JP2005509403A - Control of glycoforms in IgG - Google Patents

Abstract

  The present invention provides a method of controlling the level of non-glycosylated heavy chain variants of IgG in a CHO cell culture process by adjusting temperature and batch medium osmotic pressure.

Description

Detailed Description of the Invention

This application claims the benefit of US Provisional Application No. 60 / 279,026, filed March 27, 2001.
(Field of Invention)

The present invention relates to a method for controlling glycoforms in recombinantly produced IgG.
(Technology of the Invention)

  Monoclonal antibodies (IgG isotype) are produced using various expression systems. Most of the expression systems used to produce monoclonal antibodies are mammalian and include host systems such as Chinese hamster ovary (CHO), hybridoma and myeloma cells or their derivatives.

  Antibodies differ significantly in glycosylation pattern from other recombinant proteins. Glycosylation tends to be highly conserved in IgG molecules with a single N-linked biantennary structure at Asn297. This is buried between the CH2 domains. It then makes sufficient contact with amino acid residues in CH2. Typically, recombinantly produced IgG has heterogeneous processing of the core oligosaccharide structure attached at the Asn297 site, and the IgG exists in multiple glycoforms. In contrast, non-IgG proteins produced in CHO cells can have multiple N-linked glycosylation sites. For example, there is interferon-γ (IFN-γ), which has two glycosylation sites, Asn25 and Asn97, and human tissue plasminogen activator (t-PA), which has three sites, Asn117, Asn184 and Asn448.

  IgG N-linked glycoforms can differ by site occupancy at the Asn site, i.e., macroheterogeneity, or by variations in oligosaccharide structure at the glycosylation site, i.e., microheterogeneity. This includes variations in sugar residues, oligosaccharide lengths, or the number of branches that form the oligosaccharide (biantennary and triantennary structures). See Jenkins, Parekh, James, (1996) Nature Biotechnology, 14: 975-981.

  More specifically, glycoforms produced in the CHO cell process are observed in three major variations in terminal oligosaccharide form in structure, structure length and non-glycosylated heavy chain (NGHC). NGHC formation in the CHO system can be associated with glucose deficiency as has been demonstrated in other cell culture systems. Stark, Heath, (1979) Archives of Biochemistry and Biophysics, 192 (2): 599-609, Turco, (1980) Archives of Biochemistry and Biophysics, 205 (2): 330-339 and Davidson, Hunt, (1985) Journal of General Virology, 66: 1457-1468.

  Glycoform regulation in IgG preparations was observed under conditions different from those described in the present invention by several researchers. For example, galactosylation was reduced in IgG produced by a murine B-lymphocyte hybridoma cell line cultured in low dissolved oxygen. See Kunkel, Jan, Butler, Jamieson, (2002) Biotechnol. Prog., 16: 462-470. In other IgG-producing hybridoma lines, various glycosylation patterns were obtained by culture methods in either ascites or serum-free or serum-supplemented media. See Patel, Parekh, Moellering, (1992) Biochem. J., 285: 839-845.

  US Pat. No. 5,705,364 describes a method for controlling the sialic acid content in glycoproteins produced in mammalian host cells such as CHO by controlling temperature and osmotic pressure in the presence of alkanoic acid. Is described.

  For non-IgG proteins, culture pH and ammonia concentration affected glycosylation of recombinant mouse placental lactogen protein produced in CHO cells. See Borys, Linzer, Papoutsakis, (1993) Bio / Technology, 11: 720-724 and Borys, Linzer, Papoutsakis, (1993) Biotechnology and Bioengineering, 43: 505-514. In CHO-producing human follicle stimulating hormone, changing the steady state of dissolved oxygen and increasing specific productivity by adding butyrate was consistent with an increase in sialic acid content. See Chotigeat, Watanapokasin, Mahler, Gray, (1994) Cytotechnology, 15: 217-221. Glycosylation of the artificial N-glycosylation recognition site in recombinant hu-IL-2 produced by BHK-2 was affected by ammonia and glucosamine. Large differences were observed in sialylation, proximal fucosylation and antennaity. See Gawlitzek, Valley, Nimtz, Wagenr, Conradt, Animal Cell Technology: Developments towards the 21st Century, 379-384. Site occupancy in CHO producing tissue plasminogen activator increased with longer batch cultures and was also affected by butyrate and temperature. See Anderson, Bridges, Gawlitzek, Hoy, (2000) Biotechnology and Bioengineering, 70: 25-31.

  It has been shown that glycosylation of IFN-γ produced in CHO is sensitive to multiple culture factors. In batch culture, the non-glycosylated form increased from 3-5% of total IFN-γ at 3 hours to 30% at 195 hours. See Curling, Hayter, Baines, Bull, Gull, Strange, Jenkins, (1990) Biochem. J. 272: 333-337. Long-term culture has also been shown to increase oligomannose and truncated structures with Asn97. See Hooker, Goldman, Markham, James, Ison, Bull, Strange, Slamon, Baines, Jenkins, (1995) Biotechnology and Bioengineering, 48: 639-648. It was also shown that ExCyte, a lipid supplement, affects the percentage of fully glycosylated IFN-γ in the culture. Also, glycosylation was improved by partially replacing bovine serum albumin (BSA) in the medium with fatty acid-free BSA. See Jenkins, Castro, Menon, Ison, Bull, (1994) Cytotechnology, 15: 209-215. It has also been suggested that Lutrol F68 may affect IFN-γ glycosylation. See Castro, Ison, Hayer, Bull, (1995) Biotechnol. Appl. Biochem. 21: 87-100. Primateone RL, an animal tissue hydrolyzate, affects the sialylation of IFN-γ in batch and additive modes. See Gu, Zie, Harmon, Wang, (1997) Biotechnology and Bioengineering, 56: 353-360. Glucose restriction has been suggested to affect IFN-γ site occupancy due to reduced nucleotide biosynthesis. See Nyberg, Balcarcel, Follstad, Stephanopoulos, Wang, (1998) Biotechnology and Bioengineering, 62: 336-347.

  The contribution of oligosaccharide side chains to IgG function was greatly debated. One important function of the carbohydrate structure is in the complement binding pathway, and the degree of glycosylation was directly related to antibody-dependent cytotoxic activity (ADCC) and complement mobilization. See Jeffries, Lund, Pound, (1998) Immunology Review, 163: 50-76. Fc receptor binding was decreased in chimeric mouse-human IgG that was aglycosylated. See Tao, Morrison, (1989) The Journal of Immunology, 143: 2595-2601. Carbohydrate structure also affects protein folding, oligomer assembly and secretion, and in vivo clearance of glycoproteins. Some glycoforms can be antigenic, so regulatory authorities are requiring increased analysis of carbohydrate structure in recombinant proteins. See Jenkins, Parekh, James, (1996) Nature Biotechnology, 14: 975-981. In addition, regulatory agency requirements for therapeutic IgG require consistency in product preparation. Thus, there is a need for a process that minimizes the production of multiple glycoforms of IgG protein.

(Summary of Invention)
One aspect of the present invention is a method for controlling the level of IgG NGHC in a cell culture, the method comprising adjusting the culture temperature of the culture.
Another aspect of the invention is a method of controlling the level of IgG NGHC in a cell culture, comprising adjusting the batch medium osmotic pressure of the culture.
Another aspect of the invention is a method of controlling the level of IgG NGHC in a cell culture, comprising adjusting the culture temperature and the batch medium osmotic pressure of the culture.

(Detailed description of the invention)
All publications cited herein, including but not limited to patents and patent applications, are hereby incorporated by reference as if fully set forth.

  The present invention provides a method of controlling the level of IgG aglycosylated heavy chain (NGHC) in a cell culture process that produces recombinant monoclonal antibodies. By adjusting the culture temperature, adjusting the batch medium osmotic pressure, or adjusting both the temperature and batch medium osmotic pressure of a cell culture such as a production culture, the level of NGHC can be reduced or increased. The batch media osmotic pressure of the final seed culture used to seed the production culture can be arbitrarily adjusted to be comparable to that of the production culture.

  In one embodiment of the invention, NGHC can be reduced by raising the temperature of the cell culture. Similarly, NGHC can be increased by lowering the temperature of the cell culture. Preferred temperatures for the process are in the range of about 33 ° C to about 35 ° C.

  In another embodiment of the invention, NGHC can be reduced by lowering the batch medium osmotic pressure in the cell culture. Similarly, NGHC can be increased by increasing the batch culture osmotic pressure of the cell culture. Preferred osmotic pressure for the process is in the range of about 285 mOsm to about 417 mOsm.

  In another embodiment of the invention, NGHC levels can be reduced by a combination of high cell culture temperature and low batch medium osmotic pressure.

  In yet another embodiment of the invention, NGHC levels can be increased by combining the low temperature of the cell culture and the high batch medium osmotic pressure.

  The process of the present invention provides a level of NGHC control at any temperature within the preferred range, provided that the batch medium osmotic pressure is constant. Similarly, if the temperature is kept constant, NGHC can be controlled to a constant level at any batch medium osmotic pressure within the preferred range.

  The invention will now be described with reference to the following detailed non-limiting examples.

(Example)
Medium formulation For Examples 1-6, the medium composition consisted of a defined formulation containing amino acids, salts, trace elements, and vitamins similar to those described in PCT International Publication No. WO 92/05246. . Yeast hydrolyzate such as TC yeastate was added in an amount of 5 g / L or 10 g / L. Additional glucose was added, either 4.5 g / L or 9 g / L. Fructose ferric or ferric EDTA, recombinant insulin and lipid mixture were added to the medium. Sodium bicarbonate was added to the batch medium as a buffer, and methotrexate was added to the batch medium as a selective agent to maintain recombinant protein expression. Surfactant Lutrol F68 was also added to the batch medium. Batch medium osmotic pressure for seed and production bioreactors is described in each example. The osmotic pressure of the batch medium was adjusted by adding NaCl and KCl. During culture in the bioreactor, sodium carbonate was added when pH control was required. The medium was sterilized by either 0.1 or 0.2 micron filtration.

  In Example 7, the above-described exclusive medium was used. In addition, the cells were adapted to two commercial media CD-CHO (Invitrogen, Rockville, MO) and EX-CELL 325 (JRH Biosciences, Renexa, Kansas). It was. All cultures were then evaluated for NGHC production at various temperatures and batch medium osmotic pressure.

Scale-up and production conditions for Examples 1-5 Data were obtained by culturing recombinant CHO cell lines producing IgG1 anti-CD4 monoclonal antibodies in shake flasks and 3 L Applikon bioreactors. This antibody is described as CE9.1 in US Pat. No. 6,136,310. Shake flask scale-up cultures were grown in medium with an osmotic pressure in the range of 370-380 mOsm and shaken at 37 RPM in a 5% carbon dioxide incubator at 150 RPM. Cells were passaged on a 3-4 day schedule at a target seeding concentration of 600,000 viable cells / mL. On the passage day, the cells were trypsinized and counted for% viability using a trypan blue exclusion method and a hemocytometer, and for total cell number using a ZM Coulter Counter.

  To maintain dissolved oxygen at a settling value, as soon as there is a request, while spraying O2 at 5 mL / min, or to control the pH to a settling band of 6.9-7.0, go to the headspace on demand. A 3 L seed bioreactor was operated at 37 RPM and 200 RPM while sparging 100% carbon dioxide at 5 mL / min or adding 1.5 M Na2CO3 on demand. The bioreactor had a constant 20 mL / min air overlay.

  A 3 L production bioreactor was operated in the same manner as the seed reactor except for different temperatures and pH settling values depending on the examples. Every 1 or 2 days, the cells in the seed and production bioreactors are trypsinized and counted for% viability using the trypan blue exclusion method and a hemocytometer, and the total cell number for the ZM Coulter Counter. Was counted using. Batch medium osmotic pressure was as detailed in each example.

Scale-up and production conditions of Example 6 Shake flask scale-up culture of the recombinant cell line was performed as described above. The cells were then scaled up to an 80 L seed bioreactor with a 17 L culture volume. After culturing with 48 mmHg of dissolved oxygen at 37 ° C. and pH 6.9-7.0 for several days, the bioreactor was fed up to 71 L of fresh medium. After culturing with 48 mmHg dissolved oxygen at 37 ° C. and pH 6.9-7.0 for several days, the culture 71 L was used to inoculate a 750 L ABEC seed bioreactor with a culture volume of 300 L. After culturing with dissolved oxygen of 48 mmHg at 37 ° C. and pH 6.9-7.0 for several days, the 300 L was used to inoculate a 1500 L ABEC production bioreactor having a culture volume of 1200 L. The production reactor was operated at 48 mm Hg dissolved oxygen at the temperature and pH 6.9-7.0 detailed in the examples. The batch media osmotic pressure for the 80 L seed reactor was 370-380 mOsm. Batch medium osmotic pressure for the 750 L ABEC seed reactor and the 1500 L ABEC production reactor were as detailed in the examples.

Scale-up and production conditions of Example 7 Recombinant cell line shake flask scale-up cultures were shaken at 37 RPM in a 5% carbon dioxide incubator at 150 RPM. Cells were passaged on a 3-4 day schedule at a target seeding concentration of 800,000 VCC / mL. On the passage day, the cells were trypsinized and counted for% viability using trypan blue exclusion and a hemocytometer, and for total cell number using a Z1 Coulter Counter.

  Cell vials were thawed and scaled up using the above medium for 4 passages. This medium was used to scale up a subset of these cells and the two other parts were adapted to two commercial media CD-CHO (Invitrogen) and EXCELL-325 (JR Biosciences) I let you. In-house medium versus commercial medium was used in the following proportions for 5 passages: 100/0, 50/50, 0/100. The batch media osmotic pressure for scale-up and adaptation was 358 mOsm for autologous media and 353 mOsm for commercial media. For the last seed passage at 37 ° C., the cells in each medium were subcultured in the same medium batched at low, medium and high osmotic pressure. These were 310, 358 and 405 mOsm for the autologous medium, respectively. These were 300, 353 and 405 mOsm, respectively, for the commercial medium. At the end of this last seed passage, each medium / osmotic flask was used to inoculate a similar medium / osmotic production shake flask. The production flask for each medium / osmotic treatment was then incubated at 33 ° C, 34 ° C or 35 ° C. Production cultures were collected when viability was about 60% or less. This occurred after about 14-17 days of culture. The flask was monitored for residual glucose to ensure that glucose was not deficient on the day of collection.

Analysis for NGHC levels In Examples 1-5, cells were filtered from production cultures. For each batch, the product was captured and concentrated on a protein A affinity column. The product eluate was then analyzed for NGHC% by densitometric scanning of a reduced SDS-PAGE gel. NGHC was reported as a percentage of the total number of heavy chains or as a percentage of the total number of heavy and light chains detailed in each example.

  In Example 6, cells were collected from the production culture. For each batch, the product was captured and concentrated on an affinity column and then processed by two additional chromatographic steps. The final product was then analyzed for NGHC% by densitometric scanning of an SDS-PAGE gel.

  In Example 7, cells were collected from the production culture. For each batch, the product was captured and concentrated on an affinity column. The product eluate was then analyzed for NGHC% by capillary SDS-PAGE separation performed on a microcapillary array using an Agilent Bioanalyzer.

Example 1
Effect of culture temperature on NGHC formation First, the effect of culture temperature on NGHC formation in cultures was observed in a 3 L Applikon bioreactor with a 2 L culture volume. Standard process settings for the production reactor were a temperature of 34 ° C., pH 6.9-7.0 and 30% dissolved oxygen. The factorial test was designed to assess the effects on seed culture quality, culture temperature, pH and dissolved oxygen titer. The quality of the seed cultures was varied by changing the seeding concentration in the seed reactor and the age of the seed material from that used in a typical seed reactor. The combinations of factors tested and the resulting NGHC levels as a percentage of heavy chain are shown in Table 1. Statistical analysis showed that the culture temperature was statistically significant in the formation of NGHC (p <0.002). The adjusted R-sq is 61.5%, indicating that the temperature only accounts for some of the NGHC variations. FIG. 1 shows linear regression.

Example 2
Effects of culture temperature and batch medium osmotic pressure on NGHC formation The effects of culture temperature and batch medium osmotic pressure on titer and NGHC formation were measured. Culture temperatures between 33.4 ° C and 34.2 ° C and osmotic pressures of 340mOsm, 370mOsm and 400mOsm were evaluated in a 3L bioreactor with 2L culture volume. NGHC as a percentage of heavy chain is shown in Table 2. The data confirms the effect of the culture temperature described in Example 1 on NGHC formation and suggests that high batch medium osmotic pressure contributes to NGHC formation. IVC represents the integral of live cells.

  All cultures ended with glucose left, indicating that increased NGHC levels were not correlated with glucose deficiency. FIG. 2 shows NGHC versus temperature and osmotic pressure.

Example 3
NGHC enrichment and reduction by manipulation of culture temperature and batch medium osmotic pressure In an attempt to enrich and minimize NGHC based on the data obtained in Example 1 and Example 2, Four 3 L bioreactors with culture volumes were operated at various temperatures (33 ° C, 33.2 ° C, 35 ° C and 34.4 ° C) and batch medium osmotic pressure (340, 370 and 400 mOsm). NGHC levels as a percentage of heavy chain are shown in Table 3.

Example 4
Confirmation of conditions producing low NGHC Eight 3 L bioreactors with a 2 L culture volume were operated at temperatures between 34.2 ° C. and 35.0 ° C. and batch media osmotic pressure of 285 mOsm, 304 mOsm or 331 mOsm. The NGHC for all batches was less than 3.5%. NGHC as a percentage of total protein is shown in Table 4.

Example 5
Further evaluation of culture temperature and batch medium osmotic effect on NGHC formation Twenty-two 2L bioreactors were raised between 33.6 ° C and 34.4 ° C in increments of 0.2 ° C and between 337mOsm and 417mOsm. Six batch medium osmotic pressures were operated. NGHC is shown in Table 5 as a percentage of total protein. The data show that the glucose deficiency the day after or on the last day of culture did not correlate with high NGHC levels.

^*生産培養の最終日の翌日または最終日のグルコースレベル
^**標準的なプロセス条件についての内部対照

^* Glucose level the day after or on the last day of production culture
^** Internal controls for standard process conditions

The data from Example 4 was combined with the data from Example 5 for statistical analysis. From this analysis, the following regression equation:
NGHC = 3.13-2.57 * (temperature) + 1.89 * (osmotic pressure) -4.8 (temperature * osmotic pressure)
was gotten. P <0.001 for culture temperature, batch medium osmotic pressure, and interaction of culture temperature and batch medium osmotic pressure. Degree of freedom corrected R ² = 87.8% and overall p = 0.000. This model is shown graphically in FIG. 3 and anticipates that NGHC can be maintained at a constant level at temperatures between 34.6 ° C. and 34.7 ° C. Similarly, with a batch media osmotic pressure of 316 mOsm, NGHC can be maintained at a constant level for all temperatures between 33.5 ° C and 35.1 ° C.

Example 6
Increasing culture temperature and decreasing batch medium osmotic pressure reduce NGHC formation at the 1200 L pilot plant scale.
It has been shown that NGHC can be reduced in 1200 L bioreactor cultures by increasing the culture temperature and decreasing the batch medium osmotic pressure. The standard production bioreactor culture temperature was 33.9 ° C., and the osmotic pressure for the seed flask, seed bioreactor and production reactor was 370-380 mOsm. In two of the 20 batches, the culture temperature of the production bioreactor was raised to 34.5 ° C. and the batch medium osmotic pressure was lowered to 305-315 mOsm. For these two batches, the batch osmotic pressure of the final 750 L seed bioreactor was also lowered to 305-315 mOsm. The average NGHC for the 18 standard batches was 5.7%. The NGHC values as a percentage of total protein for the two “low NGHC” batches were 1.7% and “not detected”. FIG. 4 shows NGHC levels as a percentage of total protein in all batches.

Example 7
Effect of culture temperature and batch medium osmotic pressure on NGHC formation in autologous medium and two commercial cell culture media Autogenous medium and two commercial media CD-CHO (Invitrogen) and EXCELL-325 by adjusting temperature and osmotic pressure (JR H Biosciences) was used to show that NGHC can be reduced. For each medium, three temperatures and three osmotic pressures were evaluated in shake flasks. The temperatures were 33 ° C, 34 ° C and 35 ° C. The osmotic pressure for the proprietary media was 310, 358 and 405 mOsm. The osmotic pressure for the commercial medium was 300, 353 and 405 mOsm. Table 6 and FIG. 5 showed the same tendency as that shown in the above examples. At low temperatures, NGHC formation as a percentage of total protein is high and sensitive to batch medium osmotic pressure compared to medium and high temperatures. Glucose was not deficient under any of the test conditions.

  The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics, and therefore, reference should be made to the appended claims rather than the foregoing specification as indicating the scope of the invention. It is.

FIG. 1 is a graph of experimental results showing the effect of culture temperature on NGHC formation. FIG. 2 is a graph of experimental results showing the effects of culture temperature and batch medium osmotic pressure on NGHC formation. FIG. 3 is a graph of statistical regression of experimental results predicting the effect of culture temperature and batch medium osmotic pressure on NGHC formation. FIG. 4 is a graph of experimental results showing NGHC reduction in two 1200 L pilot plant batches with increasing culture temperature and decreasing batch medium osmotic pressure. FIG. 5 is a graph of experimental results showing the reduction of NGHC using autologous media and two commercial media by manipulating temperature and batch media osmotic pressure.

Claims (14)

  A method of controlling the level of IgG non-glycosylated heavy chain (NGHC) produced in a mammalian cell culture, comprising adjusting the temperature of the culture.   A method of controlling the level of IgG NGHC produced in a mammalian cell culture comprising adjusting the batch medium osmotic pressure of the culture.   A method of controlling the level of IgG NGHC produced in a mammalian cell culture comprising adjusting the temperature of the culture and the batch medium osmotic pressure.   4. The method according to claim 1, 2 or 3, wherein the cell culture host cell is a Chinese hamster ovary (CHO) cell.   The method of claim 4, wherein the cell culture is a production culture.   5. The method of claim 4, wherein the level of NGHC is decreased by lowering the batch medium osmotic pressure and increasing the temperature.   5. The method of claim 4, wherein the level of NGHC is reduced by reducing batch medium osmotic pressure.   5. The method of claim 4, wherein the level of NGHC is increased by increasing batch medium osmotic pressure.   5. The method of claim 4, wherein the level of NGHC is increased by decreasing the temperature and increasing the batch medium osmotic pressure.   5. The method of claim 4, wherein the level of NGHC is decreased by increasing the temperature.   The method of claim 4, wherein the level of NGHC is increased by lowering the temperature.   The method of claim 4, wherein the temperature of the culture is adjusted to about 33 ° C to about 35 ° C.   The method of claim 4, wherein the osmotic pressure is adjusted from about 285 mOsm to about 417 mOsm. The method of claim 4, wherein the temperature of the culture is adjusted to about 33 ° C to about 35 ° C and the osmotic pressure is adjusted to about 285 mOsm to about 417 mOsm.

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