Methods for producing Anti-CD38 antibodies

Abstract

The present disclosure relates to methods of controlling Mn ion concentration during the preparation of anti-CD38 antibodies to improve oligosaccharide content.

Description

Technical Field

[0001] The present disclosure relates to a method for preparing an anti-CD38 antibody, and in particular, to a method for controlling the Mn ion concentration of a culture medium during the production of an anti-CD38 antibody. Background Art

[0002] Cell culture medium is an important factor in the production of antibody drugs because it affects cell growth, productivity and product quality, which in turn affects the safety and efficacy of drugs. The composition of chemically defined culture medium is clear, which can avoid the quality variation caused by different batches of serum, improve reproducibility, and avoid the toxic effects of serum on cells and the risk of serum-derived contamination. It is conducive to the differentiation of in vitro cultured cells, can improve the expression level of cell products and facilitate 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 about 2% to 3% of the total mass; although the proportion is relatively small, N-glycosylation is involved in many important processes, plays an important role in the pharmacokinetics and pharmacodynamic regulation of monoclonal antibodies, and depends on cell culture conditions; CN113015805A discloses that the trace metal Mn in the culture medium is less than 8.5 ppb to produce daratumumab antibodies with a suitable range of G1F oligosaccharide content; therefore, it is necessary to develop a method for predictable sugar profiles of antibodies obtained by cell culture in a chemically defined culture medium. Summary of the invention

[0004] The present disclosure provides a method for producing an anti-CD38 antibody expressed by a polynucleotide encoding a heavy chain variable region (VH) of SEQ ID NO: 1 and a 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) preparing a culture medium comprising between about 8.5 parts per billion (ppb) and about 100 ppb manganese (Mn);

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

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

[0008] Cultivating a host cell expressing a polynucleotide encoding the VH of SEQ ID NO: 1 and the VL of SEQ ID NO: 2 under conditions where an anti-CD38 antibody is produced; and controlling the G1F oligosaccharide content of the anti-CD38 antibody by monitoring the concentration of Mn in the culture medium during biosynthesis of the anti-CD38 antibody and adjusting the concentration of Mn in the culture medium during biosynthesis of the anti-CD38 antibody, wherein the concentration of Mn in the culture medium is adjusted to comprise between about 8.5 ppb and about 100 ppb, thereby producing an anti-CD38 antibody having a G1F oligosaccharide content between about 17% and about 26%.

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

[0010] a) preparing a culture medium comprising between about 8.5 ppb and about 100 ppb Mn;

[0011] b) culturing a host cell comprising a polynucleotide 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 having a G1F oligosaccharide content between about 15% and about 27%;

[0012] c) formulating the anti-CD38 antibody into a pharmaceutical product.

[0013] The advantage of the present disclosure is that, unlike the teachings of the prior art, the present disclosure provides a predictable sugar profile for producing a desired anti-CD38 antibody in a culture medium containing between about 8.5 ppb and about 100 ppb of Mn, which is beneficial to the industrial production of anti-CD38 antibodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1A and Figure 1B The correlation of the anti-CD38 antibody G1F oligosaccharide percentage (G1F%) with the calculated manganese (Mn) concentration of the culture medium (Mn between about 8.5 ppb and about 80 ppb) is shown; Figure 1C and Figure 1D The correlation of the anti-CD38 antibody G0F oligosaccharide percentage (G0F%) with the calculated manganese (Mn) concentration of the culture medium (Mn between about 8.5 ppb and about 80 ppb) is shown; Figure 1E The relationship of the calculated Mn concentrations of the medium components used in each group is shown.

[0015] Figure 2A and Figure 2B The correlation of the anti-CD38 antibody G1F oligosaccharide percentage (G1F%) with the calculated manganese (Mn) concentration of the culture medium (Mn between about 10 ppb and about 100 ppb) is shown; Figure 2C and Figure 2D The correlation of the anti-CD38 antibody G0F oligosaccharide percentage (G0F%) with the calculated manganese (Mn) concentration of the culture medium (Mn between about 10 ppb and about 100 ppb) is shown; Figure 2E The relationship of the calculated Mn concentrations of the medium components used in each group is shown.

[0016] Figure 3A The correlation of the anti-CD38 antibody G1F oligosaccharide percentage (G1F%) with the calculated manganese (Mn) concentration of the culture medium (Mn between about 8.5 ppb and about 100 ppb) is shown; Figure 3B The correlation of the anti-CD38 antibody G0F oligosaccharide percentage (G0F%) with the calculated manganese (Mn) concentration of the culture medium (Mn between about 8.5 ppb and about 100 ppb) is shown; Figure 3C The relationship of the calculated Mn concentrations of the medium components used in each group is shown. DETAILED DESCRIPTION

[0017] It should be understood that the terminology used herein is for the purpose of describing particular embodiments 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 belongs.

[0018] Although any methods and materials similar or equivalent to those described herein can be used in the practice of testing the present disclosure, exemplary materials and methods are described herein.In describing and claiming the present disclosure, the following terminology will be used.

[0019] definition

[0020] As used in this specification and the appended claims, the singular forms "a," "an," "the," and "said" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a cell" includes a combination of two or more cells, and so forth.

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

[0022] "Antibody" refers to an immunoglobulin molecule having two heavy chains (HC) and two light chains (LC) interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH), which is divided into CH1, hinge, CH2 and CH3 regions. Each light chain consists of a light chain variable region (VL) and a light chain 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 fragments, and is arranged from amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. Antibodies include monoclonal antibodies, including mouse, human, humanized and chimeric antibodies, bispecific or multispecific antibodies. Immunoglobulins can be designated as five major species, IgA, IgD, IgE, IgG and IgM, based on the amino acid sequence of the heavy chain constant region. IgA and IgG are further subdivided into the isotypes IgAl, IgA2, IgGl, IgG2, IgG3, and IgG4.The light chains of antibodies from any vertebrate species can be assigned to one of two clearly distinct types, kappa and lambda, based on the amino acid sequences of their constant domains.

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

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

[0025] The term "feed medium" in this article refers to additional nutrients added to the basal medium during cell culture. Feeds usually contain carbohydrates, amino acids, vitamins, organic acids, and growth factors, which are essential for maintaining cell growth and proliferation. For example, glucose is the most common energy source. Different cell lines have different nutrient requirements, so the feeding strategy needs to be adjusted according to the nutrient requirements of the specific cell line. Feeds can be added at different stages of cell culture, usually at the beginning of the culture or according to the cell growth situation. Feeds can be added in batches, continuously, or semi-continuously, and each method has its applicable scenarios and advantages and disadvantages. A suitable feeding strategy can not only increase the cell growth rate, but also improve the quality and yield of products such as recombinant proteins. Feed medium plays a vital role in cell culture. It not only provides the necessary nutrients, but also affects the growth state of cells and the quality of the final product. In practical applications, choosing the right feeding strategy and formulation 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 feed medium will continue to grow. This requires researchers and production personnel to not only have a deep understanding of the principles and mechanisms of feeding, but also to continuously explore and optimize feeding solutions to adapt to the ever-changing and improved process requirements. At the same time, considering the issues of cost and efficiency, developing more economical and effective feeding strategies is also an important direction for the future.

[0026] The transitional terms "comprising," "consisting essentially of," and "consisting of" are intended to suggest their recognized meanings in patent parlance; that is, (i) "comprising" is synonymous with "including," "containing," or "characterized by," and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps; (ii) "consisting of" excludes any element, step, or ingredient not specified in the claim; and (iii) "consisting essentially of" limits the scope of the claim to the specified materials or steps "and those that do not materially affect the basic and novel characteristics of the disclosure protected by the claims." Embodiments described with the phrase "comprising" (or its equivalent) are also provided, as are those embodiments described independently with "consisting of" and "consisting essentially of."

[0027] "Drug product" means a finished dosage form, such as a tablet, capsule, or solution, containing an active pharmaceutical ingredient (eg, a drug substance), typically but not necessarily associated with inactive ingredients.

[0028] "Expression vector" refers to a vector that can be used in a biological system or a reconstituted biological system to direct the translation of a polypeptide encoded by a polynucleotide sequence present in the expression vector.

[0029] "GOF" refers to desialylated, galactose-free core fucosylated biantennary glycan.

[0030] "G0F oligosaccharide content" refers to the percentage of G0F oligosaccharides (G0F%) in glycoprotein oligosaccharides.

[0031] "G1F" refers to a desialylated monogalactose core fucosylated biantennary glycan.

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

[0033] "GMP-compliant conditions" means manufacturing under the Good Manufacturing Practice (CGMP) regulations enforced 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 identity, potency, quality, and purity of drug products by requiring drug manufacturers to adequately control manufacturing operations. This includes establishing a strong quality management system; obtaining raw materials of appropriate quality; establishing robust operating procedures; detecting and investigating deviations in product quality; and maintaining reliable testing laboratories. This standardized control system at a pharmaceutical company, if fully put into practice, will help prevent contamination, mix-ups, deviations, failures, and errors. This ensures that the drug product meets its quality standards.

[0034] "Polynucleotide" refers to a synthetic molecule comprising a chain of nucleotides covalently linked to a phosphate sugar backbone 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 a concentration of μg / L of the metal in solution. When measured in a solid, ppb is equivalent to a concentration of μg / kg of the metal in solution.

[0036] Any one or more culture media or combinations thereof can be used in the methods of the present disclosure, including chemically defined media and or chemically undefined media, which are well known in the art and are commonly available from commercial suppliers.

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

[0038] "Fed-batch culture" refers to a cell culture method in which additional components are provided to the culture at one or more times after the culture process begins. Such provided components typically include nutritional components of the cells that have been depleted during the culture process. The fed-batch culture is usually stopped at a certain point in time, and the cells and / or components in the culture medium are harvested and optionally purified.

[0039] Methods of the present disclosure

[0040] The present disclosure provides a method for producing an anti-CD38 antibody expressed by a polynucleotide encoding a heavy chain variable region (VH) of SEQ ID NO: 1 and a 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] Preparing a culture medium comprising between about 8.5 parts per billion (ppb) and about 100 ppb manganese (Mn); and controlling the G1F oligosaccharide content of an anti-CD38 antibody by culturing a host cell comprising a polynucleotide encoding the VH of SEQ ID NO: 1 and the VL of SEQ ID NO: 2 in a culture medium comprising between about 8.5 ppb and about 100 ppb Mn, thereby producing an anti-CD38 antibody expressed by the polynucleotide encoding the VH of SEQ ID NO: 1 and the VL of SEQ ID NO: 2 and having a G1F oligosaccharide content of between about 17% and about 26%.

[0042] The present disclosure also provides a method of producing an anti-CD38 antibody expressed by a polynucleotide encoding the VH of SEQ ID NO: 1 and the 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] Preparing a culture medium comprising between about 8.5 ppb and about 100 ppb of Mn; and controlling the G1F oligosaccharide content and the G0F oligosaccharide content of the anti-CD38 antibody by culturing a host cell comprising a polynucleotide encoding the VH of SEQ ID NO: 1 and the VL of SEQ ID NO: 2 in a culture medium comprising between about 8.5 ppb and about 100 ppb of Mn, thereby producing an anti-CD38 antibody expressed by the polynucleotides encoding the VH of SEQ ID NO: 1 and the 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] The present disclosure also provides a method of producing an anti-CD38 antibody comprising the VH of SEQ ID NO: 1 and the VL of SEQ ID NO: 2 and having a G1F oligosaccharide content between about 17% and about 26%, the method comprising:

[0045] Preparing a culture medium comprising between about 8.5 ppb and about 100 ppb Mn; and controlling the G1F oligosaccharide content of the anti-CD38 antibody by culturing a host cell comprising a polynucleotide encoding the VH of SEQ ID NO: 1 and the VL of SEQ ID NO: 2 in a culture medium comprising between about 8.5 ppb and about 100 ppb Mn, thereby producing an anti-CD38 antibody comprising the VH of SEQ ID NO: 1 and the VL of SEQ ID NO: 2 and having a G1F oligosaccharide content between about 17% and about 26%.

[0046] The present disclosure also provides a method of producing an anti-CD38 antibody comprising the VH of SEQ ID NO: 1 and the 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] Preparing a culture medium comprising between about 8.5 ppb and about 100 ppb of Mn; and controlling the G1F oligosaccharide content and the G0F oligosaccharide content of the anti-CD38 antibody by culturing a host cell comprising a polynucleotide encoding the VH of SEQ ID NO: 1 and the VL of SEQ ID NO: 2 in a culture medium comprising between about 8.5 ppb and about 100 ppb of Mn, thereby producing an anti-CD38 antibody comprising the VH of SEQ ID NO: 1 and the 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] The present disclosure also provides a method of producing an anti-CD38 antibody expressed by a polynucleotide encoding the VH of SEQ ID NO: 1 and the VL of SEQ ID NO: 2 and having a G1F oligosaccharide content between about 17% and about 26%, the method comprising:

[0049] Culturing a host cell expressing a polynucleotide encoding the VH of SEQ ID NO: 1 and the VL of SEQ ID NO: 2 under conditions where an anti-CD38 antibody is produced; and controlling the G1F oligosaccharide content of the anti-CD38 antibody by monitoring the concentration of Mn in the culture medium during biosynthesis of the anti-CD38 antibody and adjusting the concentration of Mn in the culture medium during biosynthesis of the anti-CD38 antibody, wherein the concentration of Mn in the culture medium is adjusted to contain between about 8.5 ppb and about 100 ppb Mn, thereby producing an anti-CD38 antibody having a G1F oligosaccharide content between about 17% and about 26%.

[0050] The present 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 a host cell comprising a polynucleotide encoding an anti-CD38 antibody in a culture medium measured to contain between about 8.5 ppb and 100 ppb Mn, thereby producing an anti-CD38 antibody having a G1F oligosaccharide content between about 17% and about 26%.

[0051] The present 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 a host cell comprising a polynucleotide encoding an anti-CD38 antibody in a culture medium controlled to contain between about 8.5 ppb and 100 ppb Mn, thereby producing an anti-CD38 antibody having a G1F oligosaccharide content between about 17% and about 26%.

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

[0053] The present 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 comprising between about 8.5 ppb and 100 ppb Mn; and culturing a host cell comprising a polynucleotide encoding the anti-CD38 antibody in the culture medium comprising between about 8.5 ppb and 100 ppb Mn, thereby producing an anti-CD38 antibody having a G1F oligosaccharide content between about 17% and 26%.

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

[0055] monitoring the Mn level in the culture medium during biosynthesis of the antibody; and regulating the Mn level in the culture medium during biosynthesis of the antibody or antigen-binding fragment thereof.

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

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

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

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

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

[0061] In some embodiments, the GOF 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 raw material components used to prepare the culture medium and selecting those one or more batches of raw material components that, when combined, contain between about 8.5 ppb and about 100 ppb Mn.

[0063] In some embodiments, preparing the culture medium includes measuring the Mn concentration in one or more batches of raw material components used to prepare the culture medium and selecting the one or more batches of raw material components that, when combined, contain between about 8.5 ppb and 80 ppb 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 raw material components used to prepare the culture medium and selecting the one or more batches of raw material components that, when combined, contain between about 10 ppb and 100 ppb Mn to prepare the culture medium.

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

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

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

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

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

[0070] In some embodiments, the concentration of Mn in the culture medium is adjusted to contain between about 8.5 ppb and about 100 ppb 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 between about 8.5 ppb and about 80 ppb Mn.

[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 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 between about 8.5 ppb and about 100 ppb Mn.

[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 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 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 Mn.

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

[0078] In some embodiments, the culture medium is a basal medium or a feed medium.

[0079] In some embodiments, culturing comprises fed-batch culturing or perfusion culturing.

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

[0081] In some embodiments, the eukaryotic cell is a CHO cell, a PER.C6 cell, a NS0 cell, a Sp2 / 0 cell, or a BHK cell.

[0082] In some embodiments, the CHO cell is a CHO-K1 cell, a CHO-DG44 cell, a CHO-S cell, or a CHODXB11 cell.

[0083] In some embodiments, the CHO cells lack glutamine synthetase (GS).

[0084] In some embodiments, the method is performed under GMP compliant conditions.

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

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

[0087] In some embodiments, the anti-CD38 antibody comprises an IgG1 isotype.

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

[0089] In some embodiments, the anti-CD38 antibody comprises an IgG4 isotype.

[0090] In some embodiments, 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 embodiments, the anti-CD38 antibody is expressed by a polynucleotide encoding the HC of SEQ ID NO:3 and the LC of SEQ ID NO:4.

[0092] In some embodiments, 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, about 80 ppb of Mn.

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

[0095] In some embodiments, the culture medium is controlled to contain 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, about 100 ppb of Mn.

[0096] The method of the present disclosure can be used together with any cell culture method suitable for the desired process (e.g., the production of recombinant antibodies). Cells can be grown in batches or fed-batch cultures, wherein the culture is terminated after the antibody is fully expressed, and the expressed antibody is harvested afterwards. Alternatively, cells can be grown in batches again in fed-batch cultures, wherein the culture is not terminated, but new nutrients and other components are periodically or continuously added to the culture, and the expressed antibody is periodically or continuously harvested during the culture. Other suitable methods (e.g., rotary tube cultures) are known in the art and can also be used.

[0097] In some embodiments, the fed batch culture includes a basal medium supplemented with a feed medium. The cells can be grown in any convenient volume. For example, the cells can be grown in a small-scale reaction vessel in a volume range of several milliliters to several liters. Alternatively, the cells can be grown in a large-scale commercial bioreactor in a volume range of about at least 1 liter to 10, 50, 100, 250, 500, 1000, 2500, 5000, 8000, 10000, 12000, 15000, 20000 or 25000 liters or more or any volume therebetween.

[0098] A culture medium containing between about 8.5 ppb and about 80 ppb manganese can be prepared by analyzing the manganese concentration in individual batches of each raw material component of the culture medium and supplementing the basal medium with manganese so that the prepared culture medium has a manganese concentration of between about 8.5 ppb and about 80 ppb for the raw material components or a total manganese concentration of between about 8.5 ppb and about 80 ppb for the selected components when combined.

[0099] A culture medium comprising between about 10 ppb and about 100 ppb manganese can be prepared by analyzing individual batches of each raw material component of the culture medium for manganese concentration and adding equal amounts of manganese to the basal medium and feed medium so that the raw material components have a prepared culture medium manganese concentration of between about 10 ppb and about 100 ppb, or the total manganese concentration of the selected components when combined together is between about 10 ppb and about 100 ppb.

[0100] A medium comprising between about 8.5 ppb and about 100 ppb manganese can be prepared by analyzing individual batches of each raw material component of the medium for manganese concentration and adding manganese to the feed medium so that the raw material components have a prepared medium manganese concentration of between about 8.5 ppb and about 100 ppb, or the total manganese concentration of the selected components when combined together is between about 8.5 ppb and about 100 ppb.

[0101] Methods of producing antibodies

[0102] Antibodies can be produced by fed-batch culture of CHO cells, and the culture temperature is regulated. After the cell culture fluid is harvested, it is centrifuged on a disc, deep filtered and sterile filtered to obtain a clarified cell culture fluid; the clarified cell culture fluid is 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 diafiltration to form a stock solution.

[0103] Methods for measuring the oligosaccharide composition of antibodies

[0104] Oligosaccharide composition of antibodies can be determined by UPLC method using Waters Acquity H-Class Bio UPLC system and fluorescence detector (WatersFLR) software. This method uses hydrophilic interaction chromatography to determine oligosaccharides of samples. This method uses N-glycosidase F (PNGase F) to cleave N-glycans, which are purified and labeled with 2-amino-benzamide (2-AB) to carry fluorescent groups, and then separated and determined by UPLC. Comparison with the typical spectrum (derived by LC-MS identification) determines the corresponding N-glycan peaks of the test sample. The amount of each glycan is quantified by peak area integration and expressed as a percentage of the total glycan peak area (peak area %).

[0105]

[0066] While the present disclosure has been generally described, embodiments of the present disclosure will be further disclosed in the following examples, which should not be construed as limiting the scope of the claims.

[0106] Example

[0107] The present disclosure is further illustrated by the following examples.

[0108] The basal culture medium used in the embodiments of the present disclosure is an animal-free, protein-free, chemically defined culture medium, and the components include the following substances:

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

[0110] The feed medium used in the embodiments of the present disclosure is an animal-free, protein-free, chemically defined medium, and the ingredients include the following substances:

[0111] Glucose 10-25g / L, sodium pyruvate 0.1-0.6g / L, potassium chloride 2-10g / L, ferric citrate 0.01-1.5g / L, copper sulfate 0.005-0.02g / L, magnesium chloride 0.025-0.5g / L, sodium dihydrogen phosphate 0.1-6g / L, alanine 0.1-2.5g / L, asparagine 0.1-8g / L, arginine 0.6-8g / L, aspartic acid 2-15g / L, glutamine amino acid 0.1-15g / L, glycine 0.5-5g / L, histidine 0.3-5g / L, isoleucine 0.1-5g / L, leucine 0.5-20g / L, lysine 0.2-6g / L, phenylalanine 0.5-15g / L, proline 0.1-10g / L, serine 0.3-2.5g / L, tryptophan 0.05-4g / L, tyrosine 0.05-5g / L, valine 0.05-7g / L.

[0112] In the disclosed embodiment, the fed batch culture method comprises the following steps: 6 cells / ml; after inoculation, the cells were cultured at 30°C-37°C; after inoculation, feed medium was added on the 3rd or 4th day, and feed was added every other day until the end of the culture. The amount of each feed was 3-8% of the initial volume, and the total feed amount was 20-50% of the initial weight target value; when the cell density was 15×10 6 cells / ml-5×10 7 cells / ml or cultured to the 6th to 9th day after inoculation, culturing the cells at a temperature of 28°C-35°C and maintaining until the end of the culture; culturing to 10-16 days after inoculation or when the cell viability is <70%, the culture is terminated. Except for the different Mn ion concentrations, the other components of the culture medium used in the different schemes in the disclosed embodiments are consistent. The fed batch culture methods in the disclosed embodiments are consistent.

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

[0114] During the development of an anti-CD38 antibody (daratumumab mAb, having HC of SEQ ID NO: 3 and LC of SEQ ID NO: 4), it was found that trace amounts of the metal manganese (Mn) associated with the raw materials had a modulating effect on the glycosylation profile in the anti-CD38 antibody.

[0115] The expected manganese concentration in the hydrated medium was estimated based on the cumulative contribution of Mn in the chemically defined medium to use the Mn concentration as a predictive tool. The bioreactor concentration was calculated based on the mass of basal medium and feed medium added per liter, and then the adjusted Mn concentration was derived based on the basal Mn concentration plus the feed adjusted according to the feed rate.

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

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

[0118] The Mn concentration in ten hydration media and the G0F% and G1F% data of anti-CD38 antibodies were obtained. Table 1 shows the G0F%, G1F% and the Mn concentration and total Mn concentration in the basal medium and feed medium in each batch.

[0119] Table 1

[0120]

[0121] exist Figures 1A to 1D The correlation between the Mn concentration and the anti-CD38 antibody G0F percentage and G1F percentage in each analysis batch is shown in Figure 1. The data show that when the Mn concentration is between about 8.5 ppb and about 17 ppb, it is positively correlated with the anti-CD38 antibody G1F percentage and negatively correlated with the G0F percentage, and the correlation coefficients are R 2 =0.9586 and R 2 =0.6019. When the Mn concentration was between about 17 ppb and about 80 ppb, it was negatively correlated with the anti-CD38 antibody G1F percentage and positively correlated with the G0F percentage. The correlation coefficients were R 2 =0.9854 and R 2 =0.9695.

[0122] Figure 1E The Mn concentration contributed by the basal medium or feed medium in each batch is shown. Based on these data, a total contribution of Mn between about 8.5 ppb and about 80 ppb Mn concentrations yielded anti-CD38 antibodies with G1F% content between about 17% and 23% and G0F% content between about 64% and about 69%.

[0123] Example 2. Preparation of basal medium and feed medium supplemented with manganese

[0124] Mn concentration in the bioreactor: (μg / L~ppb) = basic Mn (ppb) + additional Mn (ppb) added to the base + ((feed Mn (ppb) + additional Mn (ppb) added to the feed) × feed factor).

[0125] The Mn concentration in eight hydration media and the G0F% and G1F% data of anti-CD38 antibodies were obtained. Table 2 shows the G0F%, G1F% and the Mn concentration and total concentration in the basal medium and feed medium in each batch.

[0126] Table 2

[0127]

[0128] exist FIG. 2A to FIG. 2D The correlation between the Mn concentration and the anti-CD38 antibody G0F percentage and G1F percentage in each analysis batch is shown in Figure 1. The data show that when the Mn concentration is between about 10 ppb and about 20 ppb, it is positively correlated with the anti-CD38 antibody G1F percentage and negatively correlated with the G0F percentage, and the correlation coefficients are R 2 =0.8866 and R 2 =0.8107. When the Mn concentration was between about 20 ppb and about 100 ppb, it was negatively correlated with the anti-CD38 antibody G1F percentage and positively correlated with the G0F percentage, with correlation coefficients of R 2 =0.9297 and R 2 =0.9435.

[0129] Figure 2E The Mn concentration contributed by the basal medium or feed medium in each batch is shown. Based on these data, a total contribution of Mn between about 10 ppb and about 100 ppb Mn concentrations yielded anti-CD38 antibodies with G1F% content between about 19% and 23% and G0F% amounts between about 65% and 69%.

[0130] Example 3. Preparation of feed medium supplemented with additional manganese

[0131] Mn concentration in the bioreactor: (μg / L~ppb) = basic Mn (ppb) + ((feed Mn (ppb) + additional Mn added in feed) × feed factor).

[0132] The Mn concentration in nine hydration media and the G0F% and G1F% data of anti-CD38 antibody were obtained. Table 3 shows the G0F%, G1F% and the manganese concentration and total concentration in the basal medium and feed medium in each batch.

[0133] Table 3

[0134]

[0135] exist Figure 3A and Figure 3B The correlation between the Mn concentration and the anti-CD38 antibody G0F percentage and G1F percentage in each analysis batch is shown in Figure 1. The data show that when the Mn concentration is between about 8.5 ppb and about 100 ppb, it is positively correlated with the anti-CD38 antibody G1F percentage and negatively correlated with the G0F percentage. The correlation coefficients are R 2 =0.7261 and R 2 =0.7307.

[0136] Figure 3C The Mn concentration contributed by the basal medium or feed medium in each batch is shown. Based on these data, a total contribution of Mn between about 8.5 ppb and about 100 ppb Mn concentrations yielded anti-CD38 antibodies with G1F% content between about 18% and 26% and G0F% content between about 63% and 70%.

Claims

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

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

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

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

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

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

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 between about 8.5 ppb and 100 ppb Mn.

8. The method of 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 between about 8.5 ppb and about 80 ppb of Mn.

9. The method of 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 between about 10 ppb and about 100 ppb of Mn.

10. The method of 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 between about 8.5 ppb and about 100 ppb Mn.

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

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

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

14. The method according to claim 13, wherein the eukaryotic cell is a CHO cell, a PER.C6 cell, a NSO cell, a Sp2 / 0 cell or a BHK cell. 15 . The method according to claim 14 , wherein the CHO cell is a CHO-K1 cell, a CHO-DG44 cell, a CHOS cell or a CHO-DXB11 cell.

16. The method of claim 15, wherein the CHO cells lack glutamine synthetase (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 of any one of claims 1 to 17, wherein the anti-CD38 antibody is of the IgG1 isotype.

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

4.

20. A method for producing a pharmaceutical product, comprising: carrying out the method of any one of claims 1 to 19, and further comprising, The anti-CD38 antibody is formulated as a pharmaceutical product.

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