Methods for controlling antibody heterogeneity

Abstract

The present invention provides methods for controlling the heterogeneity of Fc-containing proteins, such as antibodies, produced in cell culture, particularly mammalian cell culture, by controlling the culture pCO2, and the products produced by these methods. In particular, the present invention provides for reducing the percentage of acidic charge variants in antibody products. Proteins containing Fc portions include, but are not limited to, Fc-containing proteins, such as antibodies and antibody derivatives, and fragments of both.

Description

[Technical field]

[0001] This application claims priority to U.S. Patent Application No. 63 / 246,047, filed September 20, 2021, which is incorporated herein by reference.

[0002] The present invention provides methods for controlling the heterogeneity of Fc-containing proteins produced in cell culture, particularly mammalian cell culture, and protein products and proteins produced by these methods. Proteins that contain an Fc portion include Fc-containing proteins such as antibodies. [Background technology]

[0003] The production of Fc-containing proteins such as antibodies in cell culture can result in charge variants, which are of two types called acidic and basic variants. In addition, there is a main peak form. Fc refers to the constant region found in the antibody heavy chain found in nature, and is also contained in, for example, monoclonal antibodies, "crystallizable fragment".

[0004] Acidic variants are typically more common than basic variants in antibodies and can result in deamidation, sialylation, glycosylation and fragmentation, which alter the stability, activity and potency of proteins containing the Fc portion (the portion from the fragment crystallizable region of the antibody). Sissolak et al., J. Indust. Microbiol. Biotech. 46:1167-78 (2019). Basic variants can cause increased binding of antibodies to Fc receptors. Hintersteiner et al., MABS 8:1458-60 (2016).

[0005] Fc glycans also play a role in safety, bioactivity, pharmacodynamics and pharmacokinetics. Reusch and Tejada, Glycobiol. 25:1325-34 (2015). A possible phenomenon is known as non-glycosylated heavy chain (NGHC). NGHC variation can alter effector functions such as opsonization. Opsonization involves the Fc portion involved in ADCC (antibody-dependent cellular cytotoxicity), ADCP (antibody-dependent cellular phagocytosis) and CDC (complement-dependent cytotoxicity). NGHC variation can be of concern in some contexts (depending on the disease state, route of administration and type of Fc-containing protein) and is less important in others.

[0006] Therefore, there is a need to control charge variation and / or NGHC in proteins containing Fc portions. However, this can create a situation where optimizing one may, but not always, lead to a possibly more unfavorable state for the other, as discussed in more detail below. Due to the impact of acidic charge variants in antibodies, it is usually desirable to reduce the occurrence of such variants. Finally, charge variation may be of concern in some contexts (depending on the disease state, route of administration and type of Fc-containing protein) and is less important in others. The invention described below addresses this and other needs. Summary of the Invention

[0007] The present invention provides a method for controlling heterogeneity in Fc-containing proteins, such as antibodies, produced by mammalian cells in culture. The method may include seeding a medium with mammalian cells producing the Fc-containing protein and culturing the cells under pCO2 conditions that allow the mammalian cells to produce the Fc-containing protein. Preferably, CO2 sparging is used to increase pCO2 in the culture. Another approach is to allow pCO2 to accumulate and be controlled with air sparging. Pressure drop in the bioreactor can also be used to control pCO2. A combination of CO2 sparging, air / nitrogen sparging and pressure drop can be used. Charge variants are primarily due to changes in the Fc region.

[0008] Depending on the objectives of one of ordinary skill in the art and taking into account the teachings contained herein, a combination of CO sparging, air / nitrogen sparging and pressure reduction may be used.

[0009] The present invention also provides a method for controlling, preferably reducing, the percentage of acidic charge variants in an Fc-containing protein product, such as an antibody, produced by mammalian cells in culture, the method comprising seeding a culture medium with mammalian cells producing the Fc-containing protein, and culturing the cells under pCO2 conditions that allow the mammalian cells to produce an Fc-containing protein product having less acidic acid variants than would be obtained without pCO2 conditions, the pCO2 conditions being, for example, 120 mmHg to 140 mmHg CO2 in the culture medium, or as otherwise disclosed herein. The pCO2 conditions can be achieved by sparging, such as CO2 sparging. The charge variants can be caused by changes in the Fc region. The Fc-containing protein produced under pCO2 conditions can have, for example, 0.5% to 4% less acidic variants than would be obtained without pCO2 conditions. The Fc-containing protein can be an antibody, such as an antibody capable of binding to the PD-1 factor or the IL-4 receptor. Preferably, the antibody is a human monoclonal antibody, preferably an IgG antibody, including subclasses such as IgG1 and IgG4. The mammalian cells may be, for example, CHO, BHK, HEK293, HeLa, human amniotic fluid, Per.C6 and Sp2 / 0 cells. The cells may be cultured for 10 to 15 days, preferably about 14 days, under various pCO2 conditions disclosed herein.

[0010] The present invention further provides a method comprising seeding a medium with mammalian cells producing an Fc-containing protein, such as an antibody, and culturing the cells under pCO2 conditions that allow the mammalian cells to produce the Fc-containing protein, wherein the predominant peak form of the Fc-containing protein produced by the cells comprises about 38% to about 65% of the total Fc-containing protein, an acidic variant of the Fc-containing protein comprises about 20% to about 47% of the total Fc-containing protein, and a basic variant of the Fc-containing protein comprises up to about 36% of the total Fc-containing protein, which may be an antibody, derivative, and fragments of both. The cells may be cultured for about 10 to 15 days, preferably about 14 days. The pCO2 conditions can be about 30mmHg to about 210mmHg, 50mmHg to 200mmHg, 60mmHg to 190mmHg, 70mmHg to 180mmHg, 80mmHg to 170mmHg, 90mmHg to 160mmHg, 100mmHg to 150mmHg, 110mmHg to 140mmHg, 120mmHg to 140mmHg, 120mmHg to 130mmHg, or any value within these ranges during culture, preferably maintained by CO2 sparging and can be measured using a CO2 electrode. The cells can be any suitable mammalian cell, including CHO, BHK, HEK293, HeLa, human amniotic fluid, Per.C6, and Sp2 / 0 cells.

[0011] The Fc-containing protein may be an antibody, such as an antibody capable of binding to the PD-1 factor or the IL-4 receptor. Preferably, the antibody is a human monoclonal antibody, preferably an IgG antibody, including all subclasses such as IgG1 and IgG4.

[0012] The present invention also provides a method of controlling the heterogeneity of antibodies, antibody derivatives or antibody fragments produced by mammalian cells in culture by seeding mammalian cells producing the antibodies, antibody derivatives or antibody fragments in a medium, as well as a method of culturing cells under pCO2 conditions that allow mammalian cells to produce antibodies, antibody derivatives or antibody fragments, wherein the main peak form of the antibodies, antibody derivatives or antibody fragments produced by the cells accounts for about 50% to about 70% of the total antibodies, antibody derivatives or antibody fragments, acidic variants of the antibodies, antibody derivatives or antibody fragments account for about 20% to about 47% of the total antibodies, antibody derivatives or antibody fragments, and basic variants of the antibodies, antibody derivatives or antibody fragments account for up to about 15% of the total antibodies, antibody derivatives or antibody fragments. The basic variants of the antibodies, antibody derivatives or antibody fragments can account for up to about 6%, about 8% or about 10%, preferably about 15% or less of the total antibodies, antibody derivatives or antibody fragments. The predominant peak form of the antibody, antibody derivative or antibody fragment produced by the cells can account for about 50% to about 65% of the total antibody, antibody derivative or antibody fragment, and the acidic variant of the antibody, antibody derivative or antibody fragment can account for about 23% to about 46%, about 23% to about 39%, or about 31% to about 46% of the total antibody, antibody derivative or antibody fragment. For example, the percentage of Fc-containing protein, such as an antibody having a non-glycosylated heavy chain, can include about 5 to about 7%, with other ranges provided herein. The cells can be cultured for about 10 to 15 days, preferably about 14 days. The pCO2 conditions can be about 30mmHg to about 210mmHg, 50mmHg to 200mmHg, 60mmHg to 190mmHg, 70mmHg to 180mmHg, 80mmHg to 170mmHg, 90mmHg to 160mmHg, 100mmHg to 150mmHg, 110mmHg to 140mmHg, 120mmHg to 140mmHg, 120mmHg to 130mmHg, or any value within these ranges during the culture, and are preferably maintained by CO2 sparging and can be measured using a CO2 electrode. As determined by one of skill in the art in view of the teachings contained herein, the pCO2 can be varied during the culture process by varying the CO2 sparging, air or other sparging, and / or bioreactor pressure.

[0013] The cells can be any suitable mammalian cell, including CHO, BHK, HEK293, HeLa, human amniotic fluid, Per.C6 and Sp2 / 0 cells. The Fc-containing proteins, such as antibodies, antibody derivatives and antibody fragments produced thereby are an invention provided herein.

[0014] The Fc-containing protein may be an antibody, such as an antibody capable of binding to the PD-1 factor or the IL-4 receptor. Preferably, the antibody is a human monoclonal antibody, preferably an IgG antibody, including all subclasses such as IgG1 and IgG4.

[0015] Typically, Fc-containing proteins, such as antibodies produced according to the teachings of the invention contained herein, have acidic charge variants that constitute 20%-50% of the total Fc-containing protein, more specifically 20%-47%, 23%-45%, 25%-40%, 28%-37%, 28%-35%, 29%-34%, 30%-33%, or any integer or fractional value within these ranges. The Fc-containing protein has a predominant peak form that constitutes 38%-70% of the total Fc-containing protein, more specifically 45%-70%, 50%-65%, 55%-60%, or any integer or fractional value within these ranges. The Fc-containing protein has basic charge variants that constitute between 1% and 40% of the total Fc-containing protein, more specifically between 2% and 35%, 3% and 30%, 4% and 25%, 5% and 20%, 6% and 15%, 7% and 12%, 7.5% and 10%, 8% and 9%, or any integer or fractional value within these ranges.

[0016] The acidic charge variant fraction of the total product can be controlled, preferably reduced, in accordance with the present invention, in the range of 0.1% to 10%, or any integer or fractional value within these ranges. See, for example, Table 1. More specifically, the acidic variant fraction can be 0.2% to 9%, 0.3% to 8%, 0.4% to 7%, 0.5% to 6%, 0.6% to 5%, 0.7% to 4.75%, 0.75% to 4.5%, 0.8% to 4.25%, 0.9% to 4%, 1% to 3.75%. The reduction may be by 1% to 3.5%, 1% to 3.25%, 1% to 3%, 1% to 2.75%, 1.25% to 2.5%, 1.25% to 2.25%, 1.25% to 2%, 1.5% to 2%, 1.5% to 1.75%, or any integer or fractional value within these ranges. Additionally, other ranges include 0.1%-4%, 0.25%-4%, 0.25%-3.75%, 0.25%-3.5%, 0.25%-3%, 0.25%-2.75%, 0.25%-2.5%, 0.25%-2.25%, 0.25%-2%, 0.25%-1.75%, 0.25%-1.5%, 0.25%-1.25%, 0.25%-1%, and 0.25%-0.75%. 0.25%~0.5%, 0.5%~4%, 0.5%~3.75%, 0.5%~3.5%, 0.5%~3%, 0.5%~2.75%, 0.5%~2.5%, 0.5%~2.25%, 0.5%~2%, 0.5%~1.75%, 0.5%~1.5%, 0.5%~1.25%, 0.5 %~1%, 0.5%~0.75%, 0.75%~4%, 0.75%~3.75%, 0.75%~3.5%, 0.75%~3%, 0.75%~2.75%, 0.75%~2.5%, 0.75%~2.25%, 0.75%~2%, 0.75%~1.75%, 0.75%~1.5% , 0.75%-1.25%, 0.75%-1%, 1%-4%, 1%-3.75%, 1%-3.5%, 1%-3%, 1%-2.75%, 1%-2.5%, 1%-2.25%, 1%-2%, 1%-1.75%, 1%-1.5%, 1%-1.25%, 1.25%-4%, 1.25%-3.75%, 1.25%-3.5%, 1.25%-3%, 1.25%-2.75%, 1.25%-2.5%, 1.25%-2.25%, 1.25%-2%, 1.25%-1.75%, 1.25%-1.5%, or any integer or fractional value within these ranges.For example, the acidic charge variant fraction may be at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%. The change may be, preferably decreased, by 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4.0%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5% or more, for example, by up to 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15% or more.

[0017] The basic charge variant fraction of the total product can be controlled in accordance with the present invention in the range of 0.1% to 15%, or any integer or fractional value within these ranges. More specifically, the basic charge variant fraction can be 0.1% to 14%, 0.1% to 13%, 0.1% to 12%, 0.1% to 11%, 0.2% to 10%, 0.2% to 9%, 0.3% to 8%, 0.4% to 7%, 0.5% to 6%, 0.6% to 5%, 0.7% to 4.75%, 0.75% to 4.5%, 0.8% to 4.25%, 0.9% to 4%, 1% to 3.75%. It may vary by 1%-3.5%, 1%-3.25%, 1%-3%, 1%-2.75%, 1%-2.75%, 1.25%-2.5%, 1.25%-2.25%, 1.25%-2%, 1.5%-2%, 1.5%-1.75%, or any integer or fractional value within these ranges. Additionally, other ranges include 0.1%-4%, 0.25%-4%, 0.25%-3.75%, 0.25%-3.5%, 0.25%-3%, 0.25%-2.75%, 0.25%-2.5%, 0.25%-2.25%, 0.25%-2%, 0.25%-1.75%, 0.25%-1.5%, 0.25%-1.25%, 0.25%-1%, and 0.25%-0.75%.0.25%~0.5%, 0.5%~4%, 0.5%~3.75%, 0.5%~3.5%, 0.5%~3%, 0.5%~2.75%, 0.5%~2.5%, 0.5%~2.25%, 0.5%~2%, 0.5%~1.75%, 0.5%~1.5%, 0.5%~1.25%, 0.5 %~1%, 0.5%~0.75%, 0.75%~4%, 0.75%~3.75%, 0.75%~3.5%, 0.75%~3%, 0.75%~2.75%, 0.75%~2.5%, 0.75%~2.25%, 0.75%~2%, 0.75%~1.75%, 0.75%~1.5% , 0.75%-1.25%, 0.75%-1%, 1%-4%, 1%-3.75%, 1%-3.5%, 1%-3%, 1%-2.75%, 1%-2.5%, 1%-2.25%, 1%-2%, 1%-1.75%, 1%-1.5%, 1%-1.25%, 1.25%-4%, 1.25%-3.75%, 1.25%-3.5%, 1.25%-3%, 1.25%-2.75%, 1.25%-2.5%, 1.25%-2.25%, 1.25%-2%, 1.25%-1.75%, 1.25%-1.5%, or any integer or fractional value within these ranges. Basic charge variant fractions of at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%. The change may be 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4.0%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5% or more, for example, up to 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15% or more.

[0018] Fc-containing proteins, such as antibodies produced according to the teachings of the invention contained herein, typically have a percentage of non-glycosylated heavy chains (NGHC) present between 3% and 8% of the total Fc-containing protein, more specifically between 4% and 7%, between 5% and 7%, and between 5% and 6.5%, between 5% and 6%, between 5% and 5.75%, between 5% and 5.5%, or any integer or fractional value within these ranges.

[0019] Fc-containing proteins, such as antibodies produced by the methods of the invention, as well as derivatives and fragments of Fc-containing proteins, are also part of the invention provided herein. Antibodies include, but are not limited to, antibodies capable of binding to PD-1 factor and antibodies capable of binding to interleukin 4 receptor. [Brief description of the drawings]

[0020] [Figure 1] Figure 1 shows pCO2 levels for Examples 1 and 3. Mid pCO2 was selected as the midpoint control. [Diagram 2] Figure 1 shows pCO2 levels for Examples 2 and 4. Mid pCO2 was selected as the midpoint control. [Diagram 3] Shown are predicted pH levels during the production days for the 2 liter bioreactor with the air sparing and pH conditions shown in Table 6. A moderate pCO2 was selected as the midpoint control. [Figure 4] The actual area 1 (%) (y-axis) and the predicted area 1 (%) (x-axis) are shown. Area 1 is for the acidic charge variant. [Diagram 5] Figure 4 summarizes the fit of the data set to the analysis of variance and parameter estimates. [Figure 6] The actual Area 2 (%) (y-axis) and the expected Area 2 (%) (x-axis) are shown. Area 2 is for the main peak form. [Figure 7] Figure 6 summarizes the fit of the data set to the analysis of variance and parameter estimates. [Figure 8]The actual region 3 (%) (y-axis) and the predicted region 3 (%) (x-axis) are shown. Region 3 is for the basic charge variant. [Figure 9] Figure 8 summarizes the fit of the data set to the analysis of variance and parameter estimates. [Figure 10] Actual NGHC (y-axis) and predicted NGHC (x-axis) are shown. [Figure 11] Figure 10 summarizes the fit of the data set to the analysis of variance and parameter estimates. [Figure 12] Viable cell density values ​​over process time (days) are shown. The y-axis has values ​​up to 350x105 cells / ml. Medium pCO2 was chosen as the midpoint control. [Figure 13] Percentage of cell viability over process time (days) is shown. Medium pCO2 was selected as the midpoint control. [Figure 14] The pH values ​​over the process time (days) are shown. Medium pCO2 was chosen as the midpoint control. [Figure 15] pCO2 values ​​over process time (days) are shown. Medium pCO2 was selected as the midpoint control. [Figure 16] Glucose values ​​over process time (days) are shown. Medium pCO2 was chosen as the midpoint control. [Figure 17] Potassium values ​​over process time (days) are shown. Medium pCO2 was selected as the midpoint control. [Figure 18] Sodium values ​​over process time (days) are shown. Medium pCO2 was selected as the midpoint control. [Figure 19] Osmolality values ​​over process time (days) are shown. Medium pCO2 was selected as the midpoint control. [Figure 20] Glutamate values ​​over process time (days) are shown. Medium pCO2 was chosen as the midpoint control. [Figure 21] Lactate values ​​over process time (days) are shown. Medium pCO2 was chosen as the midpoint control. [Figure 22]Ammonia values ​​over process time (days) are shown. Medium pCO2 was selected as midpoint control. [Diagram 23] Glutamine values ​​over process time (days) are shown. Medium pCO2 was selected as the midpoint control. [Figure 24] Shows pCO2 in mmHg (y-axis) over process time (days) from Example 6. Medium pCO2 was selected as a midpoint control. TEMP refers to the physiological temperature of cells as described herein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] 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 these inventions belong.

[0022] definition The term "about" in the context of numerical values ​​and ranges refers to a value or range that is close to or near the recited value or range, such that the invention can be practiced with, for example, a determined rate, amount, density, degree, increase, decrease, percentage, form value or presence, variant, temperature or amount of time, as is clear from the teachings contained herein. Thus, the term encompasses values ​​beyond those that simply result from systematic error. For example, "about" can indicate values ​​that are either above or below the recited value, in the range of approximately + / - 10% or more or less, depending on the ability to practice.

[0023] "Antibodies" (also referred to as "immunoglobulins") are examples of proteins with multiple polypeptide chains and extensive post-translational modifications. A canonical immunoglobulin protein (e.g., IgG) contains four polypeptide chains - two light chains and two heavy chains. Each light chain is linked to one heavy chain via a cysteine ​​disulfide bond, and the two heavy chains are linked to each other via two cysteine ​​disulfide bonds. Immunoglobulins produced in mammalian systems are also glycosylated at various residues (e.g., asparagine residues) with various polysaccharides and can vary between species, which can affect the antigenicity of therapeutic antibodies. Butler and Spearman, "The choice of mammalian cell host and possibilities for glycosylation engineering", Curr. Opin. Biotech. 30:107-112 (2014).

[0024] Antibodies are often used as therapeutic biomolecules. Antibodies comprise immunoglobulin molecules composed of four polypeptide chains, two heavy (H) chains and two light (L) chains, interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains: CH1, CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region comprises one domain (CL). The VH and VL regions can be further subdivided into regions of hypervariability, called complementarity determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (the heavy chain CDRs may be abbreviated as HCDR1, HCDR2 and HCDR3, and the light chain CDRs may be abbreviated as LCDR1, LCDR2 and LCDR3). The term "high affinity" antibody refers to an antibody that has an affinity of at least 10 as measured by surface plasmon resonance, e.g., BIACORE™ or solution affinity ELISA.-9 M, at least 10 -10 M, at least 10 -11 M, or at least 10 -12 M refers to antibodies having binding affinity for their target.

[0025] An "acidic charge variant" is an Fc-containing protein (e.g., antibody) variant that has a lower pI than the predominant peak form of the Fc-containing protein. Acidic charge variants tend to have more negative charges.

[0026] A "basic charge variant" is an Fc-containing protein (e.g., antibody) variant that has a higher pI than the predominant peak form of the Fc-containing protein. Basic charge variants tend to have more positive charge or less negative charge.

[0027] The "major peak form" of an Fc-containing protein (eg, an antibody) is the predominant form of the Fc-containing protein that has a pI between the acidic and basic charge variants.

[0028] The phrase "bispecific antibodies" includes antibodies that can selectively bind to two or more epitopes. Bispecific antibodies generally include two different heavy chains, each of which specifically binds to a different epitope, either on two different molecules (e.g., antigens) or on the same molecule (e.g., on the same antigen). When a bispecific antibody can selectively bind to two different epitopes (a first epitope and a second epitope), the affinity of the first heavy chain for the first epitope will generally be at least one to two, three or four orders of magnitude lower than the affinity of the first heavy chain for the second epitope, or vice versa. The epitopes recognized by a bispecific antibody can be on the same target or on different targets (e.g., on the same protein or on different proteins). Bispecific antibodies can be generated, for example, by combining heavy chains that recognize different epitopes of the same antigen. For example, nucleic acid sequences encoding heavy chain variable sequences that recognize different epitopes of the same antigen can be fused to nucleic acid sequences encoding different heavy chain constant regions, and such sequences can be expressed in cells that express immunoglobulin light chains. A typical bispecific antibody has two heavy chains, each with three heavy chain CDRs, followed (from N-terminus to C-terminus) by a CH1 domain, a hinge, a CH2 domain, and a CH3 domain, and an immunoglobulin light chain that does not provide antigen binding specificity but can associate with each heavy chain, or that can associate with each heavy chain and bind to one or more of the epitopes bound by the heavy chain antigen binding region, or that can associate with each heavy chain and bind one or both heavy chains to one or both epitopes.

[0029] The phrase "heavy chain" or "immunoglobulin heavy chain" includes immunoglobulin heavy chain constant region sequences from any organism, including heavy chain variable domains unless otherwise specified. Heavy chain variable domains include three heavy chain CDRs and four FR regions, unless otherwise specified. Fragments of heavy chains include CDRs, CDRs and FRs, and combinations thereof. A typical heavy chain has a variable domain followed (from N-terminus to C-terminus) by a CH1 domain, a hinge, a CH2 domain, and a CH3 domain. A functional fragment of a heavy chain includes a fragment that can specifically recognize an antigen (e.g., recognize an antigen with a KD in the micromolar, nanomolar, or picomolar range), can be expressed and secreted from a cell, and includes at least one CDR.

[0030] The phrase "light chain" includes immunoglobulin light chain constant region sequences from any organism, including human kappa and lambda light chains, unless otherwise specified. A light chain variable (VL) domain typically includes three light chain CDRs and four framework (FR) regions, unless otherwise specified. In general, a full-length light chain includes a VL domain including, from amino to carboxyl terminus, FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and a light chain constant domain. Light chains that can be used in these inventions include, for example, light chains that do not selectively bind either the first or second antigen selectively bound by the antigen binding protein. Suitable light chains include those that can be identified by screening for the most commonly used light chains in existing antibody libraries (wet libraries or in silico), where the light chain does not substantially interfere with the affinity and / or selectivity of the antigen binding domain of the antigen binding protein. Suitable light chains include those that can bind to one or both epitopes bound by the antigen binding region of the antigen binding protein.

[0031] The phrase "variable domain" includes an amino acid sequence of an immunoglobulin light or heavy chain (modified as desired) that comprises, from N-terminus to C-terminus (unless otherwise indicated), the following amino acid regions: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. A "variable domain" includes an amino acid sequence that can fold into a canonical domain (VH or VL) with a double beta-sheet structure, in which the beta-sheets are connected by disulfide bonds between residues of the first beta-sheet and the second beta-sheet.

[0032] The phrase "complementarity determining region" or the term "CDR" includes amino acid sequences that are encoded by the nucleic acid sequences of an organism's immunoglobulin genes, which normally (i.e., in a wild-type organism) appear between two framework regions in the variable region of a light or heavy chain of an immunoglobulin molecule (e.g., an antibody or T cell receptor). CDRs can be encoded, for example, by germline sequences, or by sequences that are rearranged or unrearranged, for example, by naive or mature B or T cells. In some situations (e.g., in the case of CDR3), CDRs can be encoded by two or more sequences (e.g., germline sequences) that are not contiguous (e.g., in an unrearranged nucleic acid sequence) but are contiguous in the B cell nucleic acid sequence, for example, as a result of splicing or joining of sequences (e.g., VDJ recombination to form heavy chain CDR3).

[0033] "Antibody derivatives and fragments" include, but are not limited to, antibody fragments (e.g., ScFv-Fc, dAB-Fc, half antibodies), multispecifics (e.g., IgG-ScFv, IgG-dab, ScFV-Fc-ScFV, trispecifics).

[0034] The phrase "Fc-containing protein" includes antibodies, bispecific antibodies, antibody derivatives containing Fc, antibody fragments containing Fc, Fc fusion proteins, immunoadhesins, and other binding proteins that contain at least a functional portion of an immunoglobulin CH2 and CH3 region. "Functional portion" refers to the CH2 and CH3 regions that are capable of binding to an Fc receptor (e.g., FcyR, or FcRn (neonatal Fc receptor) and / or participating in complement activation. The CH2 and CH3 regions are not functional if they contain deletions, substitutions, and / or insertions or other modifications that render them incapable of binding to any Fc receptor and incapable of activating complement. Fc fusion proteins include, for example, Fc fusions (N-terminus), Fc fusions (C-terminus), single Fc fusions, and bispecific Fc fusion proteins.

[0035] "Fc" stands for fragment crystallizable and is often referred to as fragment constant. Antibodies contain an Fc region that is made up of two identical protein sequences. IgG has a heavy chain known as the gamma chain. IgA has a heavy chain known as the alpha chain, and IgM has a heavy chain known as the mu chain. IgD has a heavy chain known as the sigma chain. IgE has a heavy chain known as the epsilon chain. In nature, the Fc region is the same in all antibodies of a given class and subclass in the same species. Human IgG has four subclasses, sharing about 95% homology between the subclasses. In each subclass, the Fc sequence is the same. For example, human IgG1 antibodies have the same Fc sequence. Similarly, IgG2 antibodies have the same Fc sequence, IgG3 antibodies have the same Fc sequence, and IgG4 antibodies have the same Fc sequence. Changes in the Fc region result in charge variations.

[0036] Fc-containing proteins, e.g., antibodies, can contain modifications in the immunoglobulin domain, including where the modifications affect one or more effector functions of the binding protein (e.g., modifications that affect FcyR binding, FcRn binding, and thus half-life, and / or CDC activity). Such modifications include, but are not limited to, the following modifications and combinations thereof, with reference to the EU numbering of immunoglobulin constant regions: 238, 239, 248, 249, 250, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 297, 298, 301, 303, 305, 307, 308, 309, 311, 312, 315, 318, 320, 322, 324, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 337, 338, 339, 340, 342, 344, 356, 358, 359, 360, 361, 362, 373, 375, 376, 378, 380, 382, ​​383, 384, 386, 388, 389, 398, 414, 416, 419, 428, 430, 433, 434, 435, 437, 438, and 439.

[0037] For example, and not by way of limitation, the binding protein is an Fc-containing protein (e.g., an antibody) that exhibits enhanced serum half-life (compared to the same Fc-containing protein without the listed modification(s)) and has modifications at positions 250 (e.g., E or Q), 250 and 428 (e.g., L or F), 252 (e.g., L / Y / F / W or T), 254 (e.g., S or T), and 256 (e.g., S / R / Q / E / D or T), or modifications at positions 428 and / or 433 (e.g., L / R / SI / P / Q or K) and / or 434 (e.g., H / F or Y), or modifications at positions 250 and / or 428, or modifications at positions 307 or 308 (e.g., 308F, V308F) and 434. In another example, modifications can include a 428L (e.g., M428L) modification and a 434S (e.g., N434S) modification, a 428L modification, a 2591 (e.g., V259I) modification, and a 308F (e.g., V308F) modification, a 433K (e.g., H433K) modification and a 434 (e.g., 434Y) modification, a 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modification, a 250Q modification and a 428L modification (e.g., T250Q and M428L), a 307 modification and / or a 308 modification (e.g., 308F or 308P).

[0038] A "culture medium" is aqueous and contains minerals, buffer salts, nutrients and other additives necessary to support the growth and protein production of cells in culture, e.g., in a bioreactor.

[0039] "Peak Viable Cell Density" or "Peak VCD" refers to the peak density of cells in culture. See Figure 12.

[0040] "Sparging" refers to pumping a gas through the culture medium. The gas can be CO2, air, or other gas. CO2 sparging increases pCO2. Air sparging and nitrogen sparging decrease pCO2. Sparging rates are determined based on the size of the bioreactor, with rates typically measured in cubic centimeters per minute (ccm) in small bioreactors. In large bioreactors (typically 1,000-10,000 liters) used for commercial production, sparging rates are measured in standard liters per minute (slpm).

[0041] "Protein product" refers to a protein of interest, such as an Fc-containing protein (e.g., an antibody). Protein products can be produced by cells in culture, usually engineered mammalian cells. Typically, cells in culture, e.g., in a bioreactor, produce proteins of interest, which become the protein product. The protein product can later be subjected to purification, characterization, sterilization, formulation, and other finishing steps, such as concentration or lyophilization, and finally packaging to form a finished protein product. Protein products include drug substance formulations (FDS).

[0042] All numerical limits and ranges set forth herein include all numerical values ​​or values ​​around or between the numerical values ​​of the range or limit. Ranges and limits set forth herein expressly express and describe all integer, decimal, and fractional values ​​encompassed by the range or limit.

[0043] Detailed Description Antibody charge variants include acidic variants and basic variants. Charge variants can be caused by enzymatic modifications including deamidation and sialylation, which increase the net negative charge on the antibody, which reduces the pI value and forms acidic variants. Furthermore, lysine cleavage from the C-terminus causes loss of net positive charge, leading to the formation of acidic variants. Acidic variants can also arise by the creation of covalent moieties such as glycation, where glucose or lactose reacts with the primary amine of a lysine residue. The formation of basic variants is caused by the presence of C-terminal lysine or glycine amidation, succinimide formation, amino acid oxidation, or removal of sialic acid. These provide the addition of a positive charge or the elimination of a negative charge, thereby increasing the pI value. See Khawli et al., mAbs 2:6, 613-624 (2010).

[0044] The present invention provides an approach to control the population of charge variants (acidic and basic) of proteins and glycosylation variants produced in mammalian cell culture. Embodiments include the production of Fc-containing proteins, including antibodies and fragments and derivatives thereof. The present invention allows this control by selecting the carbon dioxide concentration (pCO2) of the medium during production. NGHC can also be controlled via pH.

[0045] Apart from the pCO2 levels taught herein, standard conditions and media may be used. Typically, cells are cultured under physiological conditions, such as at a temperature of about 36°C to 38°C, preferably 36°C to 37°C.

[0046] Typically, Fc-containing proteins (e.g., antibodies) produced according to the teachings of the invention contained herein have acidic charge variants that constitute 20%-50% of the total Fc-containing protein, more specifically 20%-47%, 23%-45%, 25%-40%, 28%-37%, 28%-35%, 29%-34%, 30%-33%, or any integer or fractional value within these ranges. The Fc-containing protein has a predominant peak form that constitutes 38%-70% of the total Fc-containing protein, more specifically 45%-70%, 50%-65%, 55%-60%, or any integer or fractional value within these ranges. The Fc-containing protein has basic charge variants that constitute between 1% and 40% of the total Fc-containing protein, more specifically between 2% and 35%, 3% and 30%, 4% and 25%, 5% and 20%, 6% and 15%, 7% and 12%, 7.5% and 10%, 8% and 10%, 8% and 9%, or any integer or fractional value within these ranges.

[0047] Fc-containing proteins (e.g., antibodies) produced according to the teachings of the invention contained herein typically have a percentage of non-glycosylated heavy chain (NGHC) present between 3% and 8% of the total Fc-containing protein, more specifically, between 4% and 7%, between 5% and 7%, and between 5% and 6.5%, between 5% and 6%, between 5% and 5.75%, between 5% and 5.5%, or any integer or fractional value within these ranges.

[0048] The acidic charge variant fraction of the total product can be controlled, preferably reduced, in accordance with the present invention, in the range of 0.1% to 10%, or any integer or fractional value within these ranges. See, for example, Table 1. More specifically, the acidic variant fraction can be 0.2% to 9%, 0.3% to 8%, 0.4% to 7%, 0.5% to 6%, 0.6% to 5%, 0.7% to 4.75%, 0.75% to 4.5%, 0.8% to 4.25%, 0.9% to 4%, 1% to 3.75%. It may vary from 1% to 3.5%, 1% to 3.25%, 1% to 3%, 1% to 2.75%, 1% to 2.75%, 1.25% to 2.5%, 1.25% to 2.25%, 1.25% to 2%, 1.5% to 2%, 1.5% to 1.75%, or any integer or fractional value within these ranges. Additionally, other ranges include 0.1%-4%, 0.25%-4%, 0.25%-3.75%, 0.25%-3.5%, 0.25%-3%, 0.25%-2.75%, 0.25%-2.5%, 0.25%-2.25%, 0.25%-2%, 0.25%-1.75%, 0.25%-1.5%, 0.25%-1.25%, 0.25%-1%, and 0.25%-0.75%. 0.25%~0.5%, 0.5%~4%, 0.5%~3.75%, 0.5%~3.5%, 0.5%~3%, 0.5%~2.75%, 0.5%~2.5%, 0.5%~2.25%, 0.5%~2%, 0.5%~1.75%, 0.5%~1.5%, 0.5%~1.25%, 0.5 %~1%, 0.5%~0.75%, 0.75%~4%, 0.75%~3.75%, 0.75%~3.5%, 0.75%~3%, 0.75%~2.75%, 0.75%~2.5%, 0.75%~2.25%, 0.75%~2%, 0.75%~1.75%, 0.75%~1.5% , 0.75%-1.25%, 0.75%-1%, 1%-4%, 1%-3.75%, 1%-3.5%, 1%-3%, 1%-2.75%, 1%-2.5%, 1%-2.25%, 1%-2%, 1%-1.75%, 1%-1.5%, 1%-1.25%, 1.25%-4%, 1.25%-3.75%, 1.25%-3.5%, 1.25%-3%, 1.25%-2.75%, 1.25%-2.5%, 1.25%-2.25%, 1.25%-2%, 1.25%-1.75%, 1.25%-1.5%, or any integer or fractional value within these ranges.For example, the acidic charge variant fraction may be at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%. The change may be 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4.0%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5% or more, for example, up to 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15% or more.

[0049] The basic charge variant fraction of the total product can be controlled in accordance with the present invention in the range of 0.1% to 10%, or any integer or fractional value within these ranges. More specifically, the basic variant fraction can be 0.2% to 9%, 0.3% to 8%, 0.4% to 7%, 0.5% to 6%, 0.6% to 5%, 0.7% to 4.75%, 0.75% to 4.5%, 0.8% to 4.25%, 0.9% to 4%, 1% to 3.75%. It may vary by 1%-3.5%, 1%-3.25%, 1%-3%, 1%-2.75%, 1%-2.75%, 1.25%-2.5%, 1.25%-2.25%, 1.25%-2%, 1.5%-2%, 1.5%-1.75%, or any integer or fractional value within these ranges. Additionally, other ranges include 0.1%-4%, 0.25%-4%, 0.25%-3.75%, 0.25%-3.5%, 0.25%-3%, 0.25%-2.75%, 0.25%-2.5%, 0.25%-2.25%, 0.25%-2%, 0.25%-1.75%, 0.25%-1.5%, 0.25%-1.25%, 0.25%-1%, and 0.25%-0.75%. 0.25%~0.5%, 0.5%~4%, 0.5%~3.75%, 0.5%~3.5%, 0.5%~3%, 0.5%~2.75%, 0.5%~2.5%, 0.5%~2.25%, 0.5%~2%, 0.5%~1.75%, 0.5%~1.5%, 0.5%~1.25%, 0.5 %~1%, 0.5%~0.75%, 0.75%~4%, 0.75%~3.75%, 0.75%~3.5%, 0.75%~3%, 0.75%~2.75%, 0.75%~2.5%, 0.75%~2.25%, 0.75%~2%, 0.75%~1.75%, 0.75%~1.5% , 0.75%-1.25%, 0.75%-1%, 1%-4%, 1%-3.75%, 1%-3.5%, 1%-3%, 1%-2.75%, 1%-2.5%, 1%-2.25%, 1%-2%, 1%-1.75%, 1%-1.5%, 1%-1.25%, 1.25%-4%, 1.25%-3.75%, 1.25%-3.5%, 1.25%-3%, 1.25%-2.75%, 1.25%-2.5%, 1.25%-2.25%, 1.25%-2%, 1.25%-1.75%, 1.25%-1.5%, or any integer or fractional value within these ranges.The basic charge variant fraction is at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9 ... %, 2.8%, 2.9%, 3.0%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4.0%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5% or more, for example, up to 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15% or more.

[0050] Typically, CO2 concentration during fermentation comes from two sources: atmospheric CO2 and CO2 produced by cells via respiration. The present invention can advantageously use additional CO2 to control charge variants. Without being bound by any theory, it is believed that increasing CO2 levels in the medium leads to an increase in intracellular CO2, which alone or jointly contributes to charge variants. This effect is separate from any decrease in pH that may result from the formation of carbonic acid or other acidic chemicals.

[0051] Carbon dioxide concentration can be increased by using CO2 sparging or by reducing air sparging. CO2 sparging increases pCO2. If a decrease in carbon dioxide concentration is desired, sparging can be done with other gases including air. Air sparging and nitrogen sparging decrease pCO2. Reducing the pressure in the production bioreactor reduces the solubility of oxygen, which in turn requires greater sparging of oxygen to maintain the dissolved oxygen (DO) set point and increased gas flow rates to remove pCO2 from the culture medium.

[0052] Carbon dioxide concentration can be measured using a CO2 electrode (also called a Severinghaus electrode). More advanced systems are commercially available, such as the BioProfile® FLEX and FLEX2 analyzers. Charge variants can be measured using imaging capillary isoelectric focusing (iCIEF) and ion exchange chromatography with elution by salt gradient. NGHC can be measured by reduced capillary electrophoresis (CE)-SDS.

[0053] The present invention is suitable for use in mammalian cell culture. Exemplary cell lines are CHO, Per.C6 cells, Sp2 / 0 cells, and HEK293 cells. CHO cells include, but are not limited to, CHO-ori, CHO-K1, CHO-s, CHO-DHB11, CHO-DXB11, CHO-K1SV, and mutants and variants thereof. HEK293 cells include, but are not limited to, HEK293, HEK293A, HEK293E, HEK293F, HEK293FT, HEK293FTM, HEK293H, HEK293MSR, HEK293S, HEK293SG, HEK293SGGD, HEK293T, and mutants and variants thereof. Other suitable cells include, but are not limited to, BHK (baby hamster kidney) cells, HeLa cells, and human amniotic fluid cells, such as human amniotic fluid epithelial cells.

[0054] The present invention can be used to manufacture biological products and pharmaceuticals, including next-generation versions of existing biological products and pharmaceuticals produced in cell culture. A wide range of protein-based therapeutics, such as monoclonal antibody-based therapeutics, can be produced according to the present invention. For example, cells containing the necessary DNA sequences encoding antibodies, including but not limited to the antibodies identified below, can be grown in culture according to the present invention.

[0055] The following identifies and describes proteins made in cell culture that can be produced according to the present invention. Cells containing the necessary DNA encoding these proteins can be cultured for production according to the present invention.

[0056] For example, in antibody production, the invention is amendable for research and production use for diagnostics and therapeutics based on all major antibody classes, i.e., IgG, IgA, IgM, IgD and IgE. IgG is the preferred class, including subclasses IgG1 (including IgG1λ and IgG1κ), IgG2, IgG3, and IgG4. Further antibody embodiments include human antibodies, humanized antibodies, chimeric antibodies, monoclonal antibodies, multispecific antibodies, bispecific antibodies, antigen-binding antibody fragments, single chain antibodies, diabodies, triabodies or tetrabodies, Fab fragments or F(ab′)2 fragments, IgD antibodies, IgE antibodies, IgM antibodies, IgG antibodies, IgG1 antibodies, IgG2 antibodies, IgG3 antibodies, or IgG4 antibodies. In one embodiment, the antibody is an IgG1 antibody. In one embodiment, the antibody is an IgG2 antibody. In one embodiment, the antibody is an IgG4 antibody. In one embodiment, the antibody is a chimeric IgG2 / IgG4 antibody. In one embodiment, the antibody is a chimeric IgG2 / IgG1 antibody. In one embodiment, the antibody is a chimeric IgG2 / IgG1 / IgG4 antibody. Also included are derivatives, components, domains, chains and fragments of the above.

[0057] Further antibody embodiments include a human antibody, a humanized antibody, a chimeric antibody, a monoclonal antibody, a multispecific antibody, a bispecific antibody, a trispecific antibody, an antigen-binding antibody fragment, a single chain antibody, a diabody, a triabody or a tetrabody, a Fab or F(ab')2 fragment, an IgD antibody, an IgE antibody, an IgM antibody, an IgG antibody, an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody. In one embodiment, the antibody is an IgG1 antibody. In one embodiment, the antibody is an IgG2 antibody. In one embodiment, the antibody is an IgG4 antibody. In another embodiment, the antibody is a chimeric IgG2 / IgG4 antibody. In another embodiment, the antibody is a chimeric IgG2 / IgG1 antibody. In another embodiment, the antibody is a chimeric IgG2 / IgG1 / IgG4 antibody.

[0058] In additional embodiments, the antibody is an anti-programmed cell death 1 antibody (e.g., an anti-PD1 antibody as described in U.S. Patent Application Publication No. 2015 / 0203579A1), an anti-programmed cell death ligand 1 (e.g., an anti-PD-L1 antibody as described in U.S. Patent Application Publication No. 2015 / 0203580A1), an anti-Dll4 antibody, an anti-angiopoietin-2 antibody (e.g., an anti-ANG2 antibody as described in U.S. Patent No. 9,402,898), an anti-angiopoietin-like 3 antibody (e.g., an anti-PD-L1 antibody as described in U.S. Patent No. 9,018,356), an anti-PD-L1 antibody (e.g., an anti-PD-L1 antibody as described in U.S. Patent Application Publication No. 2015 / 0203580A1), an anti-Dll4 antibody, an anti-angiopoietin-2 antibody (e.g., an anti-ANG2 antibody as described in U.S. Patent No. 9,402,898), an anti-angiopoietin-like 3 antibody (e.g., an anti-PD-L1 antibody as described in U.S. Patent No. 9,018,356), an anti-Dll4 antibody, ... No. 9,302,015), anti-complement 5 antibodies (e.g., anti-C5 antibodies as described in U.S. Patent Application Publication No. 2015 / 0313194A1), anti-TNF antibodies, anti-epidermal growth factor receptor antibodies (e.g., anti-EGF antibodies as described in U.S. Patent Application No. 9,132,192), anti-AngPtl3 antibodies, anti-platelet derived growth factor receptor antibodies (e.g., anti-PDGFR antibodies as described in U.S. Patent No. 9,265,827), anti-Erb3 antibodies, anti-prolactin receptor antibodies (e.g., anti-PRLR antibodies as described in U.S. Patent No. 9,302,015), anti-complement 5 antibodies (e.g., anti-C5 antibodies as described in U.S. Patent Application Publication No. 2015 / 0313194A1), anti-TNF antibodies, anti-epidermal growth factor receptor antibodies (e.g., anti-EGF antibodies as described in U.S. Patent No. 9,132,192), R antibodies, or anti-EGFRvIII antibodies as described in U.S. Patent Application Publication No. 2015 / 0259423A1), anti-precursor protein convertase subtilisin kexin-9 antibodies (e.g., anti-PCSK9 as described in U.S. Patent Application Publication No. 8,062,640 or U.S. Patent Application Publication No. 2014 / 0044730A1), anti-growth and differentiation factor-8 antibodies (e.g., anti-GDF8 antibodies, also known as anti-myostatin antibodies, as described in U.S. Patent Application No. 8,871,209 or U.S. Patent Application Publication No. 9,260,515), anti-glucagon receptor agonist antibodies (e.g., anti-glucagon receptor agonist antibodies, also known as anti-glucagon receptor agonist antibodies, as described in U.S. Patent Application Publication ... Antibodies such as anti-GCGR antibodies as described in U.S. Patent Application Publication No. 2015 / 0337045A1 or U.S. Patent Application Publication No. 2016 / 0075778A1), anti-VEGF antibodies, anti-IL1R antibodies, interleukin 4 receptor antibodies (e.g., anti-IL4R antibodies as described in U.S. Patent Application Publication No. 2014 / 0271681A1 or U.S. Patent No. 8,735,095 or U.S. Patent No. 8,945,559), anti-interleukin 6 receptor antibodies (e.g., U.S. Patent Nos. 7,582,298, 8,043,617 or 9,173,880), anti-IL1 antibodies, anti-IL2 antibodies, anti-IL3 antibodies, anti-IL4 antibodies, anti-IL5 antibodies, anti-IL6 antibodies, anti-IL7 antibodies, anti-interleukin 33 (e.g., anti-IL33 antibodies as described in U.S. Patent Application Publication No. 2014 / 0271658A1 or 2014 / 0271642A1), anti-respiratory syncytial virus antibodies (e.g., anti-RSV antibodies as described in U.S. Patent Application Publication No. 2014 / 0271653A1), anti-group of differentiation 3 antibodies (e.g., anti-IL1 antibodies as described in U.S. Patent Application Publication No. 2014 / 0271653A1), anti-IL2 antibodies, anti-IL3 antibodies, anti-IL4 antibodies, anti-IL5 antibodies, anti-IL6 antibodies, anti-IL7 antibodies, anti-IL1 antibodies, anti-IL2 antibodies, anti-IL3 antibodies, anti-IL4 antibodies, anti-IL5 antibodies, anti-IL6 antibodies, anti-IL7 antibodies, anti-IL1 antibodies, anti-IL2 antibodies, anti-IL33 antibodies, anti-IL33 antibodies, anti-IL33 antibodies, anti-IL33 antibodies, anti-IL4 antibodies, anti-IL5 antibodies, anti-IL6 antibodies, anti-IL6 antibodies, anti-IL7 antibodies, anti-IL1 antibodies, anti-IL2 antibodies, anti-IL3 ... Nos. 2014 / 0088295A1 and 2015 / 0266966A1, and U.S. Application No. 62 / 222,605), anti-group 20 (e.g., anti-CD20 antibodies as described in U.S. Patent Application Publication Nos. 2014 / 0088295A1 and 2015 / 0266966A1, and U.S. Patent No. 7,879,984), anti-CD19 antibodies, anti-CD28 antibodies, anti-group 48 (e.g., anti-CD48 antibodies as described in U.S. Patent No. 9,228,014), anti-Fel d1 antibodies (e.g., as described in U.S. Patent Application Publication No. 9,079,948), anti-Middle East Respiratory Syndrome virus (e.g., anti-MERS antibodies as described in U.S. Patent Application Publication No. 2015 / 0337029A1), anti-Ebola virus antibodies (e.g., as described in U.S. Patent Application Publication No. 2016 / 0215040), anti-Zika virus antibodies, anti-lymphocyte activation gene 3 antibodies (e.g., anti-LAG3 antibodies, or anti-CD223 antibodies), anti-nerve growth factor antibodies (e.g., as described in U.S. Patent Application Publication No. 2016 / 0017029, and U.S. Patent Nos. 8,309,088 and 9,353,176), and anti-activin A antibodies. In some embodiments, the bispecific antibody is selected from the group consisting of anti-CD3 x anti-CD20 bispecific antibodies (as described in U.S. Patent Application Publication Nos. 2014 / 0088295A1 and 2015 / 0266966A1), anti-CD3 x anti-mucin 16 bispecific antibodies (e.g., anti-CD3 x anti-Muc16 bispecific antibodies), and anti-CD3 x anti-prostate specific membrane antigen bispecific antibodies (e.g., anti-CD3 x anti-PSMA bispecific antibodies). See also U.S. Patent Application Publication No. 2019 / 0285580A1. Also included are MetxMet antibodies, agonist antibodies against NPR1, LEPR agonist antibodies, BCMAxCD3 antibodies, MUC16xCD28 antibodies, GITR antibodies, IL-2Rg antibodies, EGFRxCD28 antibodies, Factor XI antibodies, antibodies against SARS-CoC-2 variants, Feld1 multi-antibody therapy, Bet v1 multi-antibody therapy. Also included are derivatives, components, domains, chains and fragments of the above.

[0059] Cells producing exemplary antibodies can be cultured according to the present invention. Exemplary antibodies include alirocumab, atortivimab, maftivimab, odesivimab, odesivumab-ebgn, casirivimab, imdevimab, cemiplimab and cemiplimab-rwlc (human IgG4 monoclonal antibodies that bind to PD-1), dupilumab (human monoclonal antibody of IgG4 subclass that binds to the IL-4R alpha (α) subunit, thereby inhibiting interleukin 4 (IL-4) and interleukin 13 (IL-13) signaling), evinacumab, evinacumab-dgnb, fasinumab, fianlimab, galetosumab, itepekimab, nesbacumab, odronextamab, pozelimab, sarilumab, trevoglumab, and raynucumab.

[0060] Additional exemplary antibodies include ravulizumab-cwvz, abciximab, adalimumab, adalimumab-atto, ado-trastuzumab, alemtuzumab, atezolizumab, avelumab, basiliximab, belimumab, benralizumab, bevacizumab, bezlotoxumab, blinatumomab, brentuximab vedotin, brodalumab, canakinumab, capromab pendetide, certolizumab pegol, cetuximab, denosumab, dinutuximab, durvalumab, eculizumab, elotuzumab, emicizumab-kxwh, entansin alirocumab, evolocumab, golimumab, guse These include rucumab, ibritumomab tiuxetan, idarucizumab, infliximab, infliximab-abda, infliximab-dyyb, ipilimumab, ixekizumab, mepolizumab, necitumumab, nivolumab, obiltoxaximab, obinutuzumab, ocrelizumab, ofatumumab, olaratumab, omalizumab, panitumumab, pembrolizumab, pertuzumab, ramucirumab, ranibizumab, raxibacumab, reslizumab, rinukumab, rituximab, secukinumab, siltuximab, tocilizumab, trastuzumab, ustekinumab, and vedolizumab.

[0061] In addition to next generation products, the present invention is also applicable to the manufacture of biosimilars, which are defined in different ways depending on the jurisdiction, but share the common feature of comparison with a previously approved biological product in that jurisdiction (usually referred to as the "reference product"). According to the World Health Organization, a biosimilar is a biotherapeutic product that is similar in terms of quality, safety and efficacy to an already licensed reference biotherapeutic product, and is used in many countries, such as the Philippines.

[0062] Biosimilars in the United States are currently described as: (A) a biological product is highly similar to a reference product, despite minor differences in clinically inactive ingredients; and (B) there are no clinically meaningful differences between the biological product and the reference product in terms of product safety, purity, and potency. In the United States, biosimilars are interchangeable or products that can be substituted for a previous product without the intervention of the healthcare professional who prescribed the previous product. In the European Union, a biosimilar is a biological drug that is highly similar to another biological drug (called a "reference drug") already approved in the EU, including consideration of structure, biological activity, efficacy, and safety, among others, with Russia following these guidelines. In China, biosimilar products currently refer to biological preparations that contain the same active substances as the original biological drug, are similar to the original drug in terms of quality, safety, and efficacy, and have no clinically significant differences. In Japan, biosimilars are currently products that have bioequivalent / qualitatively equivalent quality, safety, and efficacy compared to a reference product already approved in Japan. In India, biosimilars are currently referred to as "similar biological products", where a similar biological product is similar in terms of quality, safety, and efficacy to a reference biological product approved on the basis of comparability. In Australia, a biosimilar drug is currently a very similar version of a reference biological product. In Mexico, Colombia, and Brazil, a biosimilar is currently a biotherapeutic product similar in terms of quality, safety, and efficacy to an already licensed reference product. In Argentina, a biosimilar is currently derived from an original product (comparator) that has common characteristics. In Singapore, a biosimilar is a biotherapeutic drug similar in terms of physicochemical properties, biological activity, safety, and efficacy to an existing biological product currently registered in Singapore. In Malaysia, a biosimilar is a new biological drug developed to be similar in terms of quality, safety, and efficacy to a well-established drug currently already registered. In Canada, a biosimilar is a biological drug that is currently very similar to a biological drug already approved for marketing.In South Africa, a biosimilar is a biological medicine that is developed to be similar to a biological medicine that is currently already approved for human use. The production of biosimilars and their synonyms under these and any revised definitions can be carried out in accordance with the present invention.

[0063] Typically, the culture can be carried out for about 10 to 15 days, preferably about 12 to 14 days. The pCO2 conditions are 30 mmHg to 210 mmHg of CO2 during the culture. 2、 50mmHg-200mmHg, 60mmHg-190mmHg, 70mmHg-180mmHg, 80mmHg-170mmHg, 90mmHg-160mmHg, 100mmHg-150mmHg, 110mmHg-140mmHg, 120mmHg-140mmHg, 120mmHg-130mmHg, or any value within these ranges. The present invention can provide an Fc-containing protein product, such as an antibody, in which the main peak (considered to be approximately neutral) form accounts for 38%-65% of the total Fc-containing protein, acidic variants of the Fc-containing protein account for 20%-47% of the total Fc-containing protein, and basic variants of the Fc-containing protein account for up to 36% of the total Fc-containing protein. In the case of antibodies, the invention can provide a product in which the predominant peak form of the antibody produced by the cells accounts for 50%-70% of the total antibody, acidic variants of the antibody account for 20%-47% of the total antibody, and basic variants of the antibody account for up to 15% of the total antibody. EXAMPLES

[0064] The present invention is further described by the following examples which illustrate various aspects of the invention but are not intended to be limiting in any way.

[0065] Example 1 - In preparation of human IgG4 monoclonal antibodies that bind to the programmed cell death protein 1 (PD-1) factor, the culture pCO2 can be increased to decrease the acidic variant and increase the main peak form. In culture medium, 18 x 10 6CHO cells were inoculated at a concentration of 10 cells / ml and grown in a fed-batch process. The cells reached a peak concentration (30 × 10 6 Once the pCO2 concentration reached 100% (cells / ml), the high pCO2 bioreactor was sparged with additional CO2 to increase the pCO2 level above 120mmHg. A control process was implemented standard manufacturing process to maintain pCO2 levels below 105mmHg. See Figure 1. The acidic heterogeneity observed is represented in Table 1 below and supports a high pCO2 range of 31%-32% for the acidic charge variant and 57%-60% for the main peak form. [Table 1]

[0066] Example 2 - In the preparation of human IgG4 monoclonal antibodies that bind to PD-1 factor, the culture pCO2 can be reduced, thereby increasing the acidic variants In the medium, 18 x 10 6 CHO cells were inoculated at a concentration of 10 ... [Table 2]

[0067] This example established that low pCO2 results in a higher percentage of acidic charge variants.

[0068] Example 3 - Increasing culture pCO2 is associated with increased prevalence of NGHC in preparations of human IgG4 monoclonal antibodies that bind to the PD-1 factor In culture medium, 18 x 10 6CHO cells were inoculated at a concentration of 10 cells / ml and grown in a fed-batch process. The cells reached a peak concentration (30 × 10 6 Once the pCO2 concentration reached 100% (cells / ml), the high pCO2 bioreactor was sparged with additional CO2 to increase the pCO2 level above 120 mmHg. A control process implemented standard manufacturing processes to maintain pCO2 levels below 105 mmHg. See Figure 1. The observed NGHC heterogeneity is presented in Table 3 below. [Table 3] It was determined that the increase in NGHC was related to the decrease in culture pH, rather than an effect of pCO2 per se. See Example 5 and Figures 10 and 11.

[0069] Example 4 - Reduction in culture pCO2 is associated with a reduction in the prevalence of NGHC in a preparation of human IgG4 monoclonal antibodies capable of binding to the PD-1 factor In the medium, 18 x 10 6 CHO cells were inoculated at a concentration of 10 ... [Table 4] The decrease in NGHC was determined to be related to the increase in culture pH, rather than an effect of pCO2 per se. See Example 5 and Figures 10 and 11.

[0070] Example 5 - Analysis of culture pCO2 and pH in a small-scale study on the production of human IgG4 monoclonal antibodies capable of binding to PD-1 factor Data from a typical large-scale production run of an Fc-containing protein (eg, an antibody) using CHO cells is shown in Table 5 below. [Table 5]

[0071] A small-scale study was conducted using 2 L fermenters to replicate the large-scale production of the drug substance (FDS). Using the results of the study described herein, 、 We demonstrate that changes in culture pCO2 and pH similar to those observed in a 10,000L production bioreactor affect the charge variant profile and occurrence of non-glycosylated heavy chains in a human IgG4 monoclonal antibody that binds PD-1.

[0072] A small-scale study determined that elevated pCO2 levels in the production bioreactor caused the observed decrease in iCIEF region 1 (acidic charge variants) and region 3 (basic charge variants) and contributed to the concomitant increase in iCIEF region 2 (main peak morphology, also known as main peak variants). The study also concluded that culture pH, and not pCO2 itself, caused the observed change in the NGHC profile. These results are discussed in more detail below. Study parameters using air sparging and pCO2 sparging are outlined below in Table 6. [Table 6]

[0073] Charge variants (iCIEF) and NGHC results for each run are listed in Table 7. *Note - Moderate pCO2#3 (considered the midpoint control) was excluded from further analysis as it represents an outlier that may confound interpretation of the data. [Table 7]

[0074] The origins of the charge variants and peak morphologies, i.e., iCIEF Region 1 (acidic charge variants), Region 2 (main peak morphology), and Region 3 (basic charge variants), are discussed in more detail below. NGHC is also discussed below.

[0075] FIG. 3 shows the predicted pH values ​​using the parameters according to Table 6.

[0076] Figure 4 shows that culture pCO2 was the only significant term (p<0.0001) in the model for iCIEF region 1 (R1, %), accounting for 87% of the variability in this charge variant (R 2 Data are presented showing that higher pCO2 was the only statistically significant term (acidic charge variant) associated with lower Region 1 (%) (P < 0.05). Culture pH was not a statistically significant term for the acidic charge variant (Region 1). See Figure 5.

[0077] FIG. 6 shows that both culture pCO2 and pH were significant terms (p<0.0001) in the model for iCIEF region 2 (R2, % main peak shape), accounting for 97% of the observed variability (R 2 : 0.97). Thus, higher culture pCO2 and lower culture pH increase the main peak form. See FIG. 7.

[0078] Figure 8 shows data indicating that culture pCO2 was a significant term in the model (p=0.0352), explaining 38% of the variability in Region 3 (R3, % basic charge variants). However, the model was not significant (p=0.0592), likely due to overleveraging of data points. See Figure 9.

[0079] The above data show that, in general, charge variants are caused in whole or in part by increasing pCO2 levels. More importantly, increasing pCO2 was the only statistically significant item for reducing the percentage of acidic charge variants, but not decreasing pH (region 1). Thus, for the IgG class, represented here by human IgG4 monoclonal antibodies, increasing pCO2 reduces the percentage of acidic charge variants, and the reduction in the percentage of acidic charge variants is not caused by decreasing pH values. See Figures 4 and 5.

[0080] Finally, Figures 10 and 11 show that decreased culture pH, but not increased pCO2 per se, was a significant term in the model (p=0.0401), accounting for 38% of the variability in NGHC (R 2 Data show that a lower culture pH (pH 7.0:0.38) can therefore affect the NGHC profile. Although pCO2 can affect pH, other medium components also affect pH and therefore pH alters NGHC, regardless of the cause. Thus, the lower acidic charge variants (%) are due to pCO2 itself, which is a different phenomenon than an increase in NGHC caused by a decrease in pH by any type of acidic molecule.

[0081] 12-23 show the following data: (a) Viable cell density (VCD) (Figure 12), (b) survival rate values ​​(Figure 13); (c) pH value - pH changed from day 6.5 according to the zone approach shown in Table 6 (Figure 14); (d) pCO2 values ​​- pCO2 changed from day 6.5 onwards according to the zone approach shown in Table 6 (Figure 15) (e) Glucose level (Figure 16), (f) Potassium level (Figure 17), (g) Sodium levels - Changes in sodium levels after day 7 may be due to changes in the sensor and were not expected to affect the study results (Figure 18). (h) Osmolality values ​​- Atypical values ​​on day 10 may be due to sample errors (Figure 19). (i) Glutamate values ​​- Atypical values ​​on day 6.5 may be due to sampling error (Figure 20), (j) Lactate value (Figure 21), (k) Ammonia levels - Ammonia levels may have been affected by pH (Figure 22); and (l) Glutamine levels (Figure 23).

[0082] These data show similarities between cells grown under different air sparging conditions, see Table 6.

[0083] Example 6 - Production of a human IgG4 monoclonal antibody that binds to the interleukin 4 (IL-4) receptor in culture using CO2 sparging The following study was conducted to evaluate the effect of culture pCO2 on the charge variant profile of a human IgG4 monoclonal antibody that binds to the IL-4R alpha (α) subunit, thereby inhibiting interleukin-4 (IL-4) and interleukin-13 (IL-13) signaling.

[0084] Approximately 12 × 10 CHO cells were added to the culture medium in the production bioreactor. 5 The cells were inoculated at a concentration of 200 × 10 cells / ml and grown in a fed-batch process. 5 Once the peak viable cell density (VCD) in cells / mL was reached, the CO sparging was modified as defined in Table 8 to vary the pCO levels within the cell culture. The resulting pCO profiles of the three experimental conditions are provided in FIG. [Table 8]

[0085] After 10.5 days of culture, the bioreactor was harvested and the monoclonal antibody was purified. Glycosylation and charge variant profiles were determined. It was noted that as pCO2 levels increased in the production bioreactor, there was a concave decrease in the level of basic variants as measured by imaging capillary isoelectric focusing (iCIEF) (Table 9). Furthermore, increasing pCO2 led to a concave acidic variant profile, peaking at moderate pCO2 conditions but declining to the lowest percentage at high pCO2 conditions (Table 10). The overall trend was a lower percentage of acidic charge variants, with the moderate pCO2 measurements likely being the result of error. [Table 9] [Table 10]

[0086] Detailed statistical analysis in Example 5 above confirmed that increasing pCO2 without decreasing pH was the only statistically significant item for reducing the percentage of acidic charge variants (region 1) by human IgG4 monoclonal antibodies. Thus, for the IgG class represented by human IgG4 monoclonal antibodies, an increase in pCO2 itself reduces the percentage of acidic charge variants, and the reduction in the percentage of acidic charge variants in IgG4 antibodies is not caused by a decrease in pH value.

[0087] It should be understood that the description, specific examples and data, while indicating exemplary embodiments, are given by way of illustration and are not intended to limit the invention. From the discussion, disclosure and data contained herein, various changes and modifications within the invention, including combining the embodiments in whole and in part, will become apparent to those skilled in the art and are therefore considered part of the invention.

Claims

1. 1. A method for reducing the percentage of acidic charge variants in an antibody product produced by mammalian cells in culture, said method comprising: seeding the culture medium with mammalian cells that produce the antibody; The mammalian cells are ２ The pCO2 allows for the production of antibody products with fewer acidic variants than would be obtained without the conditions. ２ and culturing the cells under conditions, wherein the pCO ２ The conditions are: 120 mmHg to 140 mmHg of CO in the medium; ２ and culturing the cell culture medium, wherein the cell culture medium is a soluble form of the cell culture medium.

2. The pCO ２ 10. The method of claim 1, wherein the conditions are achieved by sparging or CO2 sparging.

3. The pCO ２ The antibody produced under the conditions is ２ 10. The method of claim 1, having 0.5% to 4% less acidic variants than that obtained without the conditions.

4. The method of claim 1 , wherein the antibody is a monoclonal antibody.

5. The method of claim 1, wherein the antibody is capable of binding to PD-1 or IL-4 receptor.

6. The method of claim 4 , wherein the antibody is a human monoclonal antibody.

7. The method of claim 6 , wherein the human monoclonal antibody is an IgG antibody.

8. The method of claim 7, wherein the IgG antibody is an IgG4 antibody.

9. The method of claim 1, wherein the cells are cultured for 10 to 15 days.

10. 2. The method of claim 1, wherein the mammalian cell is a CHO cell.

11. 1. A method for controlling heterogeneity in antibodies produced by mammalian cells in culture, said method comprising: seeding the culture medium with mammalian cells that produce the antibody; pCO2 that enables the mammalian cells to produce antibodies ２ and culturing the cells under conditions wherein the major peak form of the antibody produced by the cells accounts for 38% to 65% of the total antibody, the acidic variants of the antibody account for 20% to 47% of the total antibody, and the basic variants of the antibody account for up to 36% of the total antibody.

12. The method of claim 11 , wherein the antibody is a monoclonal antibody.

13. The method of claim 12, wherein the monoclonal antibody is capable of binding to PD-1 or IL-4 receptor.

14. The method of claim 12, wherein the monoclonal antibody is a human monoclonal antibody.

15. The method of claim 14, wherein the human monoclonal antibody is an IgG antibody.

16. 16. The method of claim 15, wherein the IgG antibody is an IgG4 antibody.

17. 1. A method for controlling heterogeneity in antibodies, antibody derivatives, or antibody fragments produced by mammalian cells in culture, said method comprising: seeding the culture medium with mammalian cells that produce the antibody, antibody derivative, or antibody fragment; pCO2 that enables the mammalian cells to produce antibodies, antibody derivatives, or antibody fragments. ２ and culturing the cells under conditions wherein a predominant peak form of the antibody, antibody derivative or antibody fragment produced by the cells accounts for 50% to 70% of the total antibody, antibody derivative or antibody fragment, acidic variants of the antibody, antibody derivative or antibody fragment account for 20% to 47% of the total antibody, antibody derivative or antibody fragment, and basic variants of the antibody, antibody derivative or antibody fragment account for up to 15% of the total antibody, antibody derivative or antibody fragment.

18. 18. The method of claim 17, wherein the basic variant of the antibody, antibody derivative, or antibody fragment comprises at most 10%, at most 8%, or at most 6% of the total antibody, antibody derivative, or antibody fragment.

19. 18. The method of claim 17, wherein the major peak form of antibody, antibody derivative or antibody fragment produced by the cells accounts for 50% to 65% of the total antibody, antibody derivative or antibody fragment, and the acidic variant of the antibody, antibody derivative or antibody fragment accounts for 23% to 46% of the total antibody, antibody derivative or antibody fragment.

20. 18. The method of claim 17, wherein the acidic variant of the antibody, antibody derivative, or antibody fragment comprises 23% to 39% or 31% to 46% of the total antibody, antibody derivative, or antibody fragment.

21. 18. The method of claim 17, wherein the percentage of antibodies with non-glycosylated heavy chains is 5-7%.

22. 18. The method of claim 17, wherein the mammalian cells produce a human monoclonal antibody.

23. 23. The method of claim 22, wherein the human monoclonal antibody is an IgG antibody.

24. 24. The method of claim 23, wherein the IgG antibody is an IgG4 antibody.

25. The pCO ２ 18. The method of claim 17, wherein the conditions are between 30 mmHg and 210 mmHg during the culture.

26. The pCO ２ The condition is CO ２ 26. The method of claim 25, maintained using sparging.

27. pCO ２ But CO ２ 18. The method of claim 17, wherein the measurement is performed using electrodes.

28. 18. The method of claim 17, wherein the mammalian cell is a CHO cell.

29. 20. An antibody product, antibody derivative product, or antibody fragment product produced by the method of any one of claims 1, 11, or 17.

30. pCO ２ 30. The method of claim 29, wherein increases upon reaching a peak cell concentration.

31. Once peak cell concentration is reached, the presence of acidic variants is reduced by increasing pCO2 in the medium, said increasing to a range selected from the group consisting of: 50mmHg to 200mmHg during culture, 60mmHg to 190mmHg during culture, 70mmHg to 180mmHg during culture, 80mmHg to 170mmHg during culture, 90mmHg to 160mmHg during culture, 100mmHg to 150mmHg during culture, 110mmHg to 140mmHg during culture, 120mmHg to 140mmHg during culture, and 120mmHg to 130mmHg during culture; 12. The method of claim 11, wherein the range is 100 mmHg to 150 mmHg during culture, or the range is 110 mmHg to 140 mmHg during culture.

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