Method for increasing the secretion of recombinant proteins

Abstract

The invention relates to a method for producing a recombinant protein in cells with a cell wall, comprising the step of increasing the secretion of the recombinant protein through the cell wall by expression of the protein in the cells in a culture medium containing a combination of a surface-active polymer and monovalent metal ions and with an osmolarity at least 0.32 osmol/L, said invention further relating to culture media and nutrient mixtures for the method.

Claims

1. A method for producing a recombinant protein in plant cells, said method comprising the steps of: (a) growing cells having a cell wall in a first culture medium having an osmolarity of less than 0.1 osmol/L wherein the medium does not comprise a non-ionic surface-active polymer or comprises the non-ionic surface-active polymer in a maximum concentration, whereby protein accumulates in the interior of the cell or in the apoplastic space of the cell, and (b) then growing the cells in a second culture medium containing a combination of a nonionic surface-active polymer in a minimum concentration and monovalent metal ions and having an osmolarity of at least 0.32 osmol/L, wherein the recombinant protein is expressed in the cells and whereby the recombinant protein is secreted through the cell wall into the medium, wherein step (a) is carried out for a at least 2 days, and wherein for said polymer either (i) the maximum concentration of step (a) is 0.08% by weight and the minimum concentration of step (b) is 0.5% by weight, or (ii) the maximum concentration of step (a) is 0.01% by weight and the minimum concentration of step (b) is 0.1% by weight.

2. The method according to claim 1, characterized in that the secretion into the medium is effected from the apoplastic space of the cell.

3. The method according to claim 1 or 2, wherein the plant cell is a cell in a plant or in a plant tissue or a moss cell.

4. The method according to claim 1, characterized in that the cell is cultivated in a suspension culture.

5. The method according to claim 1, characterized in that the metal ion is an alkali metal ion and/or that the metal ion is present in a concentration of at least 20 mM in the medium.

6. The method according to claim 1, characterized in that the non-ionic surface-active polymer is a polyalkyl glycol.

7. The method according to claim 1, characterized in that the medium of step (b) comprises nitrate ions, phosphate ions, sulfate ions, calcium ions, potassium ions, sodium ions, or combinations thereof.

8. The method according to claim 1, characterized in that the culture medium in step (a) comprises nitrate ions, phosphate ions, sulfate ions, calcium ions, potassium ions, or combinations thereof; and/or comprises sodium ions in a maximum concentration 20 mM.

9. The method according to claim 1, characterized in that said step (a) is carried out over a period of 2 to 30 days.

10. The method according to claim 1, characterized in that said step (b) is carried out over a period of at least 2 days.

11. The method of claim 5, wherein the alkali metal is sodium.

12. The method of claim 6 wherein the polyalkyl glycol is a polyethylene glycol.

13. The method of claim 7, wherein the nitrate ions are in a concentration of at least 0.2 mM.

14. The method of claim 7, wherein the phosphate ions are in a concentration of at least 0.05 mM.

15. The method of claim 7, wherein the sulfate ions are in a concentration of at least 0.02 mM.

16. The method of claim 7, wherein the calcium ions are in a concentration of at least 0.1 mM.

17. The method of claim 7, wherein the potassium ions are in a concentration of at least 0.1 mM.

18. The method of claim 7, wherein the sodium ions are in a concentration of at least 20 mM.

19. The method of claim 1, wherein said step (a) is without said secretion.

20. The method of claim 8, wherein the medium of step (a) comprises nitrate ions in a concentration of at least 0.2 mM, phosphate ions in a concentration of at least 0.05 mM, sulfate ions in a concentration of at least 0.02 mM, calcium ions in a concentration of at least 0.1 mM, potassium ions in a concentration of at least 0.1 mM.

21. The method according to claim 1, characterized in that said step (b) is carried out over a period of 3 to 120 days.

Description

(1) The present invention is further illustrated by the following Figures and Examples, without being limited to these specific embodiments of the present invention, however.

FIGURES

(2) FIG. 1 shows the synergistic effects of an increase in osmolarity by the addition of NaCl and the polymer PEG on the secretion of a recombinantly produced antibody;

(3) FIG. 2 shows additional production-increasing effects of mineral substances for stimulating plant growth (Ca nitrate);

(4) FIG. 3 shows the effects of different polymer concentrations.

EXAMPLES

(5) The present invention provides a method that enables the continuous secretion of recombinant proteins from plant cultures into the culture medium. In said method, the producing plants are submerged in a liquid culture and are photoautotrophically cultivated in specific mineral media. The culture media are optimized with respect to an optimal nutritional maintenance of moss plants, but do not per se cause the secretion of recombinantly produced larger proteins into the medium, which proteins first accumulate in the apoplast and can be measured as “intracellular” following the disruption of the moss tissue.

(6) The apoplastic restraint of the proteins can be reversed by the addition of osmotically active substances (“secretion components”), such as NaCl and polyethylene glycol (PEG) 4000, to the culture medium. This leads to a quick release of the accumulated proteins into the medium and in the further course of the culture to a continuous and temporally immediate secretion of newly synthesized proteins.

Experimental Example: Comparison of the Protein Secretion in Erlenmeyer Cultures

(7) 500 ml Erlenmeyer flasks were filled with 180 ml of sterile Knop or WM01 medium, adjusted to pH 4.5 to 6 (2-(N-morpholino) ethanesulfonic acid buffer, “MES”) and inoculated with a freshly turraxed suspension culture of Physcomitrella protonema from a recombinant strain produced by a test protein. The inoculation density was 0.1 g dry weight/l. The cultures were sealed in a sterile and gas-permeable manner and cultivated in an atmosphere supplemented with 2% CO.sub.2. Upon growth to a cell density of 1 to 3 g/l, the secretion components were added in the form of a sterile concentrate. During the cultivation procedure, the parameters of culture density (g dry weight/l), intracellular and extracellular IgG titer (mg/l) were determined at regular intervals.

(8) Results:

(9) The following test proteins were produced:

(10) TABLE-US-00001 TABLE 1 Secreted test proteins Molecular Secretion w/o Secretion with weight secretion secretion medium Protein [kDa] medium (NaCl, PEG) IgG 145 no yes Alpha-Galactosidase 80 no yes VEGF.sub.121 50 yes yes Epo 30 yes yes HSA 67 yes yes

(11) TABLE-US-00002 TABLE 2 Media (all concentrations are given in mg/l. * except for MES): 1 2 3 4 5 6 Nutrit. media KNOP 1/10 BM WM01 Murashige Linsmaier KNOP & Skoog & Skoog KH.sub.2PO.sub.4  250  25  250  500 170   170   NH.sub.4NO.sub.3 1650   1650   KNO.sub.3 1900   1900   KCl  250  25  250  500 MgSO.sub.4•7H.sub.2O  250  25  250  500 180.7 Ca(NO.sub.3).sub.2 × 1000 100 1000 2000 4H.sub.2O CaCl 332.2 332.2 NaCl MES * 5 mM 5 mM

(12) In addition, microelements such as H.sub.3BO.sub.3, FeSO.sub.4, Fe-NaEDTA, COCl.sub.2, CuSO.sub.4, KI, MnCl.sub.2, MnSO.sub.4, Na.sub.2MoO.sub.4, NiCl.sub.2, Na.sub.2SeO.sub.3, Zn acetate, or vitamins such as folic acid, myo-inositol, nicotinic acid, thiamine HCl, pyridoxine HCl, biotin or also glycine may be added. For fungal cultures, an additional catabolic carbohydrate source such as glucose, mannose or mannitol is added.

Example 1: Increase in Production by Means of PEG and an Increased Osmolarity

(13) An IgG antibody was expressed as described in P. patens using the following parameters: light rhythm [h] 16/8, initial medium KNOP, temperature 22° C., culture container: flask; medium “ 1/10 KNOP NaCl” containing 100 mM of NaCl in addition to medium “ 1/10 KNOP”. Medium “KNOP PEG” and “ 1/10 KNOP NaCl PEG”, respectively, containing 4.8% by weight of PEG-4000 in addition to medium “KNOP” and “ 1/10 KNOP NaCl”, respectively. The results of the antibody production after 6 weeks are represented in Table 3 and in FIG. 1.

(14) TABLE-US-00003 TABLE 3 PEG and NaCl (100 mM for increase in osmolarity) intracellular extracellular extra/ ng IgG/mg ng IgG/mg total Flask dry weight dry weight % Knop, Experiment 1 62.3 4.8  7% Knop, Experiment 2 88.5 6.6  7% Knop, Experiment 3 84.1 6.4  7% Average 78.3 5.9  7% 1/10 Knop NaCl, Experiment 1 27.3 29.7 52% 1/10 Knop NaCl, Experiment 2 24.0 30.8 56% 1/10 Knop NaCl, Experiment 3 19.5 43.1 69% Average 23.6 34.5 59% Knop PEG, Experiment 1 35.9 4.3 11% Knop PEG, Experiment 2 25.0 4.5 15% Knop PEG, Experiment 3 33.1 4.5 12% Average 31.4 4.4 13% 1/10 Knop NaC1, PEG, Experiment 1 27.7 180.7 87% 1/10 Knop NaC1, PEG, Experiment 2 30.1 175.3 85% 1/10 Knop NaC1, PEG, Experiment 3 36.2 208.2 85% Average 31.3 188.1 86%

(15) With the combination of NaCl and PEG an increase in productivity and secretion was observed that by far exceeded the respective effects of NaCl alone and PEG alone. A synergistic effect is thus clearly obvious.

Example 2: Increase in Production by Means of PEG and an Increased Osmolarity and Nitrate

(16) A lambda chain of an IgG1 antibody was expressed as described in P. patens using the following parameters: light rhythm [h] 16/8, initial medium KNOP, temperature 25° C., light intensity 40 μE/m.sup.2s, culture container: flask; The results of the antibody production after different time intervals are represented in Table 4 and in FIG. 2.

(17) TABLE-US-00004 TABLE 4 PEG, NaCl (osmolarity) and nitrate (concentration IgG1 in μg/ml) Day of cultivation 7 14 21 28 1/10 KNOP 100 mM NaCl medium + 0.279 0.239 0.053 0.059 1/10 KNOP medium + 100 mM NaCl, 1.719 8.947 8.882 10.872 4.8% PEG, 48 mM mannitol, 12 mM calcium nitrate 1/10 KNOP medium + 50 mM NaCl, 0.717 5.04 6.125 7.001 4.8% PEG, 48 mM mannitol, 12 mM calcium nitrate 1/10 KNOP Medium + without NaCl, 0.048 0.086 0.123 0.122 4.8% PEG, 48 mM mannitol, 12 mM calcium nitrate 1/10 + 100 mM NaCl KNOP medium, 0.19 2.248 2.363 0.494 4.8% PEG 1/10 + 100 mM NaCl KNOP medium, 0.214 2.041 1.453 0.213 4.8% PEG, mannitol 48 mM 1/10 + 100 mM NaCl KNOP medium, 0.578 6.574 8.226 11.83 4.8% PEG, 12 mM calcium nitrate 1/10 + 100 mM NaCl KNOP medium, 0.043 0.132 0.12 0.088 48 mM mannitol 1/10 + 100 mM NaCl KNOP medium, 0.159 1.027 3.715 4.606 12 mM calcium nitrate 1/10 KNOP medium + 100 mM NaCl, 0.185 1.259 3.704 5.087 48 mM mannitol, 12 mM calcium nitrate

(18) According to these results, mannitol has no influence on the secretion behavior. Nitrate is advantageous due to the improved plant growth. In further experiments, potassium, phosphate, sulfate, or calcium ions, within a range of 1 to 10 mM, also showed positive effects that are based on an improved plant growth. In these low concentrations and without a significant increase in osmolarity no effect on the secretion could be observed.

Example 3: Different Polymers and Polymer Concentrations

(19) A lambda chain of an IgG1 antibody was expressed as described in P. patens using the following parameters: light rhythm [h] 16/8, initial medium KNOP/BM, temperature 25° C., light intensity 40 μE/m.sup.2s, culture container: flask; the results of the antibody production after different time intervals are represented in Tables 5 and 6 and in FIG. 3.

(20) TABLE-US-00005 TABLE 5 Medium composition and IgG production (concentrations in mM) Medium 1 2 3 4 5 6 Magnesium ions 0.1014 0.501 0.501 0.501 0.501 0.501 Calcium ions 12.4235 14.09 14.09 14.09 14.09 14.09 Chloride ion 100 100 100 100 100 100 Sulfate ions 0.1059 0.501 0.501 0.501 0.501 0.501 Nitrate ions 24.847 28.185 28.185 28.185 28.185 28.185 Potassium ions 0.1837 0.9075 0.9075 0.9075 0.9075 0.9075 Sodium ions 100 100 100 100 100 100 Phosphate ions 0.1837 0.9075 0.9075 0.9075 0.9075 0.9075 MES 0 2.4705 2.4705 2.4705 2.4705 2.4705 Mannitol 48 48 48 48 48 48 PEG [%] w/V 4.8 4.8 1 0.5 pH 5.8 5.9 5.9 5.9 5.9 5.9 Tween 20 [w/v] 0.05 0.01

(21) TABLE-US-00006 TABLE 6 Results of the IgG production in μg/ml Medium No. 7 d 14 d 21 d 1 1.26 2.98 5.43 2 1.75 5.01 7.2 3 2.05 5.95 8.66 4 1.85 5.61 8.42 5 0.41 0.46 0.57 6 1.88 7.13 14.9

(22) Based on these results it can be concluded that low concentrations of PEG do not have a negative influence on the secretion in the presence of PEG. The effect of Tween is stronger in lower concentrations (medium 6) than in high concentrations (medium 5).