Ethics statement
All experiments and procedures were approved by the Ethics Committee of Pasteur Institute of Iran (IR.PII.REC.1399.013) and performed in accordance with the approved guidelines and regulations.
Expression cassette
GCSF (Filgrastim) encoding amino acid sequence was extracted from drug bank (Accession No. DB00099). ABD094 amino acid sequence compromising of 46 amino acids (Mw: ~ 5.8 kDa) was obtained from published patents (US10206975B2 and JP2014557602A). The two amino acid sequences were fused using a flexible Gly4Ser linker peptide compromising 15 amino acids ((G4S)3). A histidine tag was designed at upstream of the ABD-GCSF gene cassette (Supplementary Fig. 7), synthesized after E. coli codon optimization and subcloned into pET28a expression vector (Novagen, USA) at NcoI and HindIII restriction sites.
Protein expression
The expression of recombinant protein was induced in E. coli BL21 (DE3) (Novagen, USA) host cells. Luria–Bertani (LB) broth medium supplemented with kanamycin 30 µg/ml was inoculated with the recombinant bacteria and incubated at 37 °C shaker incubator until optical density of the medium reached 0.5 at 600 nm. Isopropyl β-d-1-thiogalactopyranoside (IPTG) was used as expression inducer (0.25 mM) and bacteria were incubated for further 6 h at 30 °C. Bacterial pellet was collected by centrifugation at 9000 rpm for 3 min and protein expression level was quantified on 12% SDS-PAGE stained with Coomassie Brilliant Blue G250 dye.
Bacterial lysates were run on 12% SDS-PAGE and transferred to nitrocellulose membrane (Amersham, UK) in a semi-dry transfer system (Bio-RAD) (18v, 25 min). After overnight (o/n) blocking the membrane with 2% (w/v) skim milk in phosphate buffered saline (PBS) at 4 °C, 1:2000 dilution of Horse Radish Peroxidase (HRP) conjugated anti-His antibody (Sigma, USA) was added to the membrane for 2 h at room temperature (RT). The membrane was washed 4 times with PBS/Tween-20 (0.05%) and the corresponding His-tagged fusion protein band was visualized using 3,3’-diaminobenzidine (DAB) substrate (Sigma, USA).
Protein purification
To purify the recombinant protein, colonies was inoculated into 500 ml LB broth medium under the above-mentioned culture condition. Bacterial pellet was resuspended in lysis buffer I (50 mM NaH2PO4, 10 mM Imidazole, 300 mM NaCl; pH8.0) and sonicated by 20 pulses (20 s with the same interval time) and centrifuged at 10,000 rpm for 20 min at 4 °C. The pellet was resuspended in lysis buffer II (50 mM NaH2PO4, 300 mM NaCl, 10 mM Imidazole, 8 M Urea; pH8.0) and sonicated for further 3 pulses (20 s/pulse) and centrifuged again. The supernatant was filtered through 0.45 µm syringe filter and was loaded to the Ni-agarose resin (ABT Agarose Bead Technologies, Spain) under denaturing conditions. The column was washed with 30 volume of washing buffer I (50 mM NaH2PO4, 300 mM NaCl, 30 mM Imidazole, 8 M Urea, 0.1% Triton-X114; pH8.0) to remove weakly bounded proteins and bacterial lipopolysaccharides (LPS) followed by the second washing step (50 mM NaH2PO4, 300 mM NaCl, 30 mM Imidazole, 8 M Urea, pH8.0) to remove residual Triton-X114. The recombinant protein was eluted by the elution buffer (50 mM NaH2PO4, 300 mM NaCl, 250 mM Imidazole, 8 M Urea; pH8.0) at a flow rate of 1.5 ml/min.
Refolding of the eluted protein was performed through dialysis in order to gradually remove urea (from 8 M to 0). The dialyzed protein was finally kept in phosphate-buffer (PB) (8 mM Na2HPO4, 1 mM KH2PO4, 137 mM NaCl, 3 mM KCl; pH7.4). Protein concentration was performed using Centriprep-3 kDa (Amicon, USA) and the protein concentration was measured by NanoDrop™ 3000 spectrophotometer (Bio-RAD).
The pyrogenicity of the purified recombinant protein was quantified by Pyrotell gel clot LAL kit (USA; Sensitivity 0.25EU/ml of analyzed solution) according to the manufacturer’s instruction. Briefly, Limulus Amebocyte lysate was incubated with serially diluted ABD-GCSF protein samples at 37 °C for 60 min and a positive test will be indicated by the formation of gel which does not collapse when the tube is inverted.
DTNB colorimetric analysis (Ellman)
Ellman’s reagent, 5, 5’‐dithiobis‐(2‐nitrobenzoic acid) (DTNB), was used for quantification of free sulfhydryl groups in ABD-GCSF protein in comparison with Filgrastim. In brief, the reaction buffer (0.1 M sodium phosphate, 1 mM EDTA; pH8.0) containing 5 mM Filgrastim or different concentrations of ABD-GCSF (5, 3, 2, 1 mM) and DTNB was incubated at RT for 15 min. The change in optical absorbance was measured at 412 nm. The concentration of free thiol groups was measured using molar extinction coefficient of chromophore (1.415 × 104 M−1 cm−1). The negative control sample was DTNB in the absence of any protein. Different concentrations (1.5, 1.25, 1.0, 0.75 and 0.25 mM) of cysteine amino acid solution were used as positive control.
Size exclusion chromatography (SEC)
For comparing the hydrodynamic volume of commercially available GCSF protein (Filgrastim; Pooyesh Darou, Iran) and ABD-GCSF, size exclusion chromatography (SEC) was carried out according to the European Pharmacopoeia version 9.1. In brief, proteins were diluted in 0.06 M sodium acetate (pH4.0) to 200 µg/ml. In the next step, 8 µg of each protein was injected into TSK gel G3000SWxl column (Tosoh Bioscience, Japan) connected to a Shimadzu HPLC system (Kyoto, Japan) and eluted by an isocratic mobile phase of 0.03 M (NH4)2HPO4 (pH7.0) at a flow rate of 0.5 ml/min at 30ºC. The ultraviolet (UV) absorbance was recorded at 215 nm. Molecular weight of the proteins was also estimated based on the retention time of gel filtration standard protein (Bio-RAD, Cat No. 151-1901).
DLS
Dynamic light scattering experiments were performed to determine the effect of ABD tag on hydrodynamic radius of the GCSF protein. In brief, 0.5 mg/ml of each protein (Filgrastim, PEG-Filgrastim manufactured by CinnaGen, Iran, and ABD-GCSF) was prepared in double distilled water (ddH2O) and analyzed by Zetasizer ZEN3600 system (Malvern, Germany) at 25 °C. All results were reported as hydrodynamic radius in nm.
IFS
To investigate tertiary conformational changes, intrinsic fluorescence emissions of proteins were measured using Cary Eclipse Varian Spectrophotometer (Agilent Technologies, Germany) in emission wavelength ranging from 300 to 400 nm. 300 µg/ml concentration of the proteins was prepared in PBS (pH7.4) in 1 cm path length quartz cells. Fluorescence emission was excited at 295 nm. Excitation and emission slits of 5 and 10 nm were considered.
CD analysis
For finding possible secondary structural changes of ABD-GCSF protein in comparison to the commercially available GCSF molecules (Filgrastim and PEG-Filgrastim), Circular dichroism (CD) analysis was performed using J-810 Spectropolarimeter (Jasco Instruments, Japan). In brief, 0.2 mg/ml protein in double distilled water (ddH2O) was exposed to far (190–260 nm) and near (250–320 nm) UV spectra within 1 mm path length quartz cell at 25 °C. CD spectra were recorded by the average of two scans, 1 nm band width and scanning speed of 500 nm/min.
Cell proliferation assay
GCSF-dependent NFS-60 cells (murine myeloblastic cell line, Pasteur Institute of Iran) were tested in in vitro biological activity assessment of ABD-GCSF fusion protein in comparison with Filgrastim and PEG-Filgrastim. NFS-60 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 0.025 mM sodium-pyruvate, 1% penicillin/streptomycin, 0.025 mM 2-ME and 33 IU/ml IL-3 (Sigma, USA). 5 × 103 cells/well were seeded in 96-well plates and serially dilutions (0.0001, 0.001, 0.01, 0.1, 1, 10, 100, 1000, 10,000 ng/ml) of Filgrastim, PEG-Filgrastim and ABD-GCSF were added to the wells and incubated for 72 h. Culture medium alone was served as negative control. The cells were treated with 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) (Sigma, USA) and incubated for further 4 h at 37 °C. 1% SDS was added to the wells and incubated for 16 h at 37 °C. Optical densities were measured at 550 nm using microplate reader spectrophotometer (BioTeK, USA). All tests were done in triplicate. EC50 values, the concentration of substrate in which 50% of maximum proliferation was achieved, were calculated by Prism software (v. 8.0).
Albumin binding assay
A home-made ELISA assay was developed to measure the affinity of ABD-GCSF towards human serum albumin (HSA). In brief, 96-well plate was coated with HSA (1 µg/well) in carbonate-bicarbonate buffer at 4 °C (o/n). The plate was blocked with 2%w/v skim milk for 2 h at 37 °C. After washing step with PBS supplemented with 0.05% Tween-20, serial dilution of ABD-GCSF (0-1000 nM) was added to the wells and incubated for 2 h RT. HRP-conjugated anti-His antibody (1: 2000) was used as the secondary antibody. Assessment of binding reaction was done using 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate (Sigma, USA). H2SO4 was added to the wells to stop the reaction and optical density was measured at 450 nm by a microplate reader (BioTeK, USA).
Pharmacodynamics
The effect of ABD-GCSF in acceleration of neutrophil count was investigated in normal Sprague Dawley male rats (6–7 weeks, 250–300 g weight). The research protocols and animal studies were approved by the Ethics Committee of Pasteur Institute of Iran (IR.PII.REC.1399.013) and followed ARRIVE reporting guidelines62. Animals were adopted to the conditions of light and humidity for one week and were randomly divided into five groups (five rats/group). The animals except control group (Group 1) intraperitoneally (i.p) received 100 mg/kg of cyclophosphamide (CPA) for induction of neutropenia on day zero12,63. On day 1, the rats in Groups 2 to 4 subcutaneously (s.c) received 100 µg/kg of Filgrastim, PEG-Filgrastim, or ABD-GCSF, respectively and the 5th group received PBS alone as control12,30,64. Blood samples were collected from tail vein in non-vacuumed K2EDTA Nex tubes (Nexamo Technoplast, India) according to the designed time schedule on 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 11 days post GCSF injection. Complete blood counting (CBC) of neutrophils, lymphocytes, eosinophils, monocytes, red and white blood cells was done by hematology analyzer Celltac alpha (Nihon Kohden, Japan).
Pharmacokinetics
To determine the basic pharmacokinetic parameters of Filgrastim, PEG-Filgrastim and recombinant ABD-GCSF, plasma samples were used to quantitate GCSF concentration in the above-mentioned rat groups using human Quantikine GCSF ELISA kit (R&D Systems, USA). Briefly, blood samples were collected at 0, 0.5, 1, 2, 4, 8, 10, 24, 48, 76, 92, 120, 144, 168, 192 and 240 h post s.c drug injection and centrifuged at 3,000 rpm for 15 min to separate plasma. GCSF level was measured using the kit. Pharmacokinetic parameters were calculated using the log linear trapezoidal method. Half-life (t1/2), maximum concentration (Cmax), time of maximum concentration (Tmax), apparent total clearance rate (CL/F), area under the plasma concentration–time curve (AUC) and AUC from time zero to the time that protein is detectable in the blood (AUC0-t), AUC up to unlimited time (AUC0-∞), AUC up to the last measurable concentration (AUMC0-t), and mean residence time (MRT) were calculated. Linear regression of terminal log-linear phase and residual method were respectively used for obtaining of terminal rate constant (Kel).
Statistical analysis
GraphPad Prism (v. 8.0) and SPSS software (v. 21) were used. All data were reported as mean ± SD. P values less than 0.05 were considered significant.
