a lambda light chain. [Section 21] 21. The nucleic acid according to any one of items 1 to 20 above, which is codon-optimized. [Section 22] 22. The nucleic acid according to any one of items 18 to 21, wherein the expressed immunoglobulin has an isotype of IgG1, IgG2, IgG3, or IgG4. [Section 23] 23. The nucleic acid of claim 22, wherein the immunoglobulin is a human, humanized, chimeric, or resurfaced immunoglobulin. [Section 24] 24. The nucleic acid according to any one of items 1 to 23, which is deoxyribonucleic acid (DNA). [Section 25] A vector comprising the nucleic acid according to any one of items 1 to 24 above. [Section 26] 26. An expression vector comprising the nucleic acid according to any one of items 1 to 25 above. [Section 27] A host cell comprising the vector according to item 25 above. [Section 28] A host cell comprising the expression vector according to item 26. [Section 29] 29. The host cell according to item 27 or 28 above, which is a eukaryotic cell. [Section 30] 30. The host cell according to item 29, wherein the eukaryotic cell is a Chinese hamster ovary (CHO) cell. [Section 31] A method for producing an immunoglobulin, the method comprising a step of culturing host cells in a medium under conditions in which the cells express the immunoglobulin, the host cells comprising the nucleic acid encoding the immunoglobulin heavy chain and the nucleic acid encoding the immunoglobulin light chain described in Item 1 above, and the host cells express the immunoglobulin at a higher titer than host cells comprising a nucleic acid encoding an immunoglobulin heavy chain in which all or none of introns 1 to 3 of an immunoglobulin heavy chain constant region are present and a nucleic acid encoding an immunoglobulin light chain. [Section 32] A method for producing an immunoglobulin, the method comprising a step of culturing host cells in a medium under conditions in which the cells express the immunoglobulin, the host cells comprising the nucleic acid encoding the immunoglobulin heavy chain described in Item 2 above and a nucleic acid encoding an immunoglobulin light chain, and the host cells express the immunoglobulin at a higher titer than host cells comprising a nucleic acid encoding an immunoglobulin heavy chain in which all or none of introns 1 to 3 of an immunoglobulin heavy chain constant region are present and a nucleic acid encoding an immunoglobulin light chain. [Section 33] 33. The method according to item 32, wherein the nucleotide sequence of intron 1 of the immunoglobulin heavy chain constant region is deleted. [Section 34] 33. The method according to item 32, wherein the nucleotide sequence of intron 2 of the immunoglobulin heavy chain constant region is deleted. [Section 35] 33. The method according to item 32, wherein the nucleotide sequence of intron 3 of the immunoglobulin heavy chain constant region is deleted. [Section 36] A method for producing an immunoglobulin, the method comprising a step of culturing host cells in a medium under conditions in which the cells express the immunoglobulin, the host cells comprising the nucleic acid encoding the immunoglobulin heavy chain described in Item 6 above and a nucleic acid encoding an immunoglobulin light chain, and the host cells express the immunoglobulin at a higher titer than host cells comprising a nucleic acid encoding an immunoglobulin heavy chain in which all or none of introns 1 to 3 of an immunoglobulin heavy chain constant region are present and a nucleic acid encoding an immunoglobulin light chain. [Section 37] 37. The method according to item 36, wherein introns 1 and 2 of the immunoglobulin heavy chain constant region are deleted. [Section 38] 37. The method according to item 36, wherein introns 1 and 3 of the immunoglobulin heavy chain constant region are deleted. [Section 39] A method for producing an immunoglobulin, the method comprising a step of culturing host cells in a medium under conditions in which the cells express the immunoglobulin, the host cells comprising the nucleic acid encoding the immunoglobulin heavy chain described in Item 9 above and a nucleic acid encoding an immunoglobulin light chain, the host cells expressing the immunoglobulin at a higher titer than host cells comprising a nucleic acid encoding an immunoglobulin heavy chain in which all or none of introns 1 to 3 of the immunoglobulin heavy chain constant region are present and a nucleic acid encoding an immunoglobulin light chain. [Section 40] 40. The method according to claim 39, wherein the nucleotide sequence of intron 2 of the immunoglobulin heavy chain constant region is replaced with the nucleotide sequence of intron 1. [Section 41] 40. The method according to claim 39, wherein the nucleotide sequence of intron 3 of the immunoglobulin heavy chain constant region is replaced with the nucleotide sequence of intron 1. [Section 42] 13. A method for producing an immunoglobulin, the method comprising a step of culturing host cells in a medium under conditions in which the cells express the immunoglobulin, the host cells comprising the nucleic acid encoding the immunoglobulin heavy chain of item 12 above and a nucleic acid encoding an immunoglobulin light chain, the host cells expressing the immunoglobulin at a higher titer than host cells comprising a nucleic acid encoding an immunoglobulin heavy chain in which all or none of introns 1 to 3 of the immunoglobulin heavy chain constant region are present and a nucleic acid encoding an immunoglobulin light chain. [Section 43] 43. The method according to item 42, wherein the nucleotide sequence of intron 2 of the immunoglobulin heavy chain constant region is replaced with the nucleotide sequence of intron 1. [Section 44] 43. The method according to item 42, wherein the nucleotide sequence of intron 3 of the immunoglobulin heavy chain constant region is replaced with the nucleotide sequence of intron 1. [Section 45] A method for producing an immunoglobulin, the method comprising a step of culturing host cells in a medium under conditions in which the cells express the immunoglobulin, the host cells comprising the nucleic acid encoding the immunoglobulin heavy chain of item 15 above and a nucleic acid encoding an immunoglobulin light chain, the host cells expressing the immunoglobulin at a higher titer than host cells comprising a nucleic acid encoding an immunoglobulin heavy chain in which all or none of introns 1 to 3 of an immunoglobulin heavy chain constant region are present and a nucleic acid encoding an immunoglobulin light chain. [Section 46] A method for producing an immunoglobulin, the method comprising a step of culturing host cells in a medium under conditions in which the cells express the immunoglobulin, the host cells comprising the nucleic acid encoding the immunoglobulin heavy chain of Item 16 and a nucleic acid encoding an immunoglobulin light chain, and the host cells express the immunoglobulin at a higher titer than host cells comprising a nucleic acid encoding an immunoglobulin heavy chain in which all or none of introns 1 to 3 of an immunoglobulin heavy chain constant region are present and a nucleic acid encoding an immunoglobulin light chain. [Section 47] 47. The method according to item 45 or 46, wherein the nucleotide sequence of intron 1 of the immunoglobulin heavy chain constant region is deleted. [Section 48] 48. The method according to any one of items 31 to 47, wherein the nucleic acid encoding the immunoglobulin heavy chain is deoxyribonucleic acid (DNA). [Section 49] 48. The method according to any one of items 31 to 47, wherein the immunoglobulin light chain is a kappa light chain or a lambda light chain. [Section 50] 50. The method according to any one of items 31 to 49, wherein the nucleic acid encoding the immunoglobulin heavy chain is codon-optimized. [Section 51] 51. The method according to any one of items 31 to 50 above, wherein the expressed immunoglobulin has an isotype of IgG1, IgG2, IgG3, or IgG4. [Section 52] 52. The method of claim 51, wherein the expressed immunoglobulin is a human, humanized, chimeric, or resurfaced immunoglobulin. [Section 53] 53. The method of any one of paragraphs 31 to 52, wherein the expressed immunoglobulins produced from the pool of clones have a recovered titer of at least 1,000 mg / L, at least 1,500 mg / L, at least 2,000 mg / L, at least 2,500 mg / L, or at least 3,000 mg / L. [Section 54] 54. The method of any one of paragraphs 31 to 53, wherein the immunoglobulins produced from the highest expressing clones have a recovered titer of at least 1,000 mg / L, at least 1,500 mg / L, at least 2,000 mg / L, at least 3,000 mg / L, at least 4,000 mg / L, at least 5,000 mg / L, at least 6,000 mg / L, at least 7,000 mg / L, at least 8,000 mg / L, at least 9,000 mg / L, at least 10,000 mg / L, at least 11,000 mg / L, or at least 12,000 mg / L. [Section 55] 55. The method according to any one of items 31 to 54 above, wherein the host cell is a eukaryotic cell. [Section 56] 56. The method according to claim 55, wherein the eukaryotic cell is a CHO cell. The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described will become apparent to those skilled in the art from the foregoing description and the accompanying drawings. All such modifications are intended to be included within the scope of the appended claims.

[0122] All references (e.g., publications, or patents or patent applications) cited in this specification are herein incorporated by reference in their entirety for all purposes to the same extent as if each individual reference (e.g., publication, or patent or patent application) was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

[0123] Other aspects are within the scope of the following claims.

Claims

1. An isolated nucleic acid comprising a nucleotide sequence encoding a mammalian IgG1 immunoglobulin heavy chain, wherein the nucleotide sequences of intron 2 and intron 3 of the immunoglobulin heavy chain constant region are deleted, and the nucleotide sequence of intron 1 of the immunoglobulin heavy chain constant region is removed from its original position and substituted in the position of the nucleotide sequence of intron 3.

2. A method for producing a mammalian IgG1 immunoglobulin, said method comprising culturing mammalian host cells in a culture medium under conditions in which the cells express said immunoglobulin, said host cells comprising nucleic acid encoding an immunoglobulin heavy chain according to claim 1 and nucleic acid encoding an IgG1 immunoglobulin light chain, said host cells expressing an intact immunoglobulin heavy chain at a higher titer than host cells comprising nucleic acid encoding an immunoglobulin heavy chain and an immunoglobulin light chain that include all or none of introns 1 to 3 of the immunoglobulin heavy chain constant region nucleic acid.

3. 3. The method of claim 2, wherein the nucleic acid encoding the immunoglobulin heavy chain is deoxyribonucleic acid (DNA).

4. 3. The method of claim 2, wherein the immunoglobulin light chain is a kappa light chain or a lambda light chain.

5. The method of any one of claims 2 to 4, wherein the nucleic acid encoding the immunoglobulin heavy chain is codon-optimized.

6. The method of any one of claims 2 to 5, wherein the expressed immunoglobulin is a human, humanized, chimeric, or resurfaced immunoglobulin.

7. 7. The method of any one of claims 2 to 6, wherein the expressed immunoglobulins produced from a pool of clones have a recovered titer of at least 1,000 mg / L, at least 1,500 mg / L, at least 2,000 mg / L, at least 2,500 mg / L, or at least 3,000 mg / L.

8. 8. The method of any one of claims 2-7, wherein the immunoglobulins produced from the highest expressing clones have a recovered titer of at least 1,000 mg / L, at least 1,500 mg / L, at least 2,000 mg / L, at least 3,000 mg / L, at least 4,000 mg / L, at least 5,000 mg / L, at least 6,000 mg / L, at least 7,000 mg / L, at least 8,000 mg / L, at least 9,000 mg / L, at least 10,000 mg / L, at least 11,000 mg / L, or at least 12,000 mg / L.

9. The method of any one of claims 2 to 8, wherein the host cell is a Chinese hamster ovary (CHO) cell.

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