Method for producing Anti-CD38 antibody

Abstract

Provided in the present invention are a method for controlling the concentration of Mn ions during the preparation of an anti-CD38 antibody so as to improve the content of G1F and G0F oligosaccharide modifications of the antibody, and a method for formulating the antibody into a pharmaceutical product.

Description

Method for producing anti-CD38 antibodies Technical Field

[0001] This disclosure relates to a method for preparing anti-CD38 antibodies, and more specifically, to a method for controlling the concentration of Mn ions in the culture medium during the production of anti-CD38 antibodies. Background Technology

[0002] Cell culture medium is a crucial factor in antibody drug production because it affects cell growth, productivity, and product quality, ultimately influencing drug safety and efficacy. A well-defined culture medium with a defined chemical composition avoids quality variations caused by different batches of serum, improving reproducibility. It also avoids serum toxicity to cells and the risk of serum-derived contamination, promotes cell differentiation in vitro, enhances the expression levels of cell products, and facilitates purification.

[0003] Oligosaccharides are highly branched carbohydrate structures composed of monosaccharides, such as galactose, mannose, N-acetylglucosamine, fucose, and sialic acid. Monoclonal antibodies have only one N-glycosylation site in the Fc region, accounting for approximately 2%–3% of the total mass. Although the proportion is relatively small, N-glycosylation participates in many important processes, playing a crucial role in the pharmacokinetics and pharmacodynamic regulation of monoclonal antibodies, and is dependent on cell culture conditions. CN113015805A discloses the production of daratumumab antibodies with suitable G1F oligosaccharide content ranges by using trace metal Mn of less than 8.5 ppb in the culture medium. Therefore, there is a need to develop methods for predictable glycosylation profiles of antibodies obtained through cell culture in chemically defined media. Summary of the Invention

[0004] This disclosure provides a method for generating an anti-CD38 antibody expressed by a polynucleotide encoding the heavy chain variable region (VH) of SEQ ID NO:1 and the light chain variable region (VL) of SEQ ID NO:2 and having a G1F oligosaccharide content between about 17% and about 26%, the method comprising:

[0005] a) Prepare a culture medium containing manganese (Mn) at a concentration between approximately 8.5 parts per billion (ppb) and approximately 100 ppb;

[0006] b) By culturing host cells containing polynucleotides encoding VH of SEQ ID NO:1 and VL of SEQ ID NO:2 in the culture medium prepared in step a), the G1F oligosaccharide content of the anti-CD38 antibody is controlled, thereby producing an anti-CD38 antibody expressed by polynucleotides encoding VH of SEQ ID NO:1 and VL of SEQ ID NO:2 and having a G1F oligosaccharide content between about 17% and about 26%.

[0007] This disclosure also provides a method for generating an anti-CD38 antibody, the anti-CD38 antibody being expressed by polynucleotides encoding VH of SEQ ID NO:1 and VL of SEQ ID NO:2 and having a G1F oligosaccharide content between about 17% and about 26%, the method comprising:

[0008] The host cells expressing polynucleotides encoding VH (SEQ ID NO:1) and VL (SEQ ID NO:2) were cultured under conditions that produced anti-CD38 antibodies; and the G1F oligosaccharide content of the anti-CD38 antibody was controlled by monitoring the concentration of Mn in the culture medium during the biosynthesis of the anti-CD38 antibody and adjusting the concentration of Mn in the culture medium during the biosynthesis of the anti-CD38 antibody, wherein the concentration of Mn in the culture medium was adjusted to contain between about 8.5 ppb and about 100 ppb, thereby producing an anti-CD38 antibody with a G1F oligosaccharide content between about 17% and about 26%.

[0009] This disclosure also provides a method for producing a pharmaceutical product comprising an anti-CD38 antibody expressed by polynucleotides encoding VH of SEQ ID NO:1 and VL of SEQ ID NO:2 and having a G1F oligosaccharide content between about 17% and about 26%, the method comprising:

[0010] a) Prepare a culture medium containing Mn between approximately 8.5 ppb and approximately 100 ppb;

[0011] b) Culture host cells containing polynucleotides encoding VH of SEQ ID NO:1 and VL of SEQ ID NO:2 in the culture medium prepared in step a) to control the G1F oligosaccharide content of the anti-CD38 antibody, thereby producing an anti-CD38 antibody with a G1F oligosaccharide content between about 15% and about 27%.

[0012] c) Formulate anti-CD38 antibodies into pharmaceutical products.

[0013] The advantage of this disclosure is that, unlike the teachings of the prior art, this disclosure provides a predictable glycosylation profile for producing the desired anti-CD38 antibody in a culture medium containing Mn between about 8.5 ppb and about 100 ppb, which is beneficial for the industrial production of anti-CD38 antibodies. Attached Figure Description

[0014] Figures 1A and 1B show the correlation between the percentage of anti-CD38 antibody G1F oligosaccharides (G1F%) and the calculated manganese (Mn) concentration in the culture medium (between approximately 8.5 ppb and approximately 80 ppb); Figures 1C and 1D show the correlation between the percentage of anti-CD38 antibody G0F oligosaccharides (G0F%) and the calculated manganese (Mn) concentration in the culture medium (between approximately 8.5 ppb and approximately 80 ppb); Figure 1E shows the relationship between the calculated Mn concentrations of the culture medium components used in each group.

[0015] Figures 2A and 2B show the correlation between the percentage of anti-CD38 antibody G1F oligosaccharides (G1F%) and the calculated manganese (Mn) concentration in the culture medium (between approximately 10 ppb and approximately 100 ppb); Figures 2C and 2D show the correlation between the percentage of anti-CD38 antibody G0F oligosaccharides (G0F%) and the calculated manganese (Mn) concentration in the culture medium (between approximately 10 ppb and approximately 100 ppb); Figure 2E shows the relationship between the calculated Mn concentrations of the culture medium components used in each group.

[0016] Figure 3A shows the correlation between the percentage of anti-CD38 antibody G1F oligosaccharides (G1F%) and the calculated manganese (Mn) concentration in the culture medium (between approximately 8.5 ppb and approximately 100 ppb); Figure 3B shows the correlation between the percentage of anti-CD38 antibody G0F oligosaccharides (G0F%) and the calculated manganese (Mn) concentration in the culture medium (between approximately 8.5 ppb and approximately 100 ppb); Figure 3C shows the relationship between the calculated Mn concentrations of the culture medium components used in each group. Detailed Implementation

[0017] It should be understood that the terminology used herein is for the purpose of describing specific implementations only and is not intended to be limiting. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0018] While any methods and materials similar to or equivalent to those described herein may be used in practice to test this disclosure, exemplary materials and methods are described herein. The following terminology will be used in describing and claiming protection for this disclosure.

[0019] definition

[0020] As used in this specification and the appended claims, unless otherwise expressly stated, the singular forms “a,” “an,” “the,” and “described” include plural references. Thus, for example, a reference to “a cell” includes a combination of two or more cells, and so on.

[0021] “About” means within an acceptable range of error for a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value was measured or determined, i.e., the limitations of the measurement system. In the context of a particular measurement, result, or implementation, unless otherwise expressly stated in the embodiments or elsewhere in the specification, “about” means within one standard deviation, or up to 5%, 6%, 7%, 8%, 9%, or 10%, according to convention in the art.

[0022] An antibody is an immunoglobulin molecule consisting of two heavy chains (HC) and two light chains (LC) linked by disulfide bonds. Each heavy chain comprises a variable region (VH) and a constant region (CH), the constant region being divided into CH1, hinge, CH2, and CH3 regions. Each light chain comprises a variable region (VL) and a constant region (CL). VH and VL can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), interspersed with framework regions (FRs). Each VH and VL consists of three CDRs and four FR segments, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Antibodies include monoclonal antibodies, including mouse, human, humanized, and chimeric antibodies, as well as bispecific or multispecific antibodies. Immunoglobulins can be designated as five major classes—IgA, IgD, IgE, IgG, and IgM—based on the amino acid sequence of their heavy chain constant regions. IgA and IgG are further subdivided into isotypes IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4. Based on the amino acid sequence of their constant domains, the antibody light chain of any vertebrate species can be designated as one of two completely different types, namely κ and λ.

[0023] "CD38" refers to the differentiation cluster 38 protein, which is a glycoprotein expressed on immune cells, including plasma cells, natural killer cells, and B and T cell subsets.

[0024] In this article, the term "basal medium" refers to a medium containing the essential nutrients necessary for cell survival and proliferation. A basal medium contains the minimum nutritional requirements necessary to maintain cell growth, such as carbon sources, nitrogen sources, inorganic salts, and vitamins. Different cell types have different nutritional needs, therefore, it is necessary to select the basal medium most suitable for the growth of the cell line. Although a basal medium can meet the most basic growth needs of cells, in practical applications, researchers often need to modify and optimize it according to specific experimental objectives and cell characteristics. This may include adding special growth factors, changing the concentration of nutrients, or adjusting the pH value to achieve better culture results.

[0025] In this article, the term "feeding medium" refers to the additional nutrients added to the basal medium during cell culture. Feeds typically contain carbohydrates, amino acids, vitamins, organic acids, and growth factors, all essential for maintaining cell growth and proliferation. For example, glucose is the most common energy source. Different cell lines have different nutritional requirements, therefore feeding strategies need to be adjusted according to the specific nutritional needs of each cell line. Feeds can be added at different stages of cell culture, usually at the beginning of culture or at a time determined based on cell growth. Feeding can be done in batches, continuously, or semi-continuously, each with its own applicable scenarios and advantages and disadvantages. A suitable feeding strategy can not only improve cell growth rate but also enhance the quality and yield of products such as recombinant proteins. Feeding media play a crucial role in cell culture. It not only provides the necessary nutrients but also influences cell growth status and the quality of the final product. In practical applications, selecting appropriate feeding strategies and formulations is particularly critical for achieving efficient and high-quality cell culture. With the rapid development of fields such as biopharmaceuticals and cell therapy, the demand and application of feeding media will continue to grow. This requires researchers and production personnel to not only deeply understand the principles and mechanisms of material feeding, but also to continuously explore and optimize feeding schemes to adapt to ever-changing and increasingly demanding process requirements. At the same time, considering cost and efficiency, developing more economical and effective feeding strategies is also an important direction for the future.

[0026] The transitional terms “comprising,” “substantially consisting of,” and “consisting of” are intended to imply their accepted meaning in patent terminology; that is, (i) “comprising” is synonymous with “including,” “containing,” or “characterized by,” and is inclusive or open at the end, and does not exclude additional, unlisted elements or method steps; (ii) “consisting of” excludes any element, step, or component not specified in the claims; and (iii) “substantially consisting of” limits the scope of the claims to the specified material or step “and material or step that does not substantially affect the disclosed essential and novel features protected by the claims.” Embodiments described with the phrase “comprising” (or its equivalents) are also provided, as are those described independently with “consisting of” and “substantially consisting of”.

[0027] "Pharmaceutical products" refer to finished dosage forms containing active pharmaceutical ingredients (such as pharmaceutical substances), such as tablets, capsules, or solutions, which are usually, but not necessarily, associated with inactive ingredients.

[0028] "Expression vector" refers to a vector that can be used in biological or regenerated biological systems to guide the translation of polypeptides encoded by polynucleotide sequences present in the expression vector.

[0029] “G0F” refers to a desialylated, galactose-free, fucoidan-based, biantennary.

[0030] "GOF oligosaccharide content" refers to the percentage of GOF oligosaccharides (GOF%) in glycoprotein oligosaccharides.

[0031] “G1F” refers to a desialylated monogalactose-core fucoidan-based bianthospinan.

[0032] "G1F oligosaccharide content" refers to the percentage of G1F oligosaccharides (G1F%) in glycoprotein oligosaccharides.

[0033] "GMP compliance" refers to manufacturing under Good Manufacturing Practices (CGMP) regulations mandated by the U.S. Food and Drug Administration (FDA). CGMP provides a system to ensure the proper design, monitoring, and control of manufacturing processes and facilities. Compliance with CGMP regulations ensures the consistency, potency, quality, and purity of drug products by requiring drug manufacturers to have full control over their manufacturing operations. This includes establishing a robust quality management system; obtaining raw materials of appropriate quality; establishing robust operating procedures; detecting and investigating product quality deviations; and maintaining reliable testing laboratories. If this standardized control system is fully implemented at a pharmaceutical company, it will help prevent contamination, disorder, deviations, failures, and errors. This ensures that drug products meet their quality standards.

[0034] A "polynucleotide" is a synthetic molecule that contains a phosphate-sugar backbone covalently linked by nucleotide chains or other equivalent covalent chemicals. cDNA is a typical example of a polynucleotide.

[0035] "ppb" or "parts per billion" refers to the amount of a metal in a solution or solid. When measured in a solution, ppb is equivalent to the concentration of the metal in μg / L. When measured in a solid, ppb is equivalent to the concentration of the metal in μg / kg.

[0036] Any one or more culture media or combinations thereof may be used in the methods of this disclosure, including culture media with defined chemical composition and / or culture media with undefined chemical composition, the culture media being well known in the art and generally available from suppliers.

[0037] "Chemically defined culture media" refers to synthetic growth media in which the identity and concentration of all components are known. Chemically defined culture media do not contain bacteria, yeast, animal or plant extracts, animal serum or plasma, but they may or may not include components of a single plant or animal origin (e.g., proteins, peptides, etc.).

[0038] "Feed-batch culture" refers to a cell culture method in which additional components are provided to the culture at one or more points after the culture process has begun. These provided components typically include nutrients that have been depleted from the cells during the culture process. Feed-batch culture is usually stopped at a certain point, and the cells and / or components in the culture medium are harvested and optionally purified.

[0039] The method disclosed herein

[0040] This disclosure provides a method for generating an anti-CD38 antibody expressed by a polynucleotide encoding the heavy chain variable region (VH) of SEQ ID NO:1 and the light chain variable region (VL) of SEQ ID NO:2 and having a G1F oligosaccharide content between about 17% and about 26%, the method comprising:

[0041] A culture medium containing manganese (Mn) at a concentration between about 8.5 parts per billion (ppb) and about 100 ppb was prepared; and the G1F oligosaccharide content of the anti-CD38 antibody was controlled by culturing host cells containing polynucleotides encoding VH of SEQ ID NO:1 and VL of SEQ ID NO:2 in a culture medium containing Mn at a concentration between about 8.5 ppb and about 100 ppb, thereby producing an anti-CD38 antibody expressed by polynucleotides encoding VH of SEQ ID NO:1 and VL of SEQ ID NO:2 and having a G1F oligosaccharide content between about 17% and about 26%.

[0042] This disclosure also provides a method for generating an anti-CD38 antibody, the anti-CD38 antibody being expressed by polynucleotides encoding VH of SEQ ID NO:1 and VL of SEQ ID NO:2 and having a G1F oligosaccharide content between about 17% and about 26% and a G0F oligosaccharide content between about 63% and about 70%, the method comprising:

[0043] A culture medium containing Mn between about 8.5 ppb and about 100 ppb was prepared; and the G1F oligosaccharide and G0F oligosaccharide content of the anti-CD38 antibody were controlled by culturing host cells containing polynucleotides encoding VH of SEQ ID NO:1 and VL of SEQ ID NO:2 in a culture medium containing Mn between about 8.5 ppb and about 100 ppb, thereby producing an anti-CD38 antibody expressed by polynucleotides encoding VH of SEQ ID NO:1 and VL of SEQ ID NO:2 and having a G1F oligosaccharide content between about 17% and about 26% and a G0F oligosaccharide content between about 63% and about 70%.

[0044] This disclosure also provides a method for generating an anti-CD38 antibody comprising VH of SEQ ID NO:1 and VL of SEQ ID NO:2 and having a G1F oligosaccharide content between about 17% and about 26%, the method comprising:

[0045] A culture medium containing Mn between about 8.5 ppb and about 100 ppb was prepared; and the G1F oligosaccharide content of the anti-CD38 antibody was controlled by culturing host cells containing polynucleotides encoding VH of SEQ ID NO:1 and VL of SEQ ID NO:2 in a culture medium containing Mn between about 8.5 ppb and about 100 ppb, thereby producing an anti-CD38 antibody containing VH of SEQ ID NO:1 and VL of SEQ ID NO:2 and having a G1F oligosaccharide content between about 17% and about 26%.

[0046] This disclosure also provides a method for generating an anti-CD38 antibody comprising VH of SEQ ID NO:1 and VL of SEQ ID NO:2 and having a G1F oligosaccharide content between about 17% and about 26% and a G0F oligosaccharide content between about 63% and about 70%, the method comprising:

[0047] A culture medium containing Mn between about 8.5 ppb and about 100 ppb was prepared; and the G1F oligosaccharide and G0F oligosaccharide content of the anti-CD38 antibody were controlled by culturing host cells containing polynucleotides encoding VH of SEQ ID NO:1 and VL of SEQ ID NO:2 in a culture medium containing Mn between about 8.5 ppb and about 100 ppb, thereby producing an anti-CD38 antibody containing VH of SEQ ID NO:1 and VL of SEQ ID NO:2 and having a G1F oligosaccharide content between about 17% and about 26% and a G0F oligosaccharide content between about 63% and about 70%.

[0048] This disclosure also provides a method for generating an anti-CD38 antibody, the anti-CD38 antibody being expressed by polynucleotides encoding VH of SEQ ID NO:1 and VL of SEQ ID NO:2 and having a G1F oligosaccharide content between about 17% and about 26%, the method comprising:

[0049] Host cells expressing polynucleotides encoding VH (SEQ ID NO:1) and VL (SEQ ID NO:2) were cultured under conditions that produced anti-CD38 antibodies; and the G1F oligosaccharide content of the anti-CD38 antibody was controlled by monitoring the concentration of Mn in the culture medium during the biosynthesis of the anti-CD38 antibody and adjusting the concentration of Mn in the culture medium during the biosynthesis of the anti-CD38 antibody, wherein the concentration of Mn in the culture medium was adjusted to contain between about 8.5 ppb and about 100 ppb of Mn, thereby producing an anti-CD38 antibody with a G1F oligosaccharide content between about 17% and about 26%.

[0050] This disclosure also provides a method for generating an anti-CD38 antibody having a G1F oligosaccharide content between about 17% and about 26%, the method comprising: culturing host cells containing a polynucleotide encoding an anti-CD38 antibody in a medium containing Mn measured to be between about 8.5 ppb and 100 ppb, thereby generating an anti-CD38 antibody having a G1F oligosaccharide content between about 17% and about 26%.

[0051] This disclosure also provides a method for producing an anti-CD38 antibody having a G1F oligosaccharide content between about 17% and about 26%, the method comprising: culturing host cells containing a polynucleotide encoding an anti-CD38 antibody in a medium controlled to contain Mn between about 8.5 ppb and 100 ppb, thereby producing an anti-CD38 antibody having a G1F oligosaccharide content between about 17% and about 26%.

[0052] This disclosure also provides a method for producing an anti-CD38 antibody having a G1F oligosaccharide content between about 17% and about 26%, the method comprising: culturing host cells containing a polynucleotide encoding an anti-CD38 antibody in a medium containing Mn between about 8.5 ppb and 100 ppb; and controlling the G1F oligosaccharide content by monitoring the concentration of Mn in the medium during antibody biosynthesis and adjusting the concentration of Mn in the medium during antibody biosynthesis, thereby producing the antibody.

[0053] This disclosure also provides a method for producing an anti-CD38 antibody having a G1F oligosaccharide content between about 17% and 26%, the method comprising: preparing a culture medium containing Mn between about 8.5 ppb and 100 ppb; and culturing host cells containing a polynucleotide encoding an anti-CD38 antibody in the culture medium containing Mn between about 8.5 ppb and 100 ppb, thereby producing an anti-CD38 antibody having a G1F oligosaccharide content between about 17% and 26%.

[0054] This disclosure provides a method for controlling the G1F oligosaccharide content of an anti-CD38 antibody during biosynthesis in a culture medium to have a G1F oligosaccharide content between about 17% and 26%, wherein the method includes:

[0055] The Mn level in the culture medium was monitored during the biosynthesis of the antibody; and the Mn level in the culture medium was regulated during the biosynthesis of the antibody or its antigen-binding fragment.

[0056] In some implementations, the G1F oligosaccharide content of the anti-CD38 antibody is between about 17% and about 23%.

[0057] In some implementations, the G1F oligosaccharide content of the anti-CD38 antibody is between about 19% and about 23%.

[0058] In some implementations, the G1F oligosaccharide content of the anti-CD38 antibody is between about 18% and about 26%.

[0059] In some implementations, the G0F oligosaccharide content of the anti-CD38 antibody is between about 64% and about 69%.

[0060] In some implementations, the G0F oligosaccharide content of the anti-CD38 antibody is between about 65% and about 69%.

[0061] In some implementations, the G0F oligosaccharide content of the anti-CD38 antibody is between about 63% and about 70%.

[0062] In some embodiments, preparing the culture medium includes measuring the Mn concentration in one or more batches of the raw material components used to prepare the culture medium and selecting those batches of the raw material components that, when combined, contain between about 8.5 ppb and about 100 ppb of Mn.

[0063] In some embodiments, preparing the culture medium includes measuring the Mn concentration in one or more batches of the raw material components used to prepare the culture medium and selecting those batches of the raw material components that, when combined, contain between about 8.5 ppb and 80 ppb of Mn to prepare the culture medium.

[0064] In some embodiments, preparing the culture medium includes measuring the Mn concentration in one or more batches of the raw material components used to prepare the culture medium and selecting those batches of the raw material components that, when combined, contain between about 10 ppb and 100 ppb of Mn to prepare the culture medium.

[0065] In some embodiments, the culture medium is prepared to contain Mn between about 8.5 ppb and about 80 ppb.

[0066] In some embodiments, the culture medium is prepared to contain between about 10 ppb and about 100 ppb of Mn.

[0067] In some embodiments, the culture medium is prepared to contain Mn between about 8.5 ppb and about 100 ppb.

[0068] In some implementations, the concentration of Mn in the culture medium is adjusted to contain between about 8.5 ppb and about 80 ppb of Mn.

[0069] In some implementations, the concentration of Mn in the culture medium is adjusted to contain between about 10 ppb and about 100 ppb of Mn.

[0070] In some implementations, the concentration of Mn in the culture medium is adjusted to contain between about 8.5 ppb and about 100 ppb of Mn.

[0071] In some embodiments, the G1F oligosaccharide content of the anti-CD38 antibody is between about 17% and about 23%, the G0F oligosaccharide content of the anti-CD38 antibody is between about 64% and about 69%, and the culture medium is prepared to contain Mn between about 8.5 ppb and about 80 ppb.

[0072] In some embodiments, the G1F oligosaccharide content of the anti-CD38 antibody is between about 19% and about 23%, the G0F oligosaccharide content of the anti-CD38 antibody is between about 65% and about 69%, and the culture medium is prepared to contain between about 10 ppb and about 100 ppb of Mn.

[0073] In some embodiments, the G1F oligosaccharide content of the anti-CD38 antibody is between about 18% and about 26%, the G0F oligosaccharide content of the anti-CD38 antibody is between about 63% and about 70%, and the culture medium is prepared to contain Mn between about 8.5 ppb and about 100 ppb.

[0074] In some embodiments, the G1F oligosaccharide content of the anti-CD38 antibody is between about 17% and about 23%, the G0F oligosaccharide content of the anti-CD38 antibody is between about 64% and about 69%, and the culture medium is adjusted to contain between about 8.5 ppb and about 80 ppb of Mn.

[0075] In some embodiments, the G1F oligosaccharide content of the anti-CD38 antibody is between about 19% and about 23%, the G0F oligosaccharide content of the anti-CD38 antibody is between about 65% and about 69%, and the culture medium is adjusted to contain between about 10 ppb and about 100 ppb of Mn.

[0076] In some embodiments, the G1F oligosaccharide content of the anti-CD38 antibody is between about 18% and about 26%, the G0F oligosaccharide content of the anti-CD38 antibody is between about 63% and about 70%, and the culture medium is adjusted to contain between about 8.5 ppb and about 100 ppb of Mn.

[0077] In some embodiments, the anti-CD38 antibody expressed by polynucleotides encoding VH of SEQ ID NO:1 and VL of SEQ ID NO:2 comprises VH of SEQ ID NO:1 and VL of SEQ ID NO:2.

[0078] In some implementations, the culture medium is a basal culture medium or a supplemental culture medium.

[0079] In some implementation schemes, the culture includes fed-batch culture or perfusion culture.

[0080] In some implementations, the host cell is a eukaryotic cell.

[0081] In some implementations, the eukaryotic cells are CHO cells, PER.C6 cells, NSO cells, Sp2 / O cells, or BHK cells.

[0082] In some implementations, CHO cells are CHO-K1 cells, CHO-DG44 cells, CHO-S cells, or CHODXB11 cells.

[0083] In some implementations, CHO cells lack glutamine synthase (GS).

[0084] In some implementations, the method is carried out under GMP-compliant conditions.

[0085] In some implementations, the anti-CD38 antibody comprises VH of SEQ ID NO:1 and VL of SEQ ID NO:2.

[0086] In some implementations, the anti-CD38 antibody is expressed by polynucleotides encoding VH of SEQ ID NO:1 and VL of SEQ ID NO:2.

[0087] In some implementations, the anti-CD38 antibody contains an IgG1 isotype.

[0088] In some implementations, the anti-CD38 antibody comprises the IgG2 isotype.

[0089] In some implementations, the anti-CD38 antibody contains the IgG4 isotype.

[0090] In some implementations, the anti-CD38 antibody comprises the heavy chain (HC) of SEQ ID NO:3 and the light chain (LC) of SEQ ID NO:4.

[0091] In some implementations, the anti-CD38 antibody is expressed by polynucleotides encoding HC of SEQ ID NO:3 and LC of SEQ ID NO:4.

[0092] In some implementations, the anti-CD38 antibody is a biosimilar.

[0093] In some embodiments, the culture medium is prepared to contain about 8.5 ppb, about 11 ppb, about 13 ppb, about 15 ppb, about 17 ppb, about 19 ppb, about 22 ppb, about 30 ppb, or about 80 ppb of Mn.

[0094] In some embodiments, the culture medium contains about 10 ppb, about 15 ppb, about 20 ppb, about 50 ppb, about 60 ppb, about 70 ppb, about 90 ppb, or about 100 ppb of Mn.

[0095] In some embodiments, the culture medium is controlled to contain Mn at concentrations of about 8.5 ppb, about 10 ppb, about 12 ppb, about 14 ppb, about 18 ppb, about 20 ppb, about 26 ppb, about 40 ppb, and about 100 ppb.

[0096] The methods disclosed herein can be used with any cell culture method suitable for the desired process (e.g., the production of recombinant antibodies). Cells can be grown in batch or fed-batch cultures, wherein the culture is terminated after the antibody has been adequately expressed, and the expressed antibody is then harvested. Alternatively, cells can be grown in fed-batch cultures, wherein the culture is not terminated, but new nutrients and other components are added to the culture periodically or continuously, and the expressed antibody is harvested periodically or continuously during the culture. Other suitable methods (e.g., spin-tube culture) are known in the art and may also be used.

[0097] In some embodiments, fed-batch culture includes a basal medium supplemented with fed-batch medium. Cells can be grown in any convenient volume. For example, cells can be grown in small-scale reaction vessels ranging from a few milliliters to a few liters. Alternatively, cells can be grown in large-scale commercial bioreactors ranging from about 1 liter to any volume ranging from 10, 50, 100, 250, 500, 1000, 2500, 5000, 8000, 10000, 12000, 15000, 20000, or 25000 liters or more, or between.

[0098] Culture media containing between about 8.5 ppb and about 80 ppb of manganese can be prepared by analyzing the manganese concentration in each batch of each raw material component of the culture medium and adding manganese to the basal culture medium so that the manganese concentration in the preparation medium of the raw material components is between about 8.5 ppb and about 80 ppb, or the total manganese concentration in the selected components when combined is between about 8.5 ppb and about 80 ppb.

[0099] Culture media containing between about 10 ppb and about 100 ppb of manganese can be prepared by analyzing the manganese concentration in each batch of each raw material component of the culture medium and adding equal amounts of manganese to the basal medium and the supplemental medium so that the manganese concentration in the preparation medium of the raw material components is between about 10 ppb and about 100 ppb, or the total manganese concentration in the selected components when combined is between about 10 ppb and about 100 ppb.

[0100] Culture media containing between about 8.5 ppb and about 100 ppb of manganese can be prepared by analyzing the manganese concentration in each batch of each raw material component of the culture medium and adding manganese to the feed medium so that the manganese concentration in the preparation medium of the raw material components is between about 8.5 ppb and about 100 ppb, or the total manganese concentration in the selected components when combined is between about 8.5 ppb and about 100 ppb.

[0101] Methods of generating antibodies

[0102] Antibodies can be produced through fed-batch culture of CHO cells, with temperature control as needed. After harvesting, the cell culture medium is centrifuged, subjected to deep filtration and sterile filtration to obtain a clear cell culture medium. This clear cell culture medium is then purified to produce antibodies. The purification steps include affinity chromatography, low pH inactivation, deep filtration anion exchange chromatography, anion exchange chromatography, virus removal filtration, ultrafiltration, and elution to form the stock solution.

[0103] Methods for measuring the oligosaccharide composition of antibodies

[0104] The oligosaccharide composition of antibodies can be determined using UPLC with the Waters Acquity H-Class Bio ultra-high performance liquid chromatography system and Waters FLR software. This method employs hydrophilic interaction chromatography to determine the oligosaccharides in the sample. The method involves enzymatic cleavage of N-glycans with N-glycosidase F (PNGase F), followed by purification and labeling with 2-amino-benzamide (2-AB) to acquire a fluorescent group. Ultra-high performance liquid chromatography is then used for separation and determination. The corresponding N-glycan peaks of the sample are determined by comparison with typical chromatograms (identified by LC-MS). The amount of each oligosaccharide is quantified by peak area integration and expressed as a percentage of the total oligosaccharide peak area (peak area %).

[0105] Although this disclosure has been described in general terms, embodiments of this disclosure will be further disclosed in the following examples, which should not be construed as limiting the scope of the claims.

[0106] Example

[0107] This disclosure is further described in detail through the following examples.

[0108] The basal culture medium used in this embodiment is an animal-free, protein-free culture medium with a defined chemical composition, including the following substances:

[0109] 4-Hydroxyethylpiperazine ethanesulfonic acid (HEPES) 5-7 g / L, glucose 3-8 g / L, sodium pyruvate 0.05-0.4 g / L, sodium chloride 3-5 g / L, potassium chloride 0.2-0.4 g / L, sodium selenite 0.0000005-0.000060 g / L, manganese sulfate 0.05-0.2 g / L, ethanolamine 5-25 μg / L, ferric citrate 1-10 mg / L, zinc sulfate 0.1-0.5 g / L, copper sulfate 0.02-0.1 g / L. 5 g / L, glutathione 0.05-0.3 g / L, magnesium chloride 0.025-0.15 g / L, sodium dihydrogen phosphate 0.1-0.3 g / L, sodium bicarbonate 1-5 g / L, alanine 0.015-0.035 g / L, asparagine 0.01-0.035 g / L, arginine 0.06-0.30 g / L, aspartic acid 0.02-0.2 g / L, cysteine ​​0.3-0.5 g / L, cysteine ​​0.05-0.2 g / L, glutamic acid 0.001-0.09 g / L, glycine 0-0.04 g / L, histidine 0.03-0.2 g / L, isoleucine 0.05-0.25 g / L, leucine 0.05-0.25 g / L, lysine 0.02-0.25 g / L, methionine 0.02-0.2 g / L, phenylalanine 0.055-0.075 g / L, proline 0.01-0.05 g / L, serine 0.03-0.25 g / L, threonine 0.07- 0.15 g / L, tryptophan 0.005-0.025 g / L, tyrosine 0.05-0.2 g / L, valine 0.05-0.5 g / L, biotin 0.005-0.15 mg / L, calcium pantothenate 0.002-0.006 g / L, choline chloride 0.002-0.09 g / L, folic acid 0.002-0.006 g / L, inositol 0.005-0.02 g / L, nicotinamide 0.002-0.006 g / L, vitamin B6 0.002-0.006 g / L, Vitamin B2 0.0002-0.0006 g / L, Vitamin B1 0.002-0.006 g / L, Vitamin B12 0.000005-0.000025 g / L, Linoleic acid 0.001-0.1 g / L, Block polyether F-68 (Pluronic F-68) 0.2-5 g / L.

[0110] The feeding culture medium used in this embodiment is an animal-free, protein-free, and chemically defined culture medium, and its components include the following substances:

[0111] Glucose 10-25 g / L, Sodium pyruvate 0.1-0.6 g / L, Potassium chloride 2-10 g / L, Ferric citrate 0.01-1.5 g / L, Copper sulfate 0.005-0.02 g / L, Magnesium chloride 0.025-0.5 g / L, Sodium dihydrogen phosphate 0.1-6 g / L, Alanine 0.1-2.5 g / L, Asparagine 0.1-8 g / L, Arginine 0.6-8 g / L, Aspartic acid 2-15 g / L, Glutamate The concentrations of amino acids are as follows: 0.1-15 g / L for amino acids, 0.5-5 g / L for glycine, 0.3-5 g / L for histidine, 0.1-5 g / L for isoleucine, 0.5-20 g / L for leucine, 0.2-6 g / L for lysine, 0.5-15 g / L for phenylalanine, 0.1-10 g / L for proline, 0.3-2.5 g / L for serine, 0.05-4 g / L for tryptophan, 0.05-5 g / L for tyrosine, and 0.05-7 g / L for valine.

[0112] In this embodiment of the disclosure, the fed-batch culture method includes the following steps: feeding 0.5-2×10 6 CHOK1 cells were seeded at a density of 15 × 10⁶ cells / ml; the cells were then cultured at 30℃-37℃; supplemental culture medium was added on the 3rd or 4th day after seeding, and then added every other day until the end of the culture. Each addition was 3-8% of the initial volume, and the total addition was 20-50% of the initial target weight. When the cell density reached 15 × 10⁶ cells / ml, CHOK1 cells were seeded. 6 cells / ml - 5×10 7 Cells / ml or cultured at 28℃-35℃ for 6-9 days post-inoculation, and maintained until the end of culture; culture was terminated at 10-16 days post-inoculation or when cell viability was <70%. In this disclosure, the culture medium used in different schemes is consistent except for the Mn ion concentration. The fed-batch culture method is consistent in different schemes in this disclosure.

[0113] Example 1. Preparation of a basal culture medium supplemented with additional manganese

[0114] During the development of the anti-CD38 antibody (daratumumab monoclonal antibody, HC of SEQ ID NO:3 and LC of SEQ ID NO:4), trace amounts of manganese (Mn) associated with the raw materials were found to regulate the glycosylation profile in the anti-CD38 antibody.

[0115] The expected manganese concentration in the hydrated medium was estimated by using the cumulative contribution of Mn in the culture medium determined based on its chemical composition, thus using Mn concentration as a predictive tool. The concentration in the bioreactor was calculated based on the mass of basal and feed media added per liter, and then the adjusted Mn concentration was obtained by adding the feed adjusted according to the feed rate to the basal Mn concentration.

[0116] Mn concentration in hydrated culture medium: Predicted Mn solution (μg / L~ppb) = [(component 1 (g / L) × Mn1 (ppb)) + (component 2 (g / L) × Mn2 (ppb)) + … (component n (g / L) × Mnn n (ppb))] / 1000

[0117] Mn concentration in the bioreactor: (μg / L~ppb) = basic Mn (ppb) + additional Mn (ppb) added to the basic Mn + (feed Mn (ppb) × feed factor).

[0118] Data on Mn concentration and G0F% and G1F% of anti-CD38 antibody in ten hydrated media were obtained. Table 1 shows the G0F%, G1F% and Mn concentration and total Mn concentration in each batch of basal and fed media.

[0119] Table 1

[0120] Figures 1A to 1D illustrate the correlation between Mn concentration and the percentages of anti-CD38 antibody G0F and G1F in each analytical batch. The data show that when Mn concentration is between approximately 8.5 ppb and approximately 17 ppb, it is positively correlated with the percentage of anti-CD38 antibody G1F and negatively correlated with the percentage of G0F, with correlation coefficients R0 and R1, respectively. 2 =0.9586 and R 2 =0.6019. When the Mn concentration is between approximately 17 ppb and approximately 80 ppb, it is negatively correlated with the percentage of anti-CD38 antibody G1F and positively correlated with the percentage of G0F. The correlation coefficients are R1 and R2, respectively. 2 =0.9854 and R 2 =0.9695.

[0121] Figure 1E shows the Mn concentration contributed by the basal or fed medium in each batch. Based on these data, Mn concentrations ranging from approximately 8.5 ppb to approximately 80 ppb resulted in anti-CD38 antibodies with G1F% contents ranging from approximately 17% to 23% and G0F% contents ranging from approximately 64% to approximately 69%.

[0122] Example 2. Preparation of basal and supplemental culture media with additional manganese

[0123] Mn concentration in the bioreactor: (μg / L~ppb) = basic Mn (ppb) + additional Mn (ppb) added to the basic feed + ((feed Mn (ppb) + additional Mn (ppb) added to the feed) × feed factor).

[0124] Data on Mn concentration and G0F% and G1F% of anti-CD38 antibody were obtained in eight hydration media. Table 2 shows the G0F%, G1F% and Mn concentration and total concentration in the basal and fed media for each batch.

[0125] Table 2

[0126] Figures 2A to 2D illustrate the correlation between Mn concentration and the percentages of anti-CD38 antibody G0F and G1F in each analytical batch. The data show that when Mn concentration is between approximately 10 ppb and 20 ppb, it is positively correlated with the percentage of anti-CD38 antibody G1F and negatively correlated with the percentage of G0F, with correlation coefficients R0 and R1, respectively. 2 =0.8866 and R 2 =0.8107. When the Mn concentration is between approximately 20 ppb and approximately 100 ppb, it is negatively correlated with the percentage of anti-CD38 antibody G1F and positively correlated with the percentage of G0F, with correlation coefficients R1 and R2, respectively. 2 =0.9297 and R 2 =0.9435.

[0127] Figure 2E shows the Mn concentration contributed by the basal or fed medium in each batch. Based on these data, Mn concentrations with a total contribution of approximately 10 ppb to approximately 100 ppb produce anti-CD38 antibodies with G1F% content between approximately 19% and 23% and G0F% content between approximately 65% ​​and 69%.

[0128] Example 3. Preparation of a supplemental culture medium with additional manganese

[0129] Mn concentration in the bioreactor: (μg / L~ppb) = base Mn (ppb) + ((feed Mn (ppb) + additional Mn added in the feed) × feed factor).

[0130] Data on Mn concentration and G0F% and G1F% of anti-CD38 antibody in nine hydrated media were obtained. Table 3 shows the G0F%, G1F% and manganese concentration and total concentration in the basal and fed media for each batch.

[0131] Table 3

[0132] Figures 3A and 3B illustrate the correlation between Mn concentration and the percentages of anti-CD38 antibody G0F and G1F in each analytical batch. The data show that when Mn concentration is between approximately 8.5 ppb and approximately 100 ppb, it is positively correlated with the percentage of anti-CD38 antibody G1F and negatively correlated with the percentage of G0F. The correlation coefficients are R0 and R1, respectively.2 =0.7261 and R 2 =0.7307.

[0133] Figure 3C shows the Mn concentration contributed by the basal or supplemental culture medium in each batch. Based on these data, Mn concentrations with a total contribution of approximately 8.5 ppb to approximately 100 ppb produce anti-CD38 antibodies with G1F% content between approximately 18% and 26% and G0F% content between approximately 63% and 70%.

Claims

1. A method for producing an anti-CD38 antibody, said anti-CD38 antibody being expressed by a polynucleotide encoding the heavy chain variable region (VH) of SEQ ID NO:1 and the light chain variable region (VL) of SEQ ID NO:2 and having a G1F oligosaccharide content between about 17% and about 26%, said method comprising: a) Prepare a culture medium containing Mn at concentrations between approximately 8.5 parts per billion (ppb) and approximately 100 parts per billion (ppb); and b) By culturing host cells containing the polynucleotides encoding VH of SEQ ID NO:1 and VL of SEQ ID NO:2 in the culture medium prepared in step a), the content of the G1F oligosaccharide in the anti-CD38 antibody is controlled, thereby producing the anti-CD38 antibody expressed by the polynucleotides encoding VH of SEQ ID NO:1 and VL of SEQ ID NO:2 and having a G1F oligosaccharide content between about 17% and about 26%.

2. The method of claim 1, wherein the G0F oligosaccharide content of the anti-CD38 antibody is between about 63% and about 70%.

3. The method according to any one of claims 1-2, wherein preparing the culture medium comprises measuring the Mn concentration in one or more batches of the raw material components used to prepare the culture medium and selecting those batches of the raw material components that, when combined, contain Mn between about 8.5 ppb and about 100 ppb.

4. The method of claim 3, wherein the culture medium is prepared to contain Mn between about 8.5 ppb and about 100 ppb.

5. The method of claim 4, wherein the culture medium is prepared to contain Mn between about 10 ppb and about 100 ppb.

6. The method of claim 5, wherein the culture medium is prepared to contain Mn between about 8.5 ppb and about 80 ppb.

7. The method according to any one of claims 1 to 11, wherein the G1F oligosaccharide content of the anti-CD38 antibody is between about 17% and about 26%, the G0F oligosaccharide content of the anti-CD38 antibody is between about 63% and about 70%, and the culture medium is prepared to contain Mn between about 8.5 ppb and 100 ppb.

8. The method according to any one of claims 1 to 7, wherein the G1F oligosaccharide content of the anti-CD38 antibody is between about 17% and about 23%, the G0F oligosaccharide content of the anti-CD38 antibody is between about 64% and about 69%, and the culture medium is prepared to contain Mn between about 8.5 ppb and about 80 ppb.

9. The method according to any one of claims 1 to 7, wherein the G1F oligosaccharide content of the anti-CD38 antibody is between about 19% and about 23%, the G0F oligosaccharide content of the anti-CD38 antibody is between about 65% and about 69%, and the culture medium is prepared to contain Mn between about 10 ppb and about 100 ppb.

10. The method according to any one of claims 1 to 7, wherein the G1F oligosaccharide content of the anti-CD38 antibody is between about 18% and about 26%, the G0F oligosaccharide content of the anti-CD38 antibody is between about 63% and about 70%, and the culture medium is prepared to contain Mn between about 8.5 ppb and about 100 ppb.

11. The method according to any one of claims 1 to 10, wherein the culture medium is a basal culture medium or a supplemental culture medium.

12. The method according to any one of claims 1 to 11, wherein the culture comprises fed-batch culture or perfusion culture.

13. The method according to any one of claims 1 to 12, wherein the host cell is a eukaryotic cell.

14. The method of claim 13, wherein the eukaryotic cell is a CHO cell, a PER.C6 cell, an NSO cell, an Sp2 / O cell, or a BHK cell.

15. The method according to claim 14, wherein the CHO cells are CHO-K1 cells, CHO-DG44 cells, CHOS cells, or CHO-DXB11 cells.

16. The method of claim 15, wherein the CHO cells are deficient in glutamine synthase (GS).

17. The method according to any one of claims 1 to 16, wherein the anti-CD38 antibody comprises the VH of SEQ ID NO:1 and the VL of SEQ ID NO:

2.

18. The method according to any one of claims 1 to 17, wherein the anti-CD38 antibody is an IgG1 isotype.

19. The method according to any one of claims 1 to 18, wherein the anti-CD38 antibody comprises the heavy chain (HC) of SEQ ID NO:3 and the light chain (LC) of SEQ ID NO:

4.

20. A method for producing a pharmaceutical product, comprising: carrying out the method according to any one of claims 1-19, And further includes, The anti-CD38 antibody was formulated into a pharmaceutical product.

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