Fusion protein inhibiting angiogenesis or growth and use thereof
09657084 ยท 2017-05-23
Assignee
Inventors
Cpc classification
C07K2319/35
CHEMISTRY; METALLURGY
C07K2319/30
CHEMISTRY; METALLURGY
A61P9/10
HUMAN NECESSITIES
A61P35/00
HUMAN NECESSITIES
International classification
C07K19/00
CHEMISTRY; METALLURGY
C12N5/10
CHEMISTRY; METALLURGY
Abstract
Provided are a fusion protein inhibiting angiogenesis or vascular growth, coding sequence thereof, vector comprising the coding sequence, host cell, pharmaceutical composition and use of the fusion protein. The fusion protein of the present invention has high thermostability, and has a dramatic decline in the protein aggregation formation rate in a fermentation process, and a significant increase in the purity and yield of the protein, and has better biological activity.
Claims
1. A fusion protein for the inhibition of angiogenesis or vascular growth, the fusion protein comprising a human VEGF receptor fragment and an Fc fragment of human immunoglobulin linked thereto, wherein the amino acid sequence of the VEGF receptor fragment is SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5, and wherein the fusion protein is KH02, the amino acid sequence of which is as shown in SEQ ID NO: 14; KH03, the amino acid sequence of which is as shown in SEQ ID NO: 16; or KH04, the amino acid sequence of which is as shown in SEQ ID NO: 18.
2. A nucleotide sequence encoding the fusion protein according to claim 1.
3. The nucleotide sequence of claim 2, wherein the nucleotide sequence is the sequence shown in SEQ ID NO: 13, SEQ ID NO: 15 or SEQ ID NO: 17.
4. An expression vector comprising a nucleotide sequence encoding the fusion protein according to claim 1.
5. The expression vector according to claim 4, wherein, the expression vector is a eukaryotic expression vector or a viral expression vector.
6. The expression vector according to claim 5, wherein, the expression vector is a mammalian cell expression vector or adeno-associated virus vector or adenovirus vector.
7. A pharmaceutical composition comprising the fusion protein according to claim 1 and pharmaceutically acceptable carrier or excipient.
8. A method for treating a disease caused by angiogenesis or vascular growth, the method comprising administering the pharmaceutical composition of claim 7 to a patient in need thereof to inhibit angiogenesis or vascular growth.
9. The method for treating disease caused by angiogenesis or vascular growth according to claim 8, wherein the disease caused by angiogenesis or vascular growth is selected from the group consisting of tumor, age-related macular degeneration, diabetic retinopathy, and chorioretinopathy.
10. The pharmaceutical composition according to claim 7, in the form of a formulation for injection, a freeze-dried injection powder, or an ophthalmic gel.
11. A host cell comprising a nucleotide sequence encoding the fusion protein according to claim 1.
12. The host cell according to claim 11, wherein, the host cell is a CHO cell or a 293 cell.
Description
DESCRIPTION OF THE DRAWINGS
(1)
(2)
(3)
(4)
EXAMPLES
(5) The present invention will be further described through the following Examples. It is to be understood that these Examples are only included to illustrate the present invention and the present invention is not limited to these Examples. Any modification a person skilled in the art could make in light of the present disclosure falls into the scope covered by the claims.
(6) Description of the Sequences
(7) Fragment 1 of human VEGF receptor: the amino acid sequence is SEQ ID NO: 1, the nucleotide sequence is SEQ ID NO: 2;
(8) Fragment 2 of human VEGF receptor: the amino acid sequence is SEQ ID NO: 3, the nucleotide sequence is SEQ ID NO: 4;
(9) Fragment 3 of human VEGF receptor: the amino acid sequence is SEQ ID NO: 5, the nucleotide sequence is SEQ ID NO: 6;
(10) Fusion protein KH01: the amino acid sequence is SEQ ID NO: 12, the nucleotide sequence is SEQ ID NO: 11;
(11) Fusion protein KH02: the amino acid sequence is SEQ ID NO: 14, the nucleotide sequence is SEQ ID NO: 13;
(12) Fusion protein KH03: the amino acid sequence is SEQ ID NO: 16, the nucleotide sequence is SEQ ID NO: 15;
(13) Fusion protein KH04: the amino acid sequence is SEQ ID NO: 18, the nucleotide sequence is SEQ ID NO: 17;
(14) Fusion protein KH05: the amino acid sequence is SEQ ID NO: 20, the nucleotide sequence is SEQ ID NO: 19.
Example 1: Preparation of Fusion Proteins
(15) Material or Reagents
(16) PCR kit (comprising 5buffer, dNTP and Phusion enzyme): M0530L, NEB Corp.
(17) Agarose gel electrophoresis: IQ300, GE Corp.
(18) Buffer 4 (lot no.: 0101201):NEB Corp.
(19) Avrll: R0174L, NEB Corp.
(20) BstZ17I: R0594L, NEB Corp.
(21) PCR product purification kit: QIAGEN Corp., CAT: 28106
(22) 10T4 buffer: B0202S, NEB Corp.
(23) T4 DNA ligase: M0202L, NEB Corp.
(24) Top10 E. coli: CB104, TIANGEN Corp.
(25) 2YT(KAN) plate culture medium: Shanghai Rui Cong Laboratory Equipment Co., Ltd.
(26) Freedom CHO-S Kit: A13696-01, LIFE TECHNOLOGIES CORPORATION.
(27) Clone Pix FL: Genetix Corp.
(28) HiTrap protein A agarose affinity chromatography column: HiTrap protein A HP, 51 ml; GE Corp.
(29) PBS buffer (20 mM phosphate, pH 7.4): SD117-500 ml, Shanghai Biotech Co., Ltd.
(30) ForteBio bio-molecular interaction detector: Octet QKe, Pall Corp.
(31) Analyzer for thermal stability of protein: Optim2, Avacta Corp.
(32) VEGF: R&D Systems Inc.
(33) NHS-LCLC-Biotin: Thermo Corp.
(34) 1. Construction of Plasmid Comprising Sequences Encoding Fusion Protein.
(35) 1.1 Synthesis of Genes and Primers
(36) Synthetic fragments 1 (SEQ ID NO: 21) and 2 (SEQ ID NO: 22) and primers P1-P10 (sequences are shown in SEQ ID NO: 23-32) were synthesized by Beijing GENEWIZ, Inc. Synthetic fragments 1 and 2 were recombined into plasmid vector pUC19 (Beijing GENEWIZ, Inc.). Synthetic fragment 1 comprises nucleotide sequence encoding human VEGF receptor fragment and signal peptide sequence, synthetic fragment 2 comprises coding sequence for human IgG1 Fc. The coding sequences for the present fusion proteins were constructed by PCR using the below primers and synthetic fragments 1 and 2 as the template for constructing respective fusion proteins.
(37) 1.2 Obtaining the Coding Sequences for Fusion Proteins.
(38) Coding sequence for each fusion protein was amplified in two parts, wherein the first part is human VEGF receptor fragment; and the second part is human IgG1 Fc fragment. The respective target fragment for each part was obtained by the specific primers, and then the human VEGF receptor fragment and human IgG1 Fc fragment were linked by overlapping PCR, giving the final full gene sequence. The reaction system for the amplification PCR of both the first and the second parts was (total volume 50 l): 10 l of 5buffer, 2 l of dNTP, 1 l of specific forward and reverse primers each, 1 l of template (the above synthetic fragment 1 or 2), and 0.5 l of Phusion enzyme (PCR fidelity enzyme), adjusted to 50 l with double distilled water. The reaction condition was as follows: initial denaturation at 98 C. for 30 s, followed by 10 cycles of 98 C. for 10 s and 68 C. for 2 min, followed by 30 cycles of 98 C. for 10 s, 55 C. for 30 s, and 72 C. for 50 s, and finally 72 C. for 5 min. Particularly, for kh02, the primers for the first part were P1 and P6, the template was synthetic fragment 1; the primers for the second part were P5 and P2, the template was synthetic fragment 2. For kh03, the primers for the first part were P1 and P8, the template was synthetic fragment 1; the primers for the second part were P7 and P2, the template was synthetic fragment 2. For kh04, the primers for the first part were P1 and P10, the template was synthetic fragment 1; the primers for the second part were P9 and P2, the template was synthetic fragment 2. For the coding sequence of protein KH01 (kh01), the primers for the first part were P1 and P4, the template was synthetic fragment 1; the primers for the second part were P3 and P2, the template was synthetic fragment 2. The gene products were tested by agarose gel electrophoresis. A total of 8 fragments, i.e. kh01-Q, kh01-H, kh02-Q, kh02-H, kh03-Q, kh03-H, kh04-Q and kh04-H, were obtained. The reaction system for overlapping PCR was (total volume 50 l): 10 l of 5buffer, 2 l of dNTP, 1 l of the above amplified first and second fragment as template respectively (for example, for amplification of the full length of kh01, 1 l of kh01-Q PCR recovery product and 1 l of kh01-H PCR recovery product were used), 1 l of forward and reverse primers (P1, P2) respectively, and 0.5 l of Phusion enzyme (PCR fidelity enzyme), adjusted to 50 l with double distilled water. The reaction condition was as follows: initial denaturation at 98 C. for 30 s, followed by 30 cycles of 98 C. for 10 s, 55 C. for 30 s, and 72 C. for 50 s, and finally 72 C. for 5 min. The gene products were tested by agarose gel electrophoresis (IQ300, GE). A total of 4 gene fragments, named as kh01-1 (corresponding to SEQ ID NO: 11, but with additional signal peptide encoding sequence), kh02-1 (corresponding to SEQ ID NO: 13, but with additional signal peptide encoding sequence), kh03-1 (corresponding to SEQ ID NO: 15, but with additional signal peptide encoding sequence), and kh04-1 (corresponding to SEQ ID NO: 17, but with additional signal peptide encoding sequence), were obtained. The amplified fragments were found to have the expected size by electrophoresis.
(39) 1.3 Enzyme Digestion of Vectors and Gene Fragments
(40) pCHO1.0 plasmid (from Life Technologies, catalog no.: A13696-01), kh01-1, kh02-1, kh03-1, and kh04-1 were subjected to double enzyme digestion respectively. The system for enzyme digestion was as follows. 40 l of pCHO1.0 plasmid or kh01-1, kh02-1, kh03-1, or kh04-1 amplified fragment, 10 l of 10buffer 4 (NEB), 5 l of Avrll (R0174L, NEB) and BstZ17I (R0594L, NEB) each, and 45 l sterile water were added to 1.5 ml EP tube, and the mixture was incubated at 37 C. for 5 h after being mixed evenly. The product was recovered by PCR product purification kit (CAT: 28106, QIAGEN).
(41) 1.4 Ligation and Transformation of Recombinant Plasmid
(42) The recovered pCHO1.0 fragment (larger fragment obtained by Avrll and BstZ17I digestion, about 13 kb) and the recovered kh01-1, kh02-1, kh03-1, or kh04-1 fragment (Avrll and BstZ17I digested), obtained from digestion by the same enzymes, were ligated together in the presence of T4 DNA ligase. The reaction system for this reaction was as follows. 2 l of pCHO1.0 fragment (Avrll and BstZ17I digested), 6 l of kh01-1, kh02-1, kh03-1, or kh04-1 (Avrll and BstZ17I digested) fragment, 1 l of 10T4 buffer (B0202S, NEB), and 1 l of T4 DNA ligase (M0202L, NEB) were added to 1.5 ml EP tube, the mixture was mixed evenly, and then incubated at room temperature (around 20 C.) for 4 h. The ligation product was transformed to competent Top 10 E. coli cell (CB104, TIANGEN) and plated on 2YT(KAN) plate (Shanghai Ruicong Laboratory Equipment Co., Ltd) for overnight incubation at 37 C. The plates were identified as kh01, kh02, kh03, and kh04.
(43) 1.5 Colony PCR Screening of Recombinant Plasmids.
(44) Single recombinant colonies were picked from kh01, kh02, kh03, and kh04 plates and were incubated at 37 C. for 3-5 h. After incubation, these colonies were used as PCR templates for PCR screening. The reaction system (total volume 20 l) for this PCR amplification was as follows. 10 L of 2Taq HS (R013A, TAKATA), 2 L of bacterial liquid as template, and 1 L of forward primer and reserve primer (P1 and P2, each has a final concentration 0.3 mol/L), adjusted to 20 L with double distilled water. The condition for the reaction was: 94 C. for 3 min, followed by 30 cycles of 94 C. for 60 s, 53 C. for 60 s, and 72 C. for 120 s, and finally 72 C. for 5 min. The results showed that a target band of about 1.6 Kbp was amplified from all colonies, suggesting that these colonies are all positive clones.
(45) 1.6 Identification of Recombinant Plasmid by Enzymatic Digestion.
(46) The colonies identified as positive by the colony PCR were inoculated, followed by plasmid extraction and identification by enzymatic digestion. Firstly, plasmids were extracted from recombinant bacteria and then analysed by enzymatic digestion. The system for enzymatic digestion was as follows. 2 l of Plasmid, 1 l of 10buffer 4, 1 l of Avrll and 1 l of BstZ17I were added to 1.5 ml EP tube, and sterile water was added to adjust the total volume to 10 l, and then the mixture was mixed evenly and then reacted at 37 C. for 4 h. Agarose gel electrophoresis confirmed that a band around 1.6 kb was obtained after enzymatic digestion for all colonies, suggesting that the picked clones are all positive clones.
(47) 1.7 Identification of Recombinant Plasmid by Sequencing
(48) Colonies identified by the colony PCR and enzymatic digestion as positive were sequenced (Suzhou GENEWIZ biotech Co. Ltd.). The results of the sequencing were as expected. These expression plasmids were stored for further usage. Clones with positive sequencing results were numbered as the following, kh01-1 as 610, kh02-1 as 711, kh03-1 as 812, kh04-1 as 915.
(49) 2. Transfection of Plasmid and Screening of Cells
(50) Transfection was conducted using host cell CHO-S in Freedom CHO-S Kit (A13696-01, LIFE TECHNOLOGIES) as suggested by the manufacturer. Four plasmids were transfected in this experiment: 610, 915, 812, and 711. Cells transfected with plasmids were incubated by shake-flask culturing. The culture was performed in CD FortiCHO (from Life Technologies) as the culture medium under 37 C., 8% CO.sub.2, and 110 rpm/min for 48 h. Viability and count of the cells were detected by cell counter.
(51) 48 hours after the transfection, the two-phase selection scheme was conducted: 10P/100M, 20P/200M (P=10 g/mL puromysin, M=nM methotrexate (MTX)); 30P/500M, 50P/1000M, with CD-FortiCHO being used as the culture medium. Cells obtained after the first screening were 610, 915, 812 and 711 (i.e., comprising the above respective plasmid). The single clone screening was performed at a seeding density of 500 viable cells/ml, and 48 hour after the transfection, each cell pool was seeded to 8 six-well plates and incubated for 1 week in incubator. The growth of cell clone in each plate was observed under fluorescence microscope. Single clone was picked by Clone Pix FL (Genetix). Protein expression and purity were detected to select clone for scale-up culture.
(52) 3. Expression, Purification and Identification of Proteins
(53) Clone cells with high yield were picked for scale-up culture from 96-well plate to 24-well plate, then to 6-well plate and then to 50 ml shake-flask.
(54) Supernatant of cell culture incubated for 4-6 days was collected and centrifuged to remove cell debris. The collected supernatant was filtered by 0.45 m filter. PH was adjusted to 7.4. Fusion proteins were purified by HiTrap protein A affinity chromatography column (HiTrap protein A HP, 51 ml; GE). The column was rinsed by 5deionized water, and balanced by 5PBS buffer (20 mM phosphate, pH 7.4) (SD117-500 ml, Shanghai Biotech Co., Ltd). The column was loaded with samples and eluate was collected for detection. The column was washed with ten column volume of PBS buffer (0.02 mol/L phosphate, pH 7.4) to remove non-target protein and target protein was eluted from the column by 0.1M glycine buffer (pH3). The purities of the proteins were all detected to be above 90% by SDS-PAGE (polyacrylamide gel electrophoresis).
Example 2: Analysis of Purity and Yield of the Protein
(55) Purities of proteins in fermentation broth were detected by SEC-HPLC. Also, the expression of each protein obtained in Example 1 was detected by ForteBio bio-molecular interaction detector (Octet QKe, Pall). The results are shown in Table 1. It can be seen that the expression levels and purities of fusion proteins KH02-KH04 are better than the same of KH01 protein. Among these proteins, fusion protein KH02 is the best in terms of purity and expression level. The purity of this protein is still maintained above 80% on day 9 of the incubation without addition of nutriment.
(56) TABLE-US-00001 TABLE 1 Comparison of purity of cell culture supernatant of the proteins Purity (%) Expression level (mg/L) Culture supernatant Day 7 Day 9 Day 7 Day 9 KH01 53.5 40.1 173.54 305.27 KH02 84.3 83.5 381.85 578.15 KH03 55.5 42.8 201.3 300.85 KH04 61.5 47.7 136.55 342.10
Example 3: Detection of Thermal Stability of VEGF Receptor Fusion Protein
(57) (1) Detection of Tendency for Thermal Denaturation of the VEGF Receptor Fusion Protein
(58) The spacial conformation of a protein will be unfolded when the protein is subjected to heat treatment. Hydrophobic amino acid residues (such as tryptophan, tyrosine, and phenylalanine) containing aromatic group will be exposed. The degree of protein unfolding can be reflected by fluorescence intensity (IF) inside the aromatic groups. During protein unfolding, fluorescence spectrum of the internal fluorophores will change. Protein with natural structure (normally folded) has lower internal fluorescence intensity and the peak is at around 330 nm. In contrast, denatured protein has significantly increased internal fluorescence intensity and the peak will be shifted to around 350 nm. Half thermal denaturation temperature Tm can be calculated by analysing the change of internal fluorescence intensity and the shift of peak of the protein, and indirectly reflects the tendency for thermal denaturation.
(59) The tendency for thermal denaturation of VEGF receptor fusion protein was detected by analyzer for thermal stability of protein (Optim2, Avacta). About 15 l of each sample to be detected (1 mg/ml PBS, pH7.2, purity above 90%) was added to Optim2 reaction tube. The range for temperature scanning was set to 25 C.-95 C. Samples were incubated at each temperature point for 60 s. The data were processed by Optim2 analysis software. The results are shown in
(60) TABLE-US-00002 TABLE 2 Parameters for thermal denaturation of the VEGF receptor fusion protein Denaturation Denaturation Denaturation Samples temperature 1 temperature 2 temperature 3 KH01 43.9 C. 61.2 C. 71.2 C. KH02 48.0 C. 63.7 C. 77.6 C. KH03 / 63.4 C. 76.7 C. KH04 / 63.3 C. 77.1 C.
(61) (2) Analysis of Tendency for Thermal Aggregation of the VEGF Receptor Fusion Protein
(62) Light scattering will occur when protein is exposed to UV light. In a certain range, intensity of static light scattering is linearly related to the size of the protein (10-600 KD). Detecting the intensity of light scattering SCS (Static Light Scattering) can show the change in protein size. Tendency for protein aggregation can be indirectly reflected by calculating aggregation onset temperature (Tagg) of the protein.
(63) Tendency for thermal aggregation of the VEGF receptor fusion protein was detected by analyzer for thermal stability of protein (Optim2, Avacta). About 15 l of each sample to be detected (1 mg/ml PBS, pH7.2, purity above 90%) was added to Optim2 reaction tube. The range for temperature scanning was set to 25 C.-95 C. Samples were incubated at each temperature point for 60 s. The data were processed by Optim2 analysis software. The results are shown in
(64) TABLE-US-00003 TABLE 3 Tendency for thermal aggregation of the VEGF receptor fusion protein Sample Tagg KH01 65.6 C. KH02 71.8 C. KH03 69.8 C. KH04 68.8 C.
Example 4: Detection of Biological Activities of VEGF Receptor Fusion Protein
(65) (1) Proliferation of HUVEC cell
(66) Human umbilical venous endothelial cells (HUVEC, ScienCell) that grew well were seeded to 96-well plate at 310.sup.3 cell/well and 100 l/well at 37 C., 5% CO.sub.2 for 20 hours. ECM medium (Endothelial Cell Medium, catalog no. 1001, Sciencell) containing 2% fetal bovine serum was used to prepare VEGF receptor fusion protein with different molar concentrations (0.0023, 0.007, 0.023, 0.065, 0.19, 0.57, 1.7, 5, 15, 45, 135 nM), which was then mixed evenly with 40 ng/ml VEGF (R&D SYSTEMS), incubated for 2 h. 100 l of the mixture was then added to HUVEC cells in 96-well plate per cell in triplet. The plate was continuously incubated with 5% CO.sub.2 for 96 hours. At the end of incubation, the CCK8 (Dojindo) was added. The inhibitory effect of VEGF receptor fusion protein was shown in EC.sub.50. The results are shown in
(67) TABLE-US-00004 TABLE 4 VEGF receptor fusion proteins inhibit the VEGF-stimulated proliferation of HUVEC cells Sample EC.sub.50 (nM) KH01 1.93 KH02 1.26 KH03 2.11 KH04 1.43
(68) (2) Migration of HUVEC Cells
(69) The effect of the present fusion protein on the migration of HUVEC cells was tested by using modified Boyden chamber (FluoroBlok Biocoat angiogenesis system: Endothelial cell migration, BD). HUVEC cells that grew well were seeded to upper compartment of the Boyden chamber at 310.sup.5 cell/ml and 75 l/well. ECM basic medium (catalog no. 1001, Sciencell) containing 2% fetal bovine serum was used to prepare VEGF receptor fusion protein with different molar concentrations (13333 nM, 4444 nM, 1481 nM, 494 nM, 164.5 nM, 54.8 nM, 18.3 nM, 6.1 nM), which was then mixed evenly with 500 M VEGF (R&D SYSTEMS), incubated for 2 h, and then added to the lower compartment-of the Boyden chamber at 225 l/well. The whole chamber was incubated at 37 C., 5% CO.sub.2 for 20-24 h. The culture medium in the lower compartment was removed. Fluorescent dye Calcein AM (Anaspec) with a final concentration of 5 g/ml formulated by using HBSS buffer (Hanks Balanced Salt Solution) was added. The chamber was incubated at 37 C. with 5% CO.sub.2 for 90 minutes in the dark, and then the fluorescence value was detected at excitation wavelength of 494 nm and detection wavelength of 517 nm on a multi-mode reader. Relative migration of the cells was calculated. As shown in
Example 5: Test of Binding Affinity Between VEGF Receptor Fusion Protein and VEGF
(70) The binding affinity between human VEGF receptor fusion protein and human VEGF was detected on ForteBio bio-molecular interaction detector (Octet QKe, Pall) by Biolayer-Interferometry (BLI). Human VEGF (catalog no. 293-VE-010, R&D SYSTEMS) and NHS-LCLC-biotin (catalog no. 21338, Thermo) were mixed evenly at 1:3 molar ratio, and placed at room temperature for 1 h, then the remaining NHS-LCLC-biotin was removed, giving the final labelled product biotin-hVEGF at 50 g/ml. 50 g/ml biotin-hVEGF was loaded on streptavidin sensor. Samples to be tested were formulated to different concentrations (600 nM, 200 nM, 66.7 nm, 22.2 nM, 7.4 nm, 2.46 nM, 0.82 nM, respectively) by sample diluting buffer (PBS, 0.1% BSA, 0.02% Tween-20, 0.003% NaN.sub.3). The sample diluting buffer was served as blank control. Parameters for binding kinetics between hVEGF and receptor fusion protein were detected under kinetics analysis mode. The results are shown in Table 5. It is shown that the VEGF receptor fusion proteins expressed in Example 1 all bind significantly with human VEGF. Among these proteins, the KD value of KH02 is about 0.33 nM. Especially, the dynamic dissociation rate of KH02 complex with VEGF is lower than other fusion proteins, suggesting the highest binding affinity.
(71) TABLE-US-00005 TABLE 5 Parameters for binding affinity between VEGF receptor fusion protein and human VEGF KD (nM) kon (1/Ms) kdis (1/s) KH01 0.51 0.04 6.91 0.09 10.sup.4 3.53 0.06 10.sup.5 KH02 0.33 0.02 5.16 0.05 10.sup.4 1.70 0.05 10.sup.5 KH03 1.17 0.05 8.67 0.02 10.sup.4 1.01 0.04 10.sup.4 KH04 0.35 0.02 2.06 0.05 10.sup.5 7.27 0.02 10.sup.5 Notes: Kon: association constant, Kids: dissociation constant
Example 6: Affinity Experiment of Fusion Proteins
(72) 1.1 Reagents
(73) TABLE-US-00006 TABLE 6 list of the reagents used Name Manufacturer Catalog no. Lot No. rhVEGF165 R&D Systems 293-VE II4113062 Inc. Human IgG-Fc BETHYL Corp. A80-104P A80-104P-78 antibody HRP TMB substrate R&D Systems DY999 308030 solution Inc. BSA BOVOGEN BSA S1.0 269 Corp. Tween 20 Riedel-deHan 63158 41620 Corp. 20xPBS buffer Biotech Co. SD8117 13042099Z
(74) TABLE-US-00007 TABLE 7 list of instruments Name Brand Device no. Microplate reader Molecular Devices Corp. JC084 Mini vortex mixer Shanghai LUXI Corp. GY227 Electronic balance Sartorius Corp. JC082
(75) 1.2 Protocol
(76) 1) Reagent Preparation
(77) i. 10PBS buffer: 80.1 g of NaCl, 2.0 g of KCl, 2.0 g of KH.sub.2PO.sub.4, and 29.0 g of Na.sub.2HPO.sub.4.12H.sub.2O were dissolved in pure water and the final volume was set to 1000 ml;
(78) ii. 1PBS buffer: 100 ml 10PBS buffer was dissolved in 850 ml pure water, pH was adjusted to pH 7.2-7.4, and the final volume was set to 1000 ml;
(79) iii. Carbonate buffer: 1.59 g of Na.sub.2CO.sub.3 and 2.93 g of NaHCO.sub.3 were dissolved in 1000 ml ultrapure water, pH was 9.6-9.8, and the solution was stored at room temperature, and filtered with 0.22 m filter before use.
(80) iv. BSA (bovine serum albumin): stored at 4 C.;
(81) v. Rinsing buffer: 1PBS containing 0.05% (v/v) polysorbate 20;
(82) vi. Blocking solution and diluent: 1PBS containing 1% (w/v) BSA;
(83) vii. Stop solution (2N H.sub.2SO.sub.4): 27.8 ml concentrated sulfuric acid was added slowly to 472.2 ml pure water. Concentrated sulfuric acid is a strong corrosive liquid and should be added with stirring by glass rod and handled with extra care;
(84) viii. rhVEGF.sub.165 (R&D SYSTEMS, 293-VE, 50 g/vial) stock solution: 3 ml 1PBS was filtered by 0.22 m filter. 800 l filtered PBS was added to a previously unopened vial of rhVEGF.sub.165. When the visible solid material in the vial was dissolved, 200 l additional filtered PBS was added. The vial was placed at room temperature for 10 min to fully dissolve rhVEGF.sub.165. The concentration of the dissolved rhVEGF.sub.165 stock solution was 50 /ml. The stock solution was divided into aliquots of 25 l/vial. The stock solution can be stored at 20 C. for 6 months.
(85) ix. Human IgG-Fc antibody HRP detection antibody (BETHYL A80-104P): 1 mg/ml, stored at 4 C.
(86) 2) Coating the Plate
(87) 20 l rhVEGF.sub.165 stock solution was added to 7980 l carbonate buffer, the mixture was mixed evenly and named as coating solution A and its concentration was 125 ng/ml. 2500 l coating solution A was added to 2500 l carbonate buffer, the mixture was mixed evenly and named as coating solution B. A microplate was coated in columns 1-6 with coating solution A, and in columns 7-12 with coating solution B, wherein the volume used in the coating was 100 l/well. The plate was sealed by sealing gel and incubated overnight at room temperature.
(88) 3) Rinsing the Plate
(89) The plate was washed with 250 l rinsing buffer per well and soaked for 120 s, which was repeated for three times. The remaining drops were removed by patting the plate on a paper towel until there was no obvious water mark on the paper towel.
(90) 4) Blocking the Plate
(91) Blocking solution was added at 300 l/well by an 8-channel pipette. Then the plate was sealed by sealing gel and incubated at 37 C. for 2 hours.
(92) 5) Preparation of Sample
(93) Samples (fusion proteins KH02 and KH05) were diluted to 1600 ng/ml according to initial protein concentration. The volume of the 1600 ng/ml sample should be at least above 800 l. A 4 serial dilution was conducted by adding 600 l diluting solution to 200 l sample, and such a diluting operation was repeated in series to obtain 8 different concentration gradients (including 1600 ng/ml).
(94) 6) Adding the Sample
(95) The plate was rinsed as described above. 100 l/well samples were added to microplate successively and the plate was sealed by sealing gel. The loading was proceeded from high concentration to low concentration in duplicate. After the loading, the plate was incubated at 37 C. for 1 h.
(96) 7) Adding Test Antibody
(97) The plate was rinsed as described above. Human IgG-Fc antibody HRP 0.5 l was diluted by 10 ml blocking solution and the mixture was mixed evenly. 100 l/well diluted test antibody was added to wells, and the plate was incubated at 37 C. for 1 h. 8) Color Development
(98) The plate was rinsed as described above. 100 l/well TMB substrate solution was added. The plate was incubated under dark and at room temperature for 5 min.
(99) 9) Stopping and Obtaining Readout
(100) 50 l/well stop solution was added and the reaction was terminated. The microplate was placed under microplate reader to get a readout under 450 nm.
(101) 1.3 Results of Affinity Test
(102) TABLE-US-00008 TABLE 8 Results of affinity test of samples Sample treatment Kd (unit: pM) KH05 Day 0 27.253 5 days at high 65.471 temperature 10 days at high 174.983 temperature KH02 Day 0 22.491 5 days at high 46.420 temperature 10 days at high 70.504 temperature
(103) From the results in Table 8, it can be seen that the affinity of KH02 after a 10-day high temperature treatment is still within acceptable range (22-84 pM). However, affinity of KH05 after a 10-day high temperature treatment exceeds far beyond the acceptable range. Thus, the stability of KH02 in terms of activity is better than KH05.
Example 7: Purity Test at High Temperature
(104) 1.1 Reagents and Devices
(105) TABLE-US-00009 TABLE 9 list of reagents used Name Catalog No. Specification Manufacturer Na.sub.2HPO.sub.412H.sub.2O 20130527 500 g/vial Guangdong Guanhua Corp. NaCl 201100905 500 g/vial Guangdong Guanhua Corp. Arg-HCl 20110318 N/A Shanghai Yuanju Biotech Corp. Concentrated HCl 20121101 N/A Chengdu Kelong Corp. Chromatographic S0534 TSK G3000 SWxl, TOSOH Corp. column 5 m, 7.8 300 mm TSK guard R1479 40 * 60 TOSOH Corp. column
(106) TABLE-US-00010 TABLE 10 list of main devices used Name Device No. Manufacturer Pattern No. High performance JC073 Agilent Technologies 1200 liquid spectrometry Inc. Mini vortex mixer GY265 Shanghai LUXI Corp. WH-3 Electronic balance JC080 Sartorius Corp. BS224S pH meter JC077 METTLER Corp. TOLEDO S40K
(107) 1.2 Protocol
(108) 1) Reagent Preparation
(109) i. PBS mobile phase: 7.16 g Na.sub.2HPO.sub.4.12H.sub.2O, 8.77 g NaCl and 42.2 g Arg were dissolved in 800 ml ultrapure water, the pH was adjusted to pH7.2 by HCl, the final volume was set to 1000 ml and the resulting solution was filtered by 0.22 m filter.
(110) ii. Guarding solution for chromatographic column (0.05% NaN.sub.3): NaN.sub.30.5 g was dissolved in 1000 ml ultrapure water and the resulting solution was filtered by 0.22 m filter.
(111) iii. Ultrapure water: it was filtered by 0.22 m filter.
(112) 2) Preparation of Sample
(113) Since the concentration of each sample (KH02, KH05) was 1 mg/ml, these samples can be loaded directly.
(114) 3) Conditions for Chromatographic Analysis
(115) Mobile phase: PBS mobile phase;
(116) Chromatographic column: TSK G3000 SW.sub.XL (5 m, 7.8*300 mm);
(117) Temperature of the column: 25 C.; flow rate: 0.5 ml/min; detection wavelength: 280 nm; loading volume: 50 l.
(118) 1.3 Results
(119) TABLE-US-00011 TABLE 11 Results of SEC-HPLC purity test of the samples Sample Treatment Detected purity (%) KH05 Day 0 99.19 5 days at high 54.06 temperature 10 days at high 25.20 temperature KH02 Day 0 96.75 5 days at high 94.29 temperature 10 days at high 65.15 temperature
(120) As can be seen from Table 11, the purity of KH05 dropped to 54% after a 5-day high temperature treatment. The purity of KH05 was only 25% after a 10-day high temperature treatment. The decrease of purity of KH02 was inapparent after a 5-day high temperature treatment. The purity of KH02 sample was up to 65% after a 10-day high temperature treatment.