WO2019077628A1 - Zinc supplementation for decreasing galactosylation of recombinant glycoproteins - Google Patents

Abstract

The present invention relates to a process for modulating glycoform of recombinant glycoproteins by altering the trace element composition of a culture medium. Further, the present invention provides a culture medium supplemented with trace elements for modulating the glycoform of recombinant glycoproteins.

Description

ZINC SUPPLEMENTATION FOR DECREASING GALACTOSYLATION OF

RECOMBINANT GLYCOPROTEINS FIELD OF THE INVENTION

The present invention relates to a process for modulating glycoform of recombinant glycoproteins by altering the trace element composition of a culture medium. More particularly, the present invention relates to a process for decreasing galactosylation of IgG expressed in recombinant host cells by supplementing zinc to a culture medium.

Further, the present invention provides a culture medium supplemented with trace elements for modulating the glycoform of recombinant glycoproteins.

BACKGROUND OF THE INVENTION

N-Glycosylation is the attachment of oligosaccharides onto proteins at specific conserved asparagine residues. The process of glycosylation involves sequential action of glycosidases and glycosyltransferases in Endoplasmic Reticulum and Golgi. Glycosylation can affect various effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and serum half-life. N-Glycosylation of therapeutic antibodies is an important product quality attribute that affects their effector functions. An optimal and consistent glycoform is hence desirable.

The ability to control glycosylation is believed to be essential for reproducibly achieving desirable properties of therapeutic proteins. Hence, optimal and consistent glycoform profile is desired. Trace element composition of culture media is one of the critical factors that modulate glycoform.

Zinc is an important trace metal used in various cell culture media. Zinc has been used as an insulin replacement in the development of serum-free culture media for cells including myeloma, hybridoma, and CHO-K1 cells.

A few patent applications disclose use of trace metals in regulating glycosylation/ galactosylation of recombinant proteins. Of particular relevance here is US Patent Publication No.2017/0058309 which teaches an increase in galactosylation of recombinant glycoproteins.

US Patent Publication No. 2014/0271622 discloses that glycan distribution was affected by the addition of copper to the medium. An increased copper led to a decrease in the level of galactosylation. Zinc supplementation at unspecified concentrations were found to have no impact on G0F (McCracken, N. A., R. Kowle, et al. (2014). "Control of galactosylated glycoforms distribution in cell culture system." Biotechnology progress 30(3): 547-553).

However, from the aforesaid prior art documents it is unclear whether the presence of zinc at higher concentrations can influence galactosylation of recombinant protein. However, there is no direct reference or suggestion in the prior art literature to a method of controlling or decreasing galactosylation using zinc

Further, chemically defined cell culture medium is increasingly used for bioprocessing and may be more susceptible to the effect of lot-to-lot variability in trace metal concentrations. Trace elements present as impurities in raw materials can cause variability in their final concentration in the culture medium and thereby in glycosylation.

The current focus of the drug industry is the development of monoclonal antibodies (mAbs) as candidates for therapeutics for various diseases. In this context, various processes for the production of mAbs and bio similars are being proposed. In the synthesis of bio similar, it may be critical to match the glycoform of the biosimilar to the innovator mAb. OBJECTS OF THE INVENTION

It is an object of the present invention to provide a process for decreasing the galactosylation of recombinant glycoproteins using zinc supplementation.

It is another object of the present invention to provide a process to nullify the effect of variability in zinc concentration on galactosylation by manganese supplementation.

SUMMARY OF THE INVENTION

To accomplish the objectives of the present invention, the inventors have provided a process for decreasing galactosylation of recombinant glycoproteins, viz. antibodies expressed in an expression system by supplementing a culture medium with Zinc, the said process comprising:

(i) culturing the expression system expressing recombinant glycoproteins in a culture medium; and

(ii) supplementing the culture medium of step (i) with zinc in a concentration ranging β-οιη 30μΜ ΐο 200 μΜ.

In a further aspect, the present invention provides a culture medium supplemented with zinc in concentrations ranging from 75 μΜ to 150 μΜ for decreasing the galactosylation of IgG expressed in CHO cells. BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS

Fig. 1 depicts the effect of Zn²⁺ on CHO cell growth, metabolism, and IgG glycoform profile. Cells were seeded at 3 x 10⁶ cells/ml density in medium containing 1.5 μΜ Zn²⁺ and supplemented with additional ZnCl₂ at different concentrations, (a) Growth profile, viable cell density is indicated by solid lines and filled symbols, percentage viability is indicated by dashed lines and hollow symbols, without Zn²⁺ supplementation (□), 30 μΜ Zn²⁺ (o), 100 μΜ Zn²⁺ (0), 150 μΜ Zn²⁺ (Δ), and 200 μΜ Zn²⁺ (x). (b) Specific glucose consumption was calculated between days 1 and 3. (c) IgG titer at harvest relative to un-supplemented culture. (d, e) Representative MALDI-TOF MS spectra of permethylated glycans of IgG harvested from cultures without and with 150 μΜ Zn²⁺ supplementation, respectively, (f) Average relative glycan abundance (%) analyzed by MALDI-MS. n = 4 for without (white bars) and withl50 μΜ (gray bars) Zn²⁺ supplementation; n = 1 for 30 μΜ (diagonal lines) and 100 μΜ (horizontal lines) Zn²⁺ supplementation. Glycan structure representation: GlcNAc, black square; Man, gray circle; Gal, white circle; Fuc, dark gray triangle, (g) Galactosylation percentage, (h) Normalized galactosyltransferase activity in CHO cell lysates from cultures with and without 150 μΜ Zn²⁺ supplementation, n = 3. Activity in each cell lysate was normalized to its protein content. Relative activity normalized to average activity in the CHO cell lysates from cultures without additional Zn²⁺ supplementation. Error bars indicate + 1 standard deviation;

Fig. 2 depicts the effect of Zn²⁺ supplementation on galactosylation in the presence of galactose and/or uridine supplementation. Cultures were seeded at 3 x 10⁶ cells/ml density and supplemented with 30 mM galactose (Gal), 1 mM uridine (Urd), and 30 mM galactose + 1 mM uridine. White bars indicate without and gray bars indicate in presence of 150 μΜ Zn²⁺. Galactosylation percentage calculated as described in methods section, n = 2. Error bars indicate + 1 standard deviation;

Fig. 3 depicts the effect of increasing Zn²⁺ or Mg²⁺ on galactosyltransferase activity in CHO cell lysate. Effect of increasing ratio of metal ions Zn²⁺or Mg²⁺ to Mn²⁺ on galactosyltransferase activity in CHO cell lysate was evaluated. Zn²⁺(A) or Mg²⁺(n) concentrations were varied from 0 to 1 mM in the presence of fixed Mn²⁺ at 1 mM concentration, n = 2. Error bars indicate + 1 standard deviation;

Fig. 4 depicts the effect of 150 μΜ Zn²⁺ supplementation on CHO cell growth and IgG glycosylation in the presence of 4 μΜ Mn²⁺. Cultures were seeded at 3 x 10⁶ cells/ml density and supplemented with 4 μΜ MnCl₂, with and without 150 μΜ ZnCl₂. (a) Growth profile, viable cell density is indicated by solid lines and filled symbols, percentage viability is indicated by dashed lines and hollow symbols, cultures without Zn²⁺ supplementation (□), with Zn²⁺ supplementation (Δ). (b) IgG titer at harvest relative to un- supplemented culture, (c) Average relative glycan abundance (%) analyzed by MALDI-MS. White bars indicate without and gray bars indicate in presence of 150 μΜ Zn²⁺. Glycan structure representation: GlcNAc, black square; Man, gray circle; Gal, white circle; Fuc, dark gray triangle, (d) Galactosylation percentage, n = 2. Error bars indicate + 1 standard deviation;

Fig. 5 depicts effect of Zn²⁺ on CHO cell growth and IgG glycoform profile in commercial chemically defined CD CHO medium. Cells were seeded at 0.5 x 10⁶ cells/ml density in CD CHO medium and supplemented with 150 μΜ Zn²⁺, 4 μΜ Mn²⁺, and 4 μΜΜη²⁺ + 150 μΜ Zn²⁺. NS indicates non- supplemented conditions (white bars). Cultures were harvested on day 4. (a) Growth profile, viable cell density is indicated by solid lines and filled symbols, percentage viability is indicated by dashed lines and hollow symbols, NS (□), 150 μΜ Zn²⁺ (Δ), 4 μΜ Mn²⁺ (o), 4 μΜ Mn²⁺ +150 μΜ Zn²⁺ (0). (b) IgG titer at harvest relative to un- supplemented culture, (c) Average relative glycan abundance (%) analyzed by MALDIMS. NS (white bars), 150 μΜ Zn²⁺ (dark gray bars), 4 μΜ Mn²⁺ (diagonal lines), and 4 μΜ Mn²⁺ + 150 μΜ Zn²⁺ (horizontal lines). Glycan structure representation: GlcNAc, black square; Man, gray circle; Gal, white circle; Fuc, dark gray triangle, (d) Galactosylation percentage, n = 1. In figures b, c, and d, error bars indicate standard deviation of analytical replicates Fig. 6 depicts total intracellular content of Mn²⁺ and Zn²⁺ in CHO cells cultured without and with additional Mn²⁺ and Zn²⁺ supplementation. CHO cells were inoculated in medium containing 1 nM and 4 μΜ Mn²⁺ and cultured without (white bars) and with additional 150 μΜ Zn²⁺ (dark gray bars). Metal ion content was measured by MP-AES. (a) Total intracellular Zn²⁺ content (ng/mg of protein), (b) Total intracellular Mn²⁺ content (ng/mg of protein), n = 3. Error bars indicate + 1 standard deviation

Fig. 7 depicts the effect of Zn²⁺ on high mannose glycans of IgG from cells cultured with galactose in the presence of 4 μΜ Mn²⁺. Cells were seeded at 3 x 10⁶ cells/ml density with 30 mM galactose as carbon source and supplemented with 4 μΜ Mn²⁺ and 4 μΜ Mn²⁺+ 150 μΜ Zn²⁺. (a) Growth profile, viable cell density indicated by solid lines and filled symbols, percentage viability indicated by dashed lines and hollow symbols, with 4 μΜ Mn²⁺ (□), with 4 μΜ Mn²⁺+150 μΜ Ζη²⁺(Δ). (b) IgG titer at harvest relative to unsupplemented culture with galactose as carbon source, (c) Average relative glycan abundance (%) analyzed by MALDI-MS. White bars indicate with 4 μΜ MnCl₂ and gray bars indicate in presence of 4 μΜ Mn²⁺ and 150 μΜ Zn²⁺. Glycan structure representation: GlcNAc, black square; Man, gray circle; Gal, white circle; Fuc, dark gray, n = 2. Error bars indicate + 1 standard deviation

Fig. 8 depicts the effect of 75 μΜ Zn²⁺ on CHO cell growth and IgG glycoform profile; Cells were seeded at 3 x 10⁶ cells/ml density in medium containing 1.5 μΜ Zn²⁺ and supplemented with additional 75 μΜ ZnCl₂ (a) Growth profile, viable cell density is indicated by solid lines and filled symbols, percentage viability is indicated by dashed lines and hollow symbols, without Zn²⁺ supplementation (o) 75 μΜ Zn²⁺ (x). (b) Average relative glycan abundance (%) analyzed by MALDI-MS. n = 1 for without (white bars) and with 75 μΜ Zn²⁺ (gray bars) supplementation (c) Galactosylation percentage.

DETAILED DESCRIPTION OF THE INVENTION

The invention will now be described in detail in connection with certain preferred and optional embodiments, so that various aspects thereof may be more fully understood and appreciated.

The present invention provides a process for decreasing galactosylation of therapeutic antibodies expressed in an expression system by altering the concentrations of trace elements in the cultivation/fermentation medium.

In a preferred embodiment, the present invention provides a process for decreasing galactosylation of IgG expressed in CHO cells by supplementing a culture medium with Zinc in concentrations ranging from 30 μΜ to 200 μΜ.

In another embodiment of the present invention, the process for decreasing galactosylation of IgG expressed in CHO cells by supplementing a culture medium with Zinc in concentrations ranging from 75 μΜ to 150 μΜ.

The present invention provides a process for decreasing galactosylation of IgG expressed in an expression system, the said process comprising;

(i) culturing the expression system expressing antibodies in a culture medium; and

(ii) supplementing the culture medium of step (i) with zinc in a concentration ranging β-οιη 30μΜ ΐο 200 μΜ.

More preferably, the concentration of zinc supplemented in the cultivation medium is in an amount ranging from 75 μΜ to 150 μΜ.

In yet another embodiment of the present application, the expression system is CHO cells.

In another embodiment, the present invention provides a culture medium is a chemically defined culture medium or a serum free culture medium. In still another embodiment of the present invention, the zinc is supplemented in form is a zinc salt.

Yet another embodiment of the present invention provides that the zinc salt is selected from the group consisting of zinc chloride, zinc sulphate, zinc carbonate, and zinc chromate. A further embodiment of the present invention provides that the process comprises addition of Mn²⁺ in concentration ranging from ΙμΜ to 5 μΜ to the culture medium for overcoming the effect of lot-to-lot variability in trace element concentrations on galactosylation.

In another embodiment of the present invention, there is provided a culture medium for decreasing galactosylation in the production of IgG in CHO cells, said culture medium comprising zinc in a concentration ranging from 75 μΜ to 150 μΜ.

The cultures of CHO cells producing IgG were supplemented with ZnCl₂ at concentrations ranging from 30 to 200 μΜ (Fig. 1). Cell growth was adversely affected at Zn²⁺ concentrations beyond 100 μΜ and cell viability decreased rapidly at 200 μΜ (Fig. la). Cultures with 200 μΜ Zn²⁺ were not further evaluated. Specific glucose consumption calculated between days 1 and 3 was higher with 100 and 150 μΜ Zn²⁺ supplementation (p = 0.03) (Fig. lb).

Glycoform analysis revealed a decrease in galactosylation above 100 μΜ Zn²⁺ in a dose-dependent manner (Fig. If). Galactosylation decreased from 30 to 26 and 19% (p = 0.001) with 100 μΜ Zn²⁺ and 150 μΜ Zn²⁺ supplementation respectively (Fig. lg). Galactosyltransferase activity measured in lysates of cells treated with and without 150 μΜ Zn²⁺ supplementation in the presence of 1 mM Mn²⁺ is not significantly different (Fig. lh) indicating that galactosyltransferase expression level has not changed with Zn²⁺ supplementation.

Galactosidases released from cells could result in extracellular cleavage of galactose from the glycan. However, CHO galactosidase in culture supernatant has been demonstrated to have optimum activity at pH 4 and very little activity near neutral pH. Hence, the decrease in galactosylation with Zn²⁺ supplementation is also not likely to be due to extracellular effect of galactosidase.

Zn²⁺ supplementation at concentrations above 100 μΜ decreases galactosylation of IgG expressed in CHO cells. Galactosyltransferase catalyzes the transfer of galactose to the precursor glycan using UDP-Gal as the nucleotide sugar donor.

The decrease in galactosylation can thus either be due to reduced galactosyltransferase expression or activity or reduced availability of UDP-Gal. Zn supplementation had no effect on galactosyltransferase expression. It decreased galactosylation even in cultures with galactose and uridine supplementation which is known to increase intracellular UDP-Gal concentration (Fig. 2). This suggests that reduced galactosylation may not be due to limitation of UDP-Gal with Zn²⁺ supplementation and may hence be due to decrease in galactosyltransferase activity.

Mn²⁺ is an essential cofactor required for optimal activity of galactosyltransferase. Measurement of galactosyltransferase activity in CHO cell lysate showed a decrease in activity with increasing Zn²⁺/Mn²⁺ ratio (Fig. 3).

In another preferred embodiment, the cultures of CHO cells producing IgG are supplemented with ZnCl₂ at various concentrations from 75 μΜ to 150 μΜ. Concentration of Zn²⁺ in the basal medium is 1.5 μΜ.

The effect of varying Zn²⁺/Mn²⁺ on galactosyltransferase activity in cell lysate prepared by detergent lysis of CHO cells cultured under control conditions, i.e., with 1 nM Mn²⁺ and 1.5 μΜ Zn²⁺ was evaluated. Galactosyltransferase activity was measured in presence of varying Zn²⁺/Mn²⁺ ratio where Mn²⁺ concentration was held constant at 1 mM concentration and Zn²⁺ was varied between 0 and 1 mM. Galactosyltransferase activity decreased with increasing Zn^2+/Mn²⁺ ratio in a dose dependent manner (Fig. 3). At equimolar concentrations of Mn²⁺ and Zn²⁺, activity reduced to 15%. To evaluate whether decrease in activity was specific to Zn²⁺, the effect of another divalent cation, magnesium, was also similarly evaluated. Mg²⁺ at equimolar concentrations did not have any effect on enzyme activity. At higher extracellular Zn²⁺ concentration, galactosyltransferase activity may be reduced due to change in intracellular distribution of Zn²⁺ and Mn²⁺ and their availability in the Golgi. It was therefore hypothesized that increasing extracellular Mn²⁺ concentration could reduce effect of Zn²⁺ on galactosylation.

In an embodiment, the present invention discloses that Galactosylation is lower with zinc supplementation even with supplementation of galactose and uridine.

Accordingly, Fig. 2 shows Galactosylation is reduced from 42% to 28% with 150 μΜ Zn²⁺ supplementation (p = 0.02) in presence of galactose while in presence of uridine, 150 μΜ Zn²⁺ supplementation decreased galactosylation from 34% to 26% (p = 0.02). Simultaneous feeding with galactose and uridine improved galactosylation from 19% to 28% in the presence of Zn²⁺ (p = 0.01). However, galactosylation levels remain lower than that observed in cultures without Zn²⁺ supplementation (43%, p = 0.004). In yet another embodiment, the invention suggests that the effect of Zn on galactosylation of IgG is substantially reversed with Mn²⁺ supplementation as shown in Fig. 4 and Fig. 5.

In a further embodiment, the invention relates to the robustness of effect of Zn²⁺ on galactosylation levels to changes in medium composition.

Fig. 5 depicts the effect of Zn²⁺ on galactosylation is robust to differences in medium formulation.

In another embodiment, the present invention discloses a process to overcome increase in high mannose glycan by manganese in presence of galactose as carbon source with Zn²⁺ supplementation.

Accordingly, Fig. 7 shows the effect of 150 μΜ Zn²⁺ supplementation to cells cultured with 4 μΜ Mn²⁺ with galactose as carbon source.

In yet another preferred embodiment, the present invention provides a culture medium supplemented with zinc in concentrations ranging from 75μΜ to 150 μΜ for decreasing the galactosylation of IgG expressed in CHO cells.

EXAMPLES

Following examples are given by way of illustration and therefore should not be construed to limit the scope of the invention.

Example 1: Materials and methods employed in the present invention

Details of animal cell cultures used: Deposition and procurement details

The suspension adapted CHO-DG44 (Chinese hamster ovary) based cell line expressing IgG was provided under an MTA from Inbiopro Solutions (Bangalore, India). Materials: CD CHO medium was purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA) and custom made DMEM:F12 based medium from HiMedia Laboratories (Mumbai, India). All chemicals were obtained from Sigma Aldrich (St. Louis, MO, USA) unless otherwise specified. Protein A Agarose-CL for IgG purification was purchased from Merck Millipore Pvt. Ltd. (Mumbai, India). PNGase F enzyme was purchased from New England Biolabs (Ipswich, MA, UK).

The DMEM:F12 based medium has following components in mg/L: Glycine-37.6, L-Alanine-8.9, L-Arginine hydrochloride-294.5, L-Asparagine monohydrate-15, L-Aspartic acid-13.3, L-Cysteine dihydrochloride-48.9, L-Cystine hydrochloride monohydrate-60.59, L-Glutamic acid- 14.7, L-Histidine hydrochloride monohydrate-52.5, L-Isoleucine-107, L- Leucine-111, L-Lysine hydrochloride- 164, L-Methionine-32.2, L-Phenylalanine-68.5, L- Proline-34.6, L-Serine-52.6, L-Threonine-101, L- Tryptophan- 17, L-Tyrosine disodium salt- 100,L-Valine-99.7, L-Glutamine-1310, Calcium-D-Pantothenic acid-2.2, Choline chloride- 9,D-Biotin-0.0035, Folic acid-2.7, Niacinamide-2,Pyridoxal hydrochloride-2, Pyridoxine hydrochloride-0.031, Riboflavin-0.2, Thiamine hydrochloride-2.2, Vitamin B 12-0.7, Myoinositol- 12.6, Ascorbic acid-20, Ammonium metavanadate-0.00058, Ammonium molybdate tetrahydrate-0.00618, Calcium chloride dihydrate-155, Copper sulphate pentahydrate- 0.0013, Ferric nitrate nonahydrate-0.05, Ferrous sulphate heptahydrate-0.4, Magnesium chloride anhydrous-29, Magnesium sulphate heptahydrate-100, Manganese chloride - 0.000125, Nickel chloride-0.00012, Potassium chloride-312, Sodium chloride-7000, Sodium metasillicate nonahydrate-0.0142, Sodium selenite-0.00624, Stannous chloride dihydrate- 0.00011, Zinc sulphate heptahydrate-0.4, Calcium nitrate tetrahydrate-90, Disodium hydrogen phosphate-71, Sodium dihydrogen phosphate-54.3, DL-Thioctic acid-0.1, Ethanolamine-13.6, Glutathione- 1.8, HEPES buffer-3574.5, Hypoxanthine-2.4, Linoleic acid-0.042, P-mercaptoethanol-1.4, Phenol red sodium salt-8.6, Putrescine hydrochloride- 0.081, Sodium Bicarbonate-2200, Protein hydrolysate-1000, Sodium pyruvate - 55,Thymidine-0.4, Glucose-6000, Glutamine- 1.17, Lipid mixture I, chemically defined-lX. Culture: CHO cells were routinely maintained in CD CHO medium supplemented with 8 mM glutamine, without any antibiotics, in shake flasks (37°C, 10% C0₂, 110 rpm). For all experiments, 20 ml cultures were cultivated in custom made DMEM:F12 based medium supplemented with 10% CD CHO medium in 100 ml capacity shake flasks on an orbital shaker.

Culture conditions for experiments:

Batch cultures of CHO cells were seeded at initial seeding density 3xl0⁶ cells/ml. Samples were withdrawn every day for cell density and glucose measurements. Cultures were harvested after three days and culture supernatants were stored at -80°C until processed for glycoform analysis. Cell density was measured by manual counting using hemocytometer. Viability was measured using the trypan blue dye exclusion method.

Example 2: Determination of IgG titer

IgG titer was determined by sandwich enzyme-linked immunosorbent assay (ELISA) using human IgG as a standard. ELISA plates were coated with rabbit anti-human IgG in bicarbonate buffer (pH 9.6), overnight at 4 °C. PBS containing 0.01%Tween 20 was used to wash plates. Plates were blocked using 1% bovine serum albumin (BSA) in phosphate- buffered saline (PBS). Culture supernatants and human IgG (Merck Millipore, Mumbai, India) were appropriately diluted in blocking buffer. HRP conjugated rabbit anti-human IgG (Merck Millipore, Mumbai, India) was used as the secondary antibody followed by addition of TMB/H₂O₂ (Genei Laboratories Pvt. Ltd., India). The enzyme substrate reaction was stopped using 1 M H₂SO₄. Absorbance was read at 450 nM on Bio-Rad iMark microplate reader (Bio-Rad Laboratories, Mumbai, India). Standard curve was generated using 4-300 ng/ml human IgG and used to quantify the IgG concentration in culture supernatant.

Example 3: IgG purification and gl can analysis

IgG was purified using Protein A agarose CL (Genei Laboratories Pvt Ltd, India) and concentrated using 10 kDa Amicon Ultra centrifugal filter (Merck Millipore, Mumbai, India). Concentrated IgG was deglycosylated using PNGase F (New England Bioscience, Ipswich, MA, UK) overnight at 37°C. Glycans were separated from deglycosylated IgG using 10 kDa Amicon Ultra centrifugal filter. The glycans were desalted using 2 ml Bio-Gel P-2 (Bio-Rad Laboratories, Mumbai, India) column. Fractions containing glycans were pooled, dried and permethylated using methyl iodide (CH₃I) in the presence of NaOH and DMSO. Glycans were then extracted in chloroform, dried and resuspended in 1 mM sodium acetate. Permethylated glycans were analysed by MALDI TOF-MS (AB SCIEX MALDI TOF/TOF 5800) in positive ion reflector mode using DHB matrix. Twenty spectra were analyzed for each glycan sample in Data Explorer Visualisation software 4.11 and processed for correction of baseline and noise removal. GlycoMod-ExPASy software was used to identify N-glycan peaks. The individual glycan area (GA,z) was calculated as area under the curve for first three monoisotopic peaks and relative percentage of each glycan (Gi) was calculated using the formula:

_ ¾_i ioo

Percentage Galactosylation was calculated as

(Λ161 ÷ A1GI i- A2G1F ÷ A2G2 x 2 ÷ A1G2F X 2) x 100

% Galacios-vlation = -

2 x Al ± A1G1 4- Λ2 4- A2F 4- A2G1 + A2G1F A2G2 4- A2G2F}

Example 4: Galactosyltransferase assay

Galactosyltransferase activity in CHO cell lysate was measured using HPLC-based assay. Cells cultured without additional zinc supplementation were used as source of cn yme. Briefly. 10 x 10⁶ cells were washed twice with phosphate-buffered saline and cell pellets were stored at -80°C until assay. Cells were lysed using 2.5% Triton X-100 (w/v) in phosphate-buffered saline in total volume of 100 μΐ at room temperature by gentle mixing. Lysate was then centrifuged at 3000 rpm for 15 min at 4°C and supernatant was used as enzyme source. Reaction mixture contained substrate: 4-nitrophenyl-N-acetyl-P-D- glucosamine (GlcNAc-β-ρΝΡ. 1 mM), donor nucleotide sugar: uridine diphosphate galactose (UDP-Gal, 1 mM), 1 mM MnCl₂ in 50 mM HEPES buffer (pH = 7.2) (Loba Chemie Pvt. Ltd., Mumbai, India). Ten microliter cell lysate (~ 200 μg of protein) was added to initiate reaction and was incubated at 37 °C for 30 min.

Reaction was terminated by heating at 90 °C for 10 min. Reaction mixtures were then appropriately diluted, filtered, and analyzed by HPLC. Control reactions contained 1 mM MnCl₂ and test conditions contained varying ratio of Zn²⁺/Mn²⁺ or Mg²⁺/Mn²⁺. HPLC analysis was carried out on Aglicnt 1200 series (Agilent Technologies, USA). Product formed was quantified using a reverse-phase CIS Purospher© STAR endcapped column (250 x 4 mm. 5 μηι particle size). The mobile phase used was 15% acetonitrile in water at 0.8 ml/min flow rate and absorbance was measured at 254 nm. Peak area was calculated using the Agilent Chem Station software. Percentage activity was calculated by comparing product peak area to the product peak area at 1 mM Mn²⁺. For measurement of galactosyltransferase activity in cell lysates from cultures with and without 150 μΜ Zn²⁺ supplementation, in vitro activity was determined in the presence of 1 mM Mn ⁺. Activity in each cell lysate was normalized to its protein content. Relative activity normalized to average activity in the CHO cell lysates from cultures without additional Zn²⁺ supplementation is reported.

Example 5: Determination of total intracellular Mn²⁺ and Zn²⁺ content

Total intracellular Mn²⁺ and Zn²⁺ content of cells cultured under different conditions as described above were analysed by microwave plasma atomic emission spectroscopy (MPAES) using Agilent 4100 MP-AES system. At the end of 3 days of culture, cell density was determined and 1.6 x 10^s cells were harvested by centrifugation. Further cells were washed twice with phosphate-buffered saline containing 2 g/L sodium EDTA (pH = 7.4). Cell pellets were then stored at - 80 °C until analysis. Cells were digested with 1.4 ml 70% nitric acid and 0.2 ml 30% hydrogen peroxide for 24 h at room temperature. Samples were analyzed in triplicate and concentrations were determined by plotting standard curve using commercially available trace element standards. Trace element concentrations were then normalized to total protein concentration in cells. For total cellular protein determination, cells were lysed and protein concentration was determined by Biuret method.

Example 6: Effect of Zn²⁺ on CHO cell growth and IgG glycosylation:

Cells were seeded at 3xl0⁶ cells/ml density and supplemented with Zinc chloride (ZnCl₂) at various concentrations from 30 μΜ to 200 μΜ. Cultures were harvested on the third day after inoculation (day 3) and culture supematants were stored at -80°C until processed for glycoform analysis. Glycoform analysis revealed decrease in galactosylation at and above 100 μΜ Zn²⁺ in a dose dependent manner (Fig. 1).

Example 7: Effect of Zn²⁺ supplementation on galactosylation in the presence of galactose and/or uridine supplementation

6

Cells were seeded at 3x10 cells/ml density and supplemented with 30 mM galactose

(Gal), 1 mM uridine (Urd) and 30 mM galactose + 1 mM uridine with and without 150 μΜ

2+

Zn · Cultures were harvested on the third day after inoculation (day 3) and culture supernatants were stored at -80°C until processed for glycoform analysis (Fig. 2).

Galactose supplementation increased galactosylation to 42% in the absence of Zn²⁺ supplementation. Galactosylation is however reduced to 28% with 150 μΜ Zn²⁺ supplementation (p = 0.02).

Uridine supplementation to control cultures did not increase galactosylation significantly. However, when uridine is supplemented to Zn²⁺ containing cultures galactosylation increased significantly (p = 0.02) to 26%.

Simultaneous supplementation with galactose and uridine increased galactosylation from 19% to 28% in the presence of Zn²⁺ (p = 0.01), but galactosylation levels continued to be lower than that observed in cultures without Zn²⁺ supplementation (43%, p = 0.004)

Example 8: Effect of Zn²⁺ supplementation on CHO cell growth, and IgG glycosylation in the presence of Mn²⁺

Cells were seeded at 3xl0⁶ cells/ml density and supplemented with 4 μΜ MnCl₂ with and without 150 μΜ ZnCl₂. Cultures were harvested on the third day after inoculation

(day 3) and culture supernatants were stored at -80°C until processed for glycoform analysis.

The basal culture medium contains 1 nM Mn²⁺. With an additional 4 μΜ Mn²⁺ supplementation, Zn²⁺ supplementation did not affect cell growth and galactosylation.

These results suggest that the effect of Zn²⁺ on galactosylation can be substantially reversed with Mn²⁺ supplementation (Fig. 4).

Example 9: Effect of Zn²⁺ on galactosylation in commercial chemically defined CD CHO medium

To test robustness of effect of Zn²⁺, batch cultures were inoculated in CD CHO medium. Cells were seeded at 0.5xl0⁶ cells/ml density and supplemented with 150 μΜ Zn²⁺. Initial glucose and glutamine concentrations were 33 mM and 8 mM, respectively. Cultures were harvested on the fourth day after inoculation (day 4) and culture supernatants were stored at -80°C until processed for glycoform analysis.

Galactosylation decreased from 23% to 16% with Zn²⁺ supplementation (Fig. 5B). In the presence of 4 μΜ Mn²⁺, galactosylation without and with Zn²⁺ supplementation was 38% and 41%, respectively. This indicated that the effect of Zn²⁺ on galactosylation is robust to differences in medium formulation.

Example 10: Effect of Zn²⁺ on high mannose glycans of IgG from cells cultured in galactose in the presence of 4 μΜ Mn²⁺

Cells were seeded at 3xl0⁶ cells/ml density with 4 μΜ Mn²⁺ and 30 mM galactose as carbon source and supplemented with 150 μΜ Zn²⁺. Cultures were harvested on the third day after inoculation (day 3) and culture supernatants were stored at -80°C until processed for glycoform analysis.

Percentage of high mannose glycans of IgG decreased from 29% to 5% with Zn²⁺ supplementation (p = 0.001).

Advantages of the Invention:

1. The present invention is useful to alter the galactosylation of a glycoprotein to attain a pre-defined target value. It is useful in drug development to develop a bio similar as close to the innovator molecule as possible. The process can be utilized if the galactosylation in the bio similar is to be reduced to make it structurally similar to the innovator molecule.

2. The process is simple to implement.

Claims

Claims:

1. A process for decreasing galactosylation of IgG expressed in an expression system, said process comprising:

(i) culturing the expression system expressing IgG antibodies in a culture medium; and

(ii) supplementing the culture medium of step (i) with zinc in a concentration ranging from 75μΜ to 150 μΜ.

2. The process as claimed in claim 1, wherein said expression system is CHO cells.

3. The process as claimed in claim 1, wherein said culture medium is a chemically defined culture medium or a serum free culture medium.

4. The process as claimed in claim 1, wherein said zinc is supplemented in form of a zinc salt.

5. The process as claimed in claim 4, wherein said zinc salt is selected from the group consisting of zinc chloride, zinc sulphate, zinc carbonate, and zinc chromate.

6. The process as claimed in claim 1, wherein said process comprises addition of Mn²⁺ in concentrations ranging from 1 μΜ to 5 μΜ to the culture medium for overcoming the effect of lot-to-lot variability in trace element concentrations on galactosylation.

7. A culture medium for decreasing galactosylation in the production of IgG in CHO cells, said culture medium comprising zinc in a concentration ranging from 75 μΜ to 150 μΜ.