Heamoglobin and uses thereof
11224218 · 2022-01-18
Assignee
Inventors
Cpc classification
A61P1/00
HUMAN NECESSITIES
A01N1/0226
HUMAN NECESSITIES
International classification
C12N5/00
CHEMISTRY; METALLURGY
Abstract
The present invention relates to isolated haemoglobin from worms belonging to the Nereididae family and its use in cell culture medium, in preservation solutions and as artificial oxygen carrier for transfusion.
Claims
1. A composition for in vitro cell culture, comprising: A) a serum-free nutritive cell culture medium solution for the maintenance, growth, propagation, and/or expansion of cells in an artificial environment outside of a multicellular organism or tissue, B) a purified and function hemoglobin isolated from Nereis virens, wherein said hemoglobin has no free cysteine and no glycosylations, said hemoglobin having: (i) a P.sub.50 from 5 to 15 mm Hg at 6-7° C., (ii) a P.sub.50 from 12 to 20 mm Hg at 16-17° C., and (iii) a P.sub.50 from 20 to 50 mm Hg at 36-37° C., C) a buffer, D) isolated cells which grow and produce a recombinant protein, and wherein components A), B), C), and D) are in mixture in the cell culture medium solution, and wherein of the hemoglobin has a concentration of at least 0.050 g/l.
2. The composition according to claim 1, wherein the concentration of the hemoglobin is at least 1 g/l.
3. The composition according to claim 1, wherein said hemoglobin has: (i) a P.sub.50 from 9 to 12 mm Hg at 6-7° C., (ii) a P.sub.50 from 15 to 18 mm Hg at 16-17° C., and (iii) a P.sub.50 from 30 to 44 mm Hg at 36-37° C.
4. The composition according to claim 1, wherein said hemoglobin has a molecular weight of from 3 to 4 million Daltons.
5. The composition according to claim 1, wherein the buffer further comprises CaCl.sub.2, NaCl, MgCl.sub.2, and KCl.
6. The composition according to claim 5, wherein the composition has a pH of 7.0 to 7.8.
7. The composition according to claim 1, wherein said cell culture medium solution comprises a medium selected from the group consisting of Dulbecco's Modified Eagle's Medium (DMEM), DMEM/F12, Minimal Essential Medium (MEM), Basal Medium Eagle (BME), RPMI 1640, F-10, F-12, alpha-Minimal Essential Medium (alpha-MEM), Glasgow's Minimal Essential Medium (G-MEM), Iscove's Modified Dulbecco's Medium, and CD-CHO medium.
8. The composition according to claim 1, wherein said cell culture medium solution comprises: a) a basal medium; b) ferric citrate; c) insulin, glucose, L-glutamine, sodium bicarbonate, HEPES, NaCl, surfactant, NaH.sub.2PO.sub.4—H.sub.2O, yeast-based hydrolysate, and plant based hydrolysate.
9. The composition according to claim 1, wherein the composition is in the form of an aqueous solution, a frozen solution, or a thawed solution.
10. The composition according to claim 1, wherein the concentration of the hemoglobin is 0.250 to 200 mg/ml.
11. The composition according to claim 8, wherein the concentration of the hemoglobin is at least 0.250 g/l.
12. The composition according to claim 1, wherein the buffer comprises at least one of HEPES and Tris, wherein the cell culture medium solution has a pH of 6.0 to 7.8.
13. The composition according to claim 1, wherein the cell culture medium solution has a pH of 6.0 to 7.8.
14. The composition according to claim 1, wherein the concentration of the hemoglobin is 0.250 to 1.25 mg/ml.
15. The composition according to claim 1, wherein the isolated cells are eukaryotes or prokaryotes, which produce an antibody.
Description
BRIEF DESCRIPTION OF THE FIGURES
(1) The file of this patent contains at least one drawing executed in color. Copies of this patent with color drawings will be provided by the Patent and Trademark Office upon request and payment of the necessary fee.
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EXAMPLES
Extraction and Purification of Nereis Haemoglobin Samples
(11) The Nereis species were obtained by a marine stockbreeder who farms these worms for shrimp and fish aquaculture. Living ragworms were bled.
(12) The blood samples are collected on ice. After cold centrifugation (15 000 g for 15 min at 4° C.) to eliminate any tissue debris, the supernatants are frozen at −20° C. or in liquid nitrogen, or immediately purified.
(13) Purification of the Haemoglobins
(14) Before purification, the thawed sample is centrifuged, at 5 000 g for 5 mM at 4° C. After centrifugation, a small residue is generally present; this is eliminated. The supernatant is purified by Low pressure filtration using a 2.5*100 cm Sephacryl S-400 HR column (GE Healthcare, separation range between 2×10.sup.4-8×10.sup.6 kDa). The samples are eluted with the storage buffer as described here above (150 mM NaCl, 2 mM CaCl2, 1.5 mM CaCl2, 5 mM KCl, 50 mM Hepes, pH 7.2). The rate used is generally 0.4 to 0.5 ml/min. The absorbance of the eluate is followed at two wave lengths: 280 nm (protein absorbance peak) and 414 nm (haemoglobin absorbance peak). The fractions containing the haem are concentrated using Centricon-100 (15 ml) tubes or using an agitation cell retaining the molecules with a weight above or equal to 10 000 Da. Two purification processes following the same protocol might be necessary to obtain pure fractions. The purified sample is filtrated on 0.2 μm before storage at −80° C.
(15) The concentration of haemoglobin obtained was about 50 mg/ml and the purity of the haemoglobin was 95%.
(16) The samples were kept either frozen or under liquid nitrogen until use.
(17) Gel Filtration LC Analysis
(18) Analytical gel filtration was performed on 5 to 200 μl of injected sample on a 1×30 cm Superose 6-C (fractionation range from 5 to 5000 kDa, GE Healthcare) using a high-pressure HPLC system (Waters, milford, MA, USA). The elution buffer is 0.1N Tris-HCl buffer at pH 7.2. The elution (flow rate: 0.5 ml/min) was monitored with a photodiod array detector (Waters 2996) over the range 250-700 nm Chromatographic data were collected and processed by the Empower software (Waters). The percentages of each subunit were determined by integrating the chromatogram at 414 nm (characteristic of heme) and 280 nm (characteristic of protein) with the Empower software.
(19) Multi-Angle-Laser-Light-Scattering
(20) The molecular weight and the gyration radius of the native haemoglobin were determined with MALLS detector (DAWN EOS system, Wyatt Technology Corp., Santa Barbara, Calif., USA) directly on-line with the HPLC system and the Superose 6 column Chromatographic data were collected and processed by the Astra software (Wyatt Technology Corp.). The Zimm fit method was used for molecular mass determinations. In this method, the concentration dependence of the refractive index was set to 0.19 ml g.sup.−1 (typical for human haemoglobin). The sample was kept at 4° C. until the elution which was performed at room temperature.
(21) Mass Spectrometry Analysis
(22) Electrospray data were acquired on an ESI-Q-TOF (Q-TOF II; Micromass, Altrincham, UK) mass spectrometer scanning over the mass-to-charge ratio (m/z) range 600-2500 in 10 s/scan. Data were accumulated over 3 mM to produce the final spectrum. The cone voltage (counter electrode to skimmer voltage) was set to 60 V. A 200-μ1 solution of purified, native Hb was desalted four times against 4 ml MilliQ water (at 4° C.) using centrifugal filters with a molecular weight cut-off of 100 kDa (Amicon Ultra-4, Millipore). ESI-MS analyses were performed on the non-reduced Hb, the reduced Hb, and on the reduced and carbamidomethylated Hb to determine the association of subunits into dissulfide-bridged structures. Samples with a protein concentration of 0.5 μg μl.sup.−1 in 1:1 (v/v) acetonitrile/water containing 0.2% (v/v) formic acid were introduced into the electrospray source at 5 μl min.sup.−1 Hb (0.9 mg ml.sup.−1) was reduced with 10 mM DTT at room temperature under alkaline conditions in the presence of 100 mM ammonium bicarbonate. After 10 min incubation, a 10-μl aliquot was mixed with 40 μl MilliQ and 50 μl acetonitrile containing 0.2% formic acid and analysed by ESI-MS. After 20 min incubation under reducing conditions, Hb (0.7 mg ml.sup.−1) was carbamidomethylated with 4 mM iodoacetamid at room temperature for 10 mM and 10 hours, respectively, after which 12.75-μl aliquots were mixed with 37.75 μl MilliQ and 50 μl acetonitrile containing 0.2% formic acid and analysed by ESI-MS. Carbamidomethylation was carried out for total cystein determination. Mass scale calibration employed the multiply charged series from horse heart myoglobin (16951.7 Da; Sigma, St Louis, Mo.). The raw multicharged ESI-MS spectra were deconvoluted using maximum-entropy-based analysis supplied with the instrument. Molecular masses are based on the atomic weights of the elements given by IUPAC.
(23) Oxygen Binding Properties
(24) Oxygen equilibrium curves were determined on 3 μl samples using a thermostated diffusion chamber (Colmorgen et al. 1997 J. Comp. Physiol. 167B:309) linked to cascaded Wosthoff gas mixing pumps (Bochum, Germany). The diffusion chamber was placed in the light path of a spectrometer (Hitachi U1100) at 436 nm Oxygenation data based on at least three equilibrium steps between 0.2 and 0.8 fractional saturation (Y) were converted to Hill plots (log [Y/1−Y)] against log PO2, where PO2 is the oxygen partial pressure) for the estimation of the half-saturation oxygen partial pressure (P.sub.50) and Hill's cooperativity coefficient at half-saturation (n.sub.50).
(25) Cell Culture Medium
(26) Cell Lines and Cell Culture Conditions
(27) CHO DXB11, a dihydrofolate reductase (dhfr-) Chinese Hamster Ovary cell line, SP2/0 and PTG2b cell lines were used. Both CHO and SP2/0 cells were seeded at 0.5*10.sup.5 cells/ml in shake flasks containing serum-free medium, specifically designed for optimal suspension growth and protein production, supplemented with 8 mM L-Glutamine and different concentrations of the haemoglobin. Cells were grown in batch culture without refeeding at 37° C. in a shaking incubator at least for 1 day, preferably at least 4 days, preferably at least 8 days.
(28) Sampling and Studied Parameters
(29) Samples were removed at days 0, 1, 4, 6 and 8 post-seeding to determined cell count, cell viability and protein production. Cell count and cell viability were measured by the tryphan blue exclusion technique using a Vi-CELL™ Analyzer (Beckman Coulter). Total antibody production was measured using an ELISA assay specific for the IgG being expressed according to manufacturer's instructions (ELISA kit, Bethyl Laboratories).
(30) Cell Preservation
(31) Cell Line and Cell Culture Conditions
(32) Experiments were carried out using an epithelial cell line originally derived from porcine kidneys: LLC-PK1.
(33) LLC-PK1 cells were cultured in M199 medium supplemented with 3% SVF, 100 U/ml penicillin, 100 μg/ml streptomycin and 2 mM L-Glutamine.
(34) LLC-PK1 cells were seeded in a 6 wells plate at a concentration of 1.6.10.sup.5 cells/ml. After 48 h of culture, the cells were washed in PBS and maintained at 4° C. during 24 h in 1.2 ml of UW solution (Viaspan®) or Custodiol® solution alone or in the presence of different concentration of the haemoglobin.
(35) Cell Viability Detection
(36) LDH Test
(37) Cell viability was determined by detection of lactate dehydrogenase (LDH) as the presence of this enzyme in the cells reflects permeabilization of the plasmic membrane and thus cell death.
(38) After 24 h of conservation in the preservation solution, supernatant was eliminated and cells were washed 3 times in PBS and then lysed in 1.2 ml of PBS containing 0.1% of Triton X-100.
(39) The quantity of LDH present in the cells was determined by a colorimetric dosage according to the manufacturer's instructions (TOX7, Sigma-Aldrich). The delta of absorbance measured at 490 nm and 630 nm is directly proportional to the quantity of enzyme present in the sample.
(40) MTT Test
(41) The MTT assay is another in vitro cell viability assay used to determine a reduction in cell viability. After 24 h of conservation in the preservation solution, supernatant was eliminated and cells were washed 2 times in PBS. The tetrazolium compound MTT (3-[4, 5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) was added to the wells at a concentration of 0.5 mg/ml in PBS and the cells were incubated 30 min at 37° C. MTT is reduced by metabolically active cells to insoluble purple formazan dye crystals. The crystals were solubilized by adding 2 ml of DMSO and the absorbance was detected with a spectrophotometer. The delta of absorbance measured at 535 nm and 690 nm is directly proportional to the metabolic activity of the cells.
(42) Results
(43) Structural Analysis
(44) Molecular weight and gyration radius were determined by MALLS (
(45) Subunit and polypeptid chains composition determined by ESI-MS (Table 1).
(46) TABLE-US-00001 TABLE 1 Subunits (non reduced Polypeptide chains conditions) (reduced conditions) Mw Free Mw Disulphide (Da) Cys name (Da) Cys name bridge Monomeres 15448.0 0 an1 0 15885.0 0 an2 2 1 intra- Trimeres 49372.5 0 T1 16145.5 1 an3 1 inter- 16494.5 1 an4 1 inter- 16734.5 4 an5 2 inter- 1 intra- Linker 50606.9
Functional Analysis
(47) The functional properties of the Nereis haemoglobin were determined at 3 different temperatures: 4° C., 15° C. and 37° C. Results are shown in Table 2.
(48) TABLE-US-00002 TABLE 2 Temperature Hemoglobin (° C.) mean stdev P50 (mmHg) stdev n50 stdev 6.65 0.40 10.98 1.27 1.19 0.04 16.51 0.13 16.57 1.96 1.17 0.10 36.56 0.29 37.11 6.00 1.47 0.40
Stability of the Nereis Haemoglobin in Rat Plasma
(49) The haemoglobin was diluted in rat plasma at a concentration of 1 mg/ml and maintained at 20° C. The solution is centrifuged at 10000 g for 2 min at 4° C. and then filtrated on a 0.45 μm filter before injection.
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(51) Effect of the Nereis Haemoglobin in Preservation Solution.
(52) 1—Analysis of LLC-PK1 Cells Viability by Detection of the Liberation of Lactate Dehydrogenase (LDH)
(53) The results are expressed in percentage of LDH quantity detected in cells maintained at 4° C. in the preservation solution compared to LDH quantity detected in cells before cold preservation (T0). Different concentrations of haemoglobin: 0.039. 0.078. 0.156. 0.312. 0.625 and 1.25 g/l were added to the preservation solution.
(54) The percentage of LDH liberation is calculated as following: 100−[(LDH cells)×100/(LDH cells at T0)].
(55) The results are shown in Table 3 and
(56) TABLE-US-00003 TABLE 3 Hb Hb Hb Hb Hb Hb UW 0.039 0.078 0.156 0.312 0.625 1.25 Mean 79 75 73 57 24 7 2 SD 5.9 4.3 4.9 7.4 6.3 4.5 2.3
(57) Conservation of LLC-PK1 cells at 4° C. during 24 h induces about 80% of cell death. The presence of Nereis haemoglobin in UW solution (Viaspan®) protects LLC-PK1 cells from cell death in a dose-dependent manner and this protection is almost complete at 0.625 g/1.
(58) 2—Analysis of LLC-PK1 Cells Metabolic Activity by MTT Test
(59) The results are expressed in percentage of metabolic activity detected in cells maintained at 4° C. in the preservation solution compared to metabolic activity detected in cells before cold preservation (T0).
(60) Different concentrations of haemoglobin: 0.04. 0.08. 0.156. 0.312. 0.625. 1.25 and 2.5 g/l were added to the preservation solution.
(61) The percentage of metabolic activity is calculated as following: (metabolic activity of cells)/(metabolic activity of cells at T0)×100.
(62) The results are shown in Table 4 and
(63) TABLE-US-00004 TABLE 4 Hb Hb Hb Hb Hb Hb Hb UW 0.039 0.078 0.152 0.312 .625 1.25 2.5 Mean 11.3 11.5 13.5 25.9 53.2 65.6 75.7 85.1 SD 5.3 5.1 4.8 13.2 9.1 5.8 8.6 12.8
(64) Results show that the presence of haemoglobin in UW solution (Viaspan®), in particular at a concentration higher than 0.625 g/l, allows the preservation of the metabolic activity of LLC-PK1 cells. Same results were obtained with haemoglobin in Custodiol® solution (data not shown).
(65) Effect of the Nereis Haemoglobin in Cell Culture Medium.
(66) 1—Stability of the Haemoglobin in Cell Culture Medium at 37° C.
(67) The haemoglobin (1.25 mg/mL) was added to the following cell culture media: DMEM, CDCHO, ExCell, GR7F, a-MEM-SVF, a-MEM-PRP, SCIVAX media.
(68) The absorbance between 250 and 700 nm was measured at different points of time.
(69) Table 5 and
(70) TABLE-US-00005 TABLE 5 DMEM CDCHO ExCell Time At/At0 (414 nm) At/At0 (414 nm) At/At0 (414 nm) 0 1.00 1.00 1.00 24 0.92 1.00 48 0.91 1.00 0.88 72 0.90 1.00 0.90 96 0.91 1.05 0.93 168 0.83 1.00 0.97 214 0.78 0.99 334 0.70 0.78 358 0.90 384 0.65 0.76 0.91
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(72) In addition, no dissociation and no oxidation of the haemoglobin during time was observed when cultured in GR7F, a-MEM-SVF, a-MEM-PRP, and SCIVAX media (data not shown).
(73) 2—Effect of the Haemoglobin on Cell Viability, Cell Growth and Protein Production
(74) CHO and Sp2/0 cell lines were cultivated during 8 to 11 days in cell culture medium alone or in the presence of 0.05 g/1, 0.25 g/l and 1 g/l of the haemoglobin. PTG2b cell lines were cultivated during 7 days in cell culture medium alone or in the presence of 0.125 g/1, 0.25 g/l and 1 g/l of the haemoglobin.
(75) CHO, SP2/0 and PTG2b cells were seeded at day 0 (DO) at a density of 0.5*10.sup.5 cells/ml. Cell count and cell viability were measured by the tryphan blue exclusion technique using a Vi-CELL™ Analyzer (Beckman Coulter). Total protein production was measured using an ELISA assay specific for the protein being expressed according to manufacturer's instructions (ELISA kit, Bethyl Laboratories).
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