CN113549667A - Method for reducing galactosylation of antibody - Google Patents
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Abstract
The invention provides a method for reducing galactosylation of an antibody, and relates to the technical field of biology. The method comprises the following steps: using a galactose analogue selected from the group consisting of compounds of formula (1) or a biologically acceptable salt thereof, wherein R is1、R2、R4Each independently selected from one of halogen or-OH; r3Is selected from-OH or-OAc. The method of the invention adds galactose analogue with a certain concentration in the cell culture process, can effectively and stably regulate galactosylation, and controls the proportion of each glycoform to be near a target level.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a method for reducing galactosylation of an antibody.
Background
Glycosylation is one of the most common post-translational modifications of proteins, and is the transfer of carbohydrate molecules to specific locations of proteins by glycosyltransferases. Starting at the endoplasmic reticulum of the cell and ending at the cell golgi apparatus. Common glycosylation modifications are mannosylation, fucosylation, galactosylation, and sialylation. The structure and composition of the sugar chain significantly affect the stability, safety and effectiveness of the protein.
The monoclonal antibody is taken as a typical glycoprotein therapeutic drug, the glycosylation of an Fc segment of the monoclonal antibody is an important index for the posttranslational modification of an antibody protein, and the physiological activity of the monoclonal antibody in vivo is regulated by 2 independent mechanisms: targeting and neutralizing antigens or triggering apoptosis, as well as antibody Fc effector functions, including antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC).
It was found that the Fc fragment effector function of the antibody is associated with the oligosaccharide chains located in this region. Galactosylation is one of the common types of glycosylation, which also has an effect on the activity of the antibody. Although it does not appear to affect antibody binding to antigen, it has been reported that increasing the proportion of IgG antibody galactose modification significantly enhances CDC effects. Also, an increase in galactose may increase antibody sialylation levels, and an increase in sialylation may increase the half-life of the glycoprotein in serum; however, an increase in galactose also reduces the ADCC effect, and therefore, reducing galactose as an antibody may prevent the ADCC effect from being reduced. This requires the cell line to be obtained in the cell construction or the development of a process that can regulate galactosylation of the antibody to a suitable level during cell culture.
In addition, in the development process of antibody biological similar drugs, the product quality requirements which are more consistent with those of the original research drugs are often required to be maintained. Glycoforms, one of the key quality attributes of antibodies, needs to be controlled at a level relatively similar to that of the original drug, while galactosylation is one of the glycosylation modifications that can affect the ratio of multiple glycoforms in the glycospectrum, e.g., G0F, G1F, G2F, G1, etc. Therefore, in the development of the upstream process, a suitable cell culture process needs to be developed to regulate the glycoform of the antibody so as to ensure that each glycoform in the glycoform is comparable to the original drug.
The glycoforms in the common antibody glycoform spectrum are Man5, G0F, G1F, G2F, G0, G1, and the like. In the production process of the antibody, the cell strain, cell culture process parameters, culture medium components and the like used can all influence the galactosylation of the monoclonal antibody. Therefore, galactosylation can be regulated and controlled by modifying cell strains, optimizing cell culture process parameters, optimizing culture medium components and the like.
In the prior art, the galactosyl group is mainly regulated and controlled by the following means: (1) in the cell construction, the coding gene of key enzyme such as galactosyltransferase and the like in the galactosylation modification process is knocked out by adopting a genetic engineering technology so as to reduce the galactosylation ratio or reduce the activity of the key enzyme; (2) optimizing cell culture media composition, e.g. by adding Mn2+、Zn2+Plasma; (3) changing process parameters in cell culture, such as reducing Dissolved Oxygen (DO) control during cell culture (Kunkel et al, 1998), adjusting the pH control range during cell culture (Muthining et al, 2003), reducing the culture temperature during culture (Sou et al, 2015); the addition of glutamine-alanine dipeptide (Ala-Gln) during the culture process reduces the galactosylation ratio; the whole acetyl galactose (alpha-2-F whole acetyl galactose or beta-2-F whole acetyl galactose) is added in the culture process to reduce galactosylation ratio. The main drawbacks of these methods are: methods for knocking out or down-regulating expression of key enzyme genes at the gene level: on one hand, the technology may relate to the related gene technology patent barriers, and on the other hand, the difficulty coefficient for controlling galactosylation to a target level is large, and the development period is long. Optimizing the components of the culture medium: not only limited by commercial media, but also limited in regulatory effect, and possibly affecting other quality attributes. Changing process parameters in cell culture: the DO control is reduced, the cell growth, the product expression and the product quality control are not facilitated, and the control is difficult to be stably carried out in the amplification production; pH control is not a common regulation method, as there may be large differences between different cells and different antibody products, and therefore the regulation effect is uncertain; reducing the culture temperature can obviously reduce the growth and metabolic rate of cells, can obviously reduce the yield of protein, and has limited regulation and control effect on the semi-lactosylation; addingGlutamine dipeptide (Ala-Gln): because the additive is an amino acid dipeptide, the effect of the additive is greatly different in different culture media, and on the other hand, the additive can be involved in amino acid metabolism and can cause the accumulation of ammonium in the metabolism, thereby influencing the growth and the metabolism of cells and even influencing certain quality attributes of antibodies; the whole acetyl galactose has negative influence on cell growth, metabolism and protein expression at the concentration higher than 60. mu.M, and the process stability is unknown, and it is uncertain whether commercial production can be carried out. Therefore, it is necessary to provide a new method for controlling galactosyl group of antibody.
In view of this, the invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a method for reducing galactosylation of antibodies, which can effectively and stably regulate galactosylation by adding a galactose analogue with a certain concentration in the cell culture process and control the ratio of each glycoform to be close to a target level.
The technical scheme provided by the invention is as follows:
a method of reducing galactosylation of an antibody comprising: using a galactose analog selected from the group consisting of compounds of formula (1) or a biologically acceptable salt thereof, in culturing cells expressing the antibody:
wherein R is1、R2And R4Each independently selected from one of halogen or OH; r3Selected from-OH and-OAc.
The method is based on the fact that in the presence of galactose analogs, the galactose analogs can be combined with UDP to form a complex, compete with UDP-Gal for the combination sites of galactosyltransferase, so as to inhibit the activity of the enzyme and block galactosylation modification, and therefore, during the cell culture process, a certain concentration of galactose analogs are added to reduce the level of galactosylation modification, so as to regulate the proportion of each glycoform in an N-glycospectrum.
In one embodiment, the halogen includes F, Cl, Br, and I.
In one embodiment, the galactose analogue comprises 2-deoxy-2-fluoro-D-galactose (2FG), 4-deoxy-4-fluoro-D-galactose (4FG) and/or 6-deoxy-6-fluoro-D-galactose (6 FG).
In a preferred embodiment, the galactose analogue is 2-deoxy-2-fluoro-D-galactose (2 FG). The structural formula of the D-galactose is shown in the formula A), and the structural formula of the 2-deoxy-2-fluoro-D-galactose is shown in the formula B).
In one embodiment, the antibody is a known monoclonal antibody; preferably, the monoclonal antibodies include rituximab, adalimumab, trastuzumab, omalizumab, bevacizumab and ranibizumab.
In one embodiment, the antibody comprises IgG1, IgG2, IgG4, preferably IgG 1.
In one embodiment, the galactose analogue is added on the day of inoculation of the cells expressing the antibody during the reactor culture phase, and/or on days 3 to 4 after inoculation.
In one embodiment, the galactose analog is added to a final concentration of 50 to 200. mu.M, including but not limited to 50. mu.M, 60. mu.M, 70. mu.M, 80. mu.M, 90. mu.M, 100. mu.M, 110. mu.M, 120. mu.M, 130. mu.M, 140. mu.M, 150. mu.M, 160. mu.M, 170. mu.M, 180. mu.M, 190. mu.M and 200. mu.M; preferably, the galactose analogue is added to a final concentration of 100. mu.M.
In the method of the invention, the concentration and time of adding 2FG can influence the reduction of galactosylation, and the invention optimizes the time and concentration of adding, so that the proportion of each glycoform of the produced antibody can be regulated to be basically consistent with the level of a reference substance.
In one embodiment, prior to the reactor culturing, further comprising a seed cell culture, the seed cell culture being performed in a basal medium free of the galactose analog.
In one embodiment, the culturing is batch culturing, fed-batch culturing, enhanced fed-batch culturing, and perfusion culturing; in fed-batch culture, feeding is periodically performed, for example, in a 14-day culture period, and feeding medium is fed on days 0, 3, 4, 5, 6, 8, 10, and 12 of the culture.
In a specific embodiment, in the fed-batch culture method, the seed cells are cultured at 0.5-5 × 106cells/mL are inoculated into the reactor at a density of 0.9. + -. 0.1X 10 for culture6The density of (3) is inoculated.
In one embodiment, during the culturing, the glucose addition is performed according to the daily residual glucose concentration. Sampling daily during the culture process to detect the density of living cells and biochemical value of cell metabolism, and adding glucose with 300g/Kg of glucose mother liquor according to daily glucose residual concentration so as to maintain normal glucose metabolism of the cells in the culture.
In a specific embodiment, the seed cells are cultured in a basal medium without 2FG (containing 4mM glutamine) at 0.5. + -. 0.1X 106cells/mL were inoculated for amplification and cultured on a shaker. When the seed density reaches 4.5-6.5 × 106culturing and inoculating 3L or 10L reactor at cell/mL with inoculation density of 0.9 + -0.1 × 106cells/mL (culture in 10L reactor requires dissolved oxygen control). The initial culture temperature is 36.5 ℃, and the temperature is reduced when the cell density reaches 15-19X 106cells/mL to continue the culture. Feeding the culture medium on 0, 3, 4, 5, 6, 8, 10 and 12 days of culture for 14 days.
In one embodiment, the scale-up culture can also be performed in a 500L disposable reactor, where the culture requires dissolved oxygen control and DO is maintained at 50%.
Has the advantages that:
(1) the method provided by the invention is simple, convenient to operate and short in process development period, and can effectively and stably regulate galactosylation, so that the proportion of each glycoform is controlled to be near the target level;
(2) the influence on cell growth and metabolism, antibody yield and other quality attributes except glycoform is very small;
(3) the developed cell culture process method is stable and reliable and can be successfully amplified to the production scale;
(4) the method reduces the galactosylation modification level, thereby regulating and controlling the proportion of each glycoform in the N-glycoform, mainly comprising the steps of reducing the proportion of G1F and G2F and improving the proportion of G0F in the glycoform.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a schematic representation of the intracellular galactosylation pathway (among others, Glucose-Glucose; Galactose-Galactose; Glc-6P-Glucose phosphate; Gal-1P-1 Galactose phosphate; UDP-Glc-uridine diphosphate Glucose; UDP-Gal-uridine diphosphate Galactose; GalT-galactosyltransferase; UDP-Glc4-epimerase: uridine diphosphate Glucose-4 isomerase);
FIG. 2 is a graph showing the growth of cells in the 3L reactor culture of example 1;
FIG. 3 is a graph showing the cell metabolism in the 3L reactor culture in example 1;
FIG. 4 is a graph showing the expression of the antibody in the 3L reactor culture in example 1;
FIG. 5 is a graph showing the growth of cells in the culture of a 10L reactor in example 2;
FIG. 6 is a graph showing the expression of the antibody in the culture in the 10L reactor in example 2;
FIG. 7 is a graph showing the growth of cells in a 500L reactor in example 3;
FIG. 8 is a graph showing the expression profile of the antibody in the 500L reactor in example 3.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Experimental materials:
1. CHO cell line (CHOK1SV, Lonsa, usa) stably expressing a monoclonal antibody against sclerostin, GS screening system;
2. commercial basal medium (Thermo, usa) and feed medium (GE, usa);
3.2FG, 2-deoxy-2-fluoro-D-galactose (Carbosynth, UK)
4. Reference (Lily clinical stock solution standard, USA)
The detection method comprises the following steps:
1. and (3) live cell density detection: viable CELL density assays were performed using Vi-CELL (Beckman, Germany);
2. and (3) detecting the biochemical value of cell metabolism: detecting biochemical values of cell metabolism such as glucose, lactic acid, ammonia, IgG, etc. during culture with Cedex Bio (Roche, Switzerland);
3. detecting the content of the antibody in the culture process: detecting the antibody content during the culture process by Cedex Bio (Roche, Switzerland);
n-glycoform detection: the N-oligosaccharide of glycoprotein is released by PNGaseF enzyme, the released N-oligosaccharide is fluorescently labeled by a 2-AB labeling reagent, the oligosaccharide is separated by adopting ultra-high performance liquid chromatography-hydrophilic chromatography, the analysis is carried out under the conditions that a fluorescence detector lambda ex is 330nm and lambda em is 420nm, and the proportion of each peak is analyzed by adopting an area normalization method. The oligosaccharide species of the sample proteins were identified by LC-MS system (Thermo, USA) using the fluorescence chromatograms obtained by mass-to-charge ratio and retention time detection.
The technical principle is as follows: FIG. 1 shows the intracellular galactosylation pathway, and generally, galactosylation modification of a protein occurs in the Golgi of a cell, and uridine diphosphate galactose (UDP-Gal) is transferred to a sugar chain by the action of galactose transferase (GalT). This plays a crucial role in the process for the substrates uridine diphosphate galactose and galactosyltransferase, where the substrate UDP-Gal has two origins, one is the de novo synthetic pathway and one is the salvage synthetic pathway, and in the presence of a galactose analog it will bind to UDP to form a complex, competing with UDP-Gal for the galactosyltransferase binding site, thereby inhibiting the enzyme activity and blocking galactosylation modification.
Based on the above principle, during cell culture, galactose analogs such as 2FG (2-deoxy-2-fluoro-D-galactose), 4FG (4-deoxy-4-fluoro-D-galactose), and 6FG (6-deoxy-6-fluoro-D-galactose) are added at a certain concentration to reduce galactosylation modification level, thereby controlling the ratio of each glycoform in the N-glycogram.
Example 1.3L fed-batch culture Process in a reactor
3L reactor culture process:
seed cells (CHO cell line stably expressing a monoclonal antibody against sclerostin) were cultured in batches by fed-batch in a 3L reactor (Thermo-Finesse G3lab, USA).
The culture was carried out in commercial basal medium (Thermo, USA) without 2FG (containing 4mM glutamine) at 0.5. + -. 0.1X 106cells/mL density inoculation for amplification, cultured in shaker (Infors, Switzerland) for 3 days at 110rpm, 36.5 deg.C, CO2Concentration 5% and humidity 80%.
When the density of the seed cells reaches 4.5-6.5 multiplied by 106cells/mL, 3L reactor culture inoculation with inoculation density of 0.9 + -0.1 × 106cells/mL, initial working volume 1.6L. The initial culture temperature is 36.5 ℃, and the cell density reaches 15-19 multiplied by 106The cells/mL are cooled for continuous culture.
On day 4 after inoculation, 2FG was added to 4 of the reactors at final concentrations of 50. mu.M, 100. mu.M, 150. mu.M, and 200. mu.M, respectively, to the control group without 2FG, and to the other reactor on day 0 after inoculation, 2FG was added at a final concentration of 100. mu.M. Feeding the culture medium on 0, 3, 4, 5, 6, 8, 10 and 12 days of culture for 14 days.
Sampling daily during the culture process to detect the density of living cells and biochemical value of cell metabolism, and adding glucose by using 300g/Kg of glucose mother liquor according to the daily residual concentration of glucose so as to maintain the normal glucose metabolism of the cells in the culture. On the 14 th day of culture, the cell culture broth was centrifuged to collect the supernatant, which was sent to the analysis department for analysis of antibody quality.
3L reactor test results:
FIG. 2 is a graph showing the growth of cells in a 3L reactor culture, FIG. 3 is a graph showing the metabolism of cells in a 3L reactor culture, and FIG. 4 is a graph showing the expression of antibodies in a 3L reactor culture. In the 3L reactor test it can be seen that even at final concentrations of 2FG up to 200. mu.M, there is little effect on cell growth, metabolism and antibody production.
In reducing galactosylation, the effect of reducing galactosylation is related to the concentration and time of 2FG addition. The addition of 2FG at day 0 of the culture was superior to the addition at day 4, the addition at day 0 at a concentration of 100. mu.M reduced the galactosylation ratio by about 8.8% and the addition at day 4 reduced the ratio by 6.1%; the galactosylation ratio was reduced by about 4.8%, 6.1%, 6.8%, 7.7% at the addition of 2FG concentrations of 50. mu.M, 100. mu.M, 150. mu.M and 200. mu.M at day 4 of the culture, respectively.
The ratio of each glycoform of the produced antibody was controlled to a level substantially consistent with that of the control reference by adding 100. mu.M 2FG on the 0 th day of culture.
TABLE 1.3 summary of antibody glycoform results in L reactor culture
Example 2.10L fed-batch culture in a bioreactor
The culture process of the 10L reactor comprises the following steps:
seed cells were cultured at 0.9. + -. 0.1X 106cells/mL were inoculated into a 10L glass reactor (Thermo-repair G3lab, USA) and a 10L SUB, i.e., disposable bioreactor (GE-XDR10, USA) for fed-batch culture.
Dissolved oxygen control for reactorThe pH value is controlled to be 6.9 +/-0.3 at 50 percent. The stirring speed of the 10L glass reactor was 112-130rpm, and the stirring speed of the 10L SUB disposable reactor was 75-85 rpm. The initial culture temperature is 37 ℃, and the cell density reaches 15-19 multiplied by 106The cells/mL are cooled for continuous culture.
On the day of inoculation (day 0), 100. mu.M of 2FG was added, and the feed medium was fed on days 0, 3, 4, 5, 6, 8, 10, and 12 of culture for 14 days.
Sampling daily during the culture process to detect the density of living cells and biochemical value of cell metabolism, and adding glucose with 300g/Kg of glucose mother liquor according to daily glucose residual concentration so as to maintain normal glucose metabolism of the cells in the culture. After the cell fluid is harvested after the culture is finished, the stock solution obtained after downstream purification is taken to a quality analysis department for analysis of antibody quality.
10L reactor test results:
FIG. 5 is a graph showing the cell growth in a 10L reactor culture, and FIG. 6 is a graph showing the expression of antibodies in a 10L reactor culture. It can be seen that the confirmation of the process on the 3L reactor was performed on 2 different types of 10L reactors, and the results showed that the process was more reproducible and scalable, cell growth and antibody expression were essentially consistent, and the N-sugar profile of the antibody was essentially consistent with the control reference.
TABLE 2.10L antibody glycoform results in reactor culture
Example 3.3 batch production Scale (500L SUB) Fed-flow fed-batch culture Process
The culture was carried out in 3 batches on a 500L disposable reactor (GE, USA). The cultivation process was as in example 2, with the reactor parameters set to: the stirring speed is 90-105rpm, the dissolved oxygen is controlled at 50 percent, and the pH is controlled at 6.9 +/-0.3. 100 μ M of 2FG was added on the day of inoculation. The culture period is 14 days, after the culture is finished, the cell sap is purified downstream to obtain stock solution, and then the stock solution is sent to a QC department for antibody quality analysis.
FIG. 7 is a graph showing the cell growth curve in a 500L reactor culture, and FIG. 8 is a graph showing the expression of the antibody in a 500L reactor culture. As shown in fig. 7, fig. 8 and table 3, 3 batches of 500L scale cells grew and antibody expression were consistent, and the N-sugar profile was also substantially consistent with the control reference. This indicates that the process is very robust.
TABLE 3.10L antibody glycoform results in reactor culture
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (10)
1. A method of reducing galactosylation of an antibody comprising: using a galactose analog selected from the group consisting of compounds of formula (1) or a biologically acceptable salt thereof, in culturing cells expressing the antibody:
wherein R is1、R2And R4Each independently selected from one of halogen or-OH; r3Is selected from-OH or-OAc.
2. The method of claim 1, wherein the halogen comprises F, Cl, Br, and I.
3. The method of claim 1, wherein the galactose analog comprises 2-deoxy-2-fluoro-D-galactose, 4-deoxy-4-fluoro-D-galactose, and/or 6-deoxy-6-fluoro-D-galactose.
4. The method of claim 1, wherein the antibody is a monoclonal antibody; preferably, the monoclonal antibodies include rituximab, adalimumab, trastuzumab, omalizumab, bevacizumab and ranibizumab.
5. The method of claim 1, wherein the galactose analogue is added on the day of inoculation of the antibody-expressing cells during the reactor culture stage, and/or on days 3-4 after inoculation.
6. The method according to claim 1, wherein the galactose analogue is added to a final concentration of 50 to 200 μ M.
7. The method according to claim 1, wherein the galactose analogue is added to a final concentration of 80 to 100. mu.M.
8. The method of claim 1, wherein the galactose analog is added to a final concentration of 100 μ Μ.
9. The method of claim 5, further comprising, prior to the reactor culturing, a seed cell culture in a basal medium free of the galactose analog.
10. The method according to any one of claims 1 to 9, wherein the culture is batch culture, fed-batch culture, enhanced fed-batch culture, and perfusion culture; feed-stream fed-batch culture is preferred.
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