Cloning of p.1.1-F8BDD
The DNA fragment encoding the FVIII-BDD ORF with Kozak consensus sequence was obtained by PCR using primers AD-8-AbsF (5’-ACC TCG AGG CCG CCA CCA TGG AAA TAG AGC TCT CC-3’) and AD-ONheI-R (5’-AAG CTA GCT CAG TAG AGG TCC TGT GCC-3’), the plasmid pOptivec/F8BDD  as a template, and Tersus polymerase mix (Evrogen, Moscow, Russia). The PCR product was subcloned into T-vector, sequenced using the primers to FVIII ORF listed in  and then inserted into the expression vector p1.1 (described in ) using AbsI (Sibenzyme, Novosibirsk, Russia) and NheI (Fermentas, Vilnius, Lithuania) restriction enzymes. Resulting expression plasmid p1.1-F8BDD for the transfection was purified by EndoFree Plasmid MaxiKit (Qiagen, Valencia, CA, USA) and sequence-verified.
CHO DG-44 (Cat. No. A1100001, Invitrogen, Carlsbad, CA, USA) cell line, adapted to suspension culture in a chemically defined medium was used. Cells were cultured in Erlenmeyer flasks (VWR International, Radnor, PA, USA) in 30 ml of CD DG-44 medium (Invitrogen) supplemented with 8 mM L-glutamine (Invitrogen) and 0.18% of surfactant Pluronic F-68 (BASF Inc., Florham Park, NJ, USA) at 37 °C, 8% CO2, with stirring on an orbital shaker at 130 rpm. Cells were passaged every 2–3 days at the culture density 1.2 x 106 live cells per ml, and diluted with fresh medium in a ratio of 1:4.
Transfection was performed using Fugene HD (Origen Biomedical, Austin, TX, USA), in a ratio of 18 μg of plasmid DNA per 1.5 × 107 cells in 30 ml of culture medium. Plasmid for the transfection was linearized by the PvuI or used in the supercoiled form. Transfection efficiency was determined by fluorescence microscopy 48 h post-transfection for control plasmid mixture (p1.1-F8BDD plasmid mixed 95:5 by weight with green fluorescent protein encoding plasmid pEGFP-N2). After transfection cells were cultured for 48 h without medium exchange, then transferred into selective medium ProCHO 5 (Lonza, Basel, Switzerland) and grown until cell viability was restored to 90% (15–20 days). During the cultivation in selective medium, cells were passaged every 3–5 days to the cell density 5 × 105 cells/ml or less until positive growth was observed. After this, cells were passaged every 3 days at the seeding density 3 × 105 cells/ml. Stably transfected cell population from the suspension culture was transferred to the 96-well plates with the culture medium Ex-Cell CHO Cloning Medium (Sigma-Aldrich, St. Louis, MO, USA), supplemented by the 8 mM of L-glutamine and 50–400 nM MTX. The growth of colonies was monitored at the 10th and 14th days of cultivation. Growth-positive wells were screened by ELISA, culture medium in antigen-positive wells was changed and colonies were grown close to the confluence. Alternatively, at 48 h post-transfection cells were seeded into 96-well plates at a density of 10,000 cells per well, 200 μl per well of AOF Ex-Cell CHO Cloning Media (Sigma-Aldrich) medium with 50 nM MTX and 8 mM of L-glutamine at 37 °C, 5% CO2. Cells were maintained as described above.
Cells from most productive colonies obtained by both techniques were transferred to 48-well plates, cultivated for 5 d and screened again by ELISA. Best secreting oligoclonal lines were further expanded and readapted to suspension growth in ProCHO 5 medium with 8 mM L-glutamine during three sequential passages in 24-, 12- and 6-well plates.
FVIII concentration in the culture medium was measured 48 h post-transfection and at the end of the selection for stable transfectants. Oligoclonal cell line with the highest level of secreted FVIII, obtained in the presence of 50 nM MTX, was used for further amplification of bicistronic FVIII-dhfr cassette. It was grown in the ProCHO 5 medium with 8 mM of L-glutamine in the presence of increasing concentrations of methotrexate (MTX). Each amplification step lasted for 14–25 days until cell viability restored to 90%. Concentration of secreted FVIII was measured by ELISA and/or clotting test at the end of each step. Transgene-amplified oligoclonal cell line, obtained by the highest MTX selection pressure possible (4 μM) was cloned by limiting dilution in AOF Ex-Cell CHO Cloning Media (Sigma-Aldrich) with 8 mM alanyl-glutamine (Invitrogen) and HT (hypoxanthine/thymidine) supplement (Invitrogen), without MTX. Cells were seeded into 96-well plates at a density of 0.5 cells per well, 200 μl of medium per well at 37 °C, 5% CO2. The growth of single colonies was monitored at the 10th and 14th days of cultivation. The colonies were screened by ELISA, culture medium in positive wells was changed and colonies were grown close to the confluence. According to the results of the subsequent ELISA of actively growing colonies the most productive of them were transferred to 48-well plates. Clonal lines with the best levels of secretion were further expanded and readapted to suspension growth in ProCHO 5 medium with 8 mM L-glutamine during three sequential passages in 24-, 12- and 6-well plates.
Conditioned medium from 6-well plates was analyzed by ELISA. The top three cell lines were moved to Erlenmeyer flasks and evaluated by growth rate and specific productivity. Preparative batch cultivation was conducted in 500 ml Erlenmeyer flasks, 125 ml of culture medium per flask. Cells were seeded at a concentration of 2.5 × 105 cells/ml and cultured without medium change to a density of 3 × 106 cells/ml (4–5 days).
Concentration of the factor VIII antigen (FVIII:Ag) was measured by ELISA using polyclonal antibodies to FVIII (LifeSpan BioSciences, Seattle, WA, USA) at 50 ng per well and specific MAb A2 to FVIII heavy chain described in . Serial dilutions of normal calibrated human plasma (NPO Renam, Moscow, Russia) in PBS with 1% bovine serum albumin (BSA) were used as a standard. Samples were also diluted in PBS with 1% BSA.
One stage clotting assay with chromogenic substrate was performed by the TECHNOCHROM® F VIII:C kit (Technoclone GmbH, Vienna, Austria); two stage clotting assay was performed by the optical coagulation analyzer ThromboScreen 400c (ThermoFisher Scientific, USA) and the Factor VIII- test reagent kit (NPO Renam), using normal calibrated human plasma (NPO Renam) as the initial standard and one stage assay-characterized sample of the FVIII-BDD preparation as the working standard. Specific activity of several FVIII-BDD samples was 1.7 times lower in the two-stage assay than in the one-stage assay if human plasma was used as the activity standard. Samples were diluted with the imidazole buffer with 1% BSA (NPO Renam).
Effect of alkanoic acids in the culture medium on FVIII secretion was assessed in batch cultures, seeded as 3.75 × 105 cells/ml and cultured for 3 days unless stated otherwise. Sodium butyrate and sodium propionate were dissolved in water at 1 M and adjusted to the pH 7.2 by HCl prior to addition to the culture medium. Antioxidant butylated hydroxyanisole was dissolved in DMSO at 2.5 M prior to addition to the culture medium, final concentration 0.1 mM; addition of dichlorofluorescein (DCF, Invitrogen) and subsequent flow cytometry analysis for oxidative stress level was performed as described in .
Oxidative stress induction and flow cytometry
Cells of the 11A4H line were seeded as 3.5 × 105 cells/ml in the protein-free ProCHO-5 medium and cultivated for 4 days in the shake flask. On 3rd and 4th day 1 × 106 cells were sampled. Then samples were stained with 5 μM of CM-H2-DCF-DA (Invitrogen) for 20 min on ice and counterstained with 1.5 μM propidium iodide just before flow cytometry analysis on Cytomics FC500 instrument (Beckman Coulter). Direct reads were gated on FS/SS scatter against intact control cells sample and then subsequently gated for propidium iodide exclusion (channel FL-3 620 nm). Generation of ROS were assessed as green fluorescence of activated DCF by measuring in FL-1 (525 nm) channel for 105 gated cell reads, every obtained FL-1 scatter histogram were unimodular, mean values were calculated for core 5 ml of intact cell culture with H202 in final concentrations 10 μM, 100 μM and 1000 μM directly prior to live staining.
Quantitative PCR, PCR, RT-PCR
Copy number of the expression cassettes in the genome was determined by the quantitative real-time-PCR (qPCR). A calibration curve was prepared using serial dilutions of highly purified p1.1-F8BDD plasmid. Genomic DNA was isolated by Wizard SV Genomic DNA Purification System (Promega, USA) and quantified by the Qubit Fluorometer (Invitrogen), using dsDNA HS kit (Invitrogen) and external DNA concentration standard, prepared in-house from the highly purified plasmid DNA, quantified by UV spectrophotometry. Concentration of the genomic DNA was validated by control qPCR with primers to PPIB gene region, presumably unique to the CHO cells genome according to the BLAST search results. Samples of genomic DNA with the determined copy numbers of PPIB less than 0.3 per haploid genome or more than 2.0 were discarded. Weight of one CHO haploid genome was established as 3 pg according to .
The mRNA levels were assayed by reverse transcription and quantitative PCR (RT-qPCR). Total RNA was isolated by the RNeasy Mini Kit (Qiagen), its concentration was determined by UV spectroscopy, purification quality was confirmed by monitoring the A260/A280 ratio (1.8–2.0); integrity was verified by electrophoresis in 1% agarose gel. The cDNA was synthesized with the reagent kit Mint (Evrogen) using 1 μg of total RNA per sample. The ΔΔCq method  was used to calculate mRNA expression relative to β-actin.
Primers (listed in Additional file 1: Table S1) were designed by the Beacon Designer v7.51 program (PREMIER Biosoft International, Palo Alto, CA). The unique primers allowing selective amplification of the expression cassette were chosen for the genome copy number analysis, and primers located in the different exons or at the exon junctions were used for mRNA expression level analysis.
QPCR was performed using qPCRmix-HS SYBR reaction mixture (Evrogen) and iCycler iQ thermocycler (Bio-Rad, USA). Calculations of threshold cycles, calibration curves, PCR efficiency and copy numbers were made by the iCycler Iq4 program. All determinations were repeated 3 times, in 3–5 replicates, sample volume 25 μl.
PCR analysis of the whole FVIII ORF area was performed for genomic DNA and cDNA in the essentially same conditions. Templates were taken as 10 ng per tube, reaction volume was 10 μl. Primers AD-8-AbsF and AD-ONheI-R, described in the Cloning subsection, were used; Encyclo PCR kit (Evrogen) was employed. Temperature gradient was from 53 °C to 68 °C. Amplification program was 3’ at 95 °C; 25 cycles as 15” at 95 °C, 53–68 °C for 15”, 72 °C for 3’. Final elongation was 72 °C for 5’. Amplification products were electrophoresed on 0.8% agarose gels and stained with ethidium bromide.
Southern blot hybridization
Biotinylated probes for Southern blotting were prepared by the Biotin DecaLabel DNA Labeling Kit (Fermentas). Template for the probes was pAL-ID plasmid (containing regions present in expression plasmids p1.1 including origin of replication, a beta-lactamase gene, EMCV IRES, DHFR ORF ) or a PCR product corresponding to the fragment of FVIII-BDD ORF. Genomic DNA was digested with EcoRI (Fermentas) for 16 h, precipitated by ethanol, separated by 0.8% agarose gel. Gel transfer to an Amersham Hybond-N+ membrane (GE Healthcare, USA) was performed according to the manufacturer’s protocol in 20x SSC buffer (3 M NaCl, 0.3 M Na3C6H5O7) for 16 h. DNA was fixed by heating the dried membranes at 80 °C for 2 h. Prehybridization and hybridization were conducted according to  in the buffer containing 7% SDS, 0.5 M Na2PO4, pH 7.2, 1% BSA for 16 h at 65 °C. Membrane was washed according to the manufacturer’s protocol and stained by Biotin Chromogenic Detection Kit (Fermentas).
SDS-PAGE and immunoblotting
Samples of conditioned medium were clarified by centrifugation and concentrated 30 times by precipitation with trichloroacetic acid. Cell lysates were prepared using a modified RIPA buffer (50 mM Tris -HCl, pH 7.4, 1% NP- 40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM Na-EDTA) with protease inhibitor cocktail (Sigma-Aldrich) and normalized to total protein concentration, determined by QuantiPro ™ BCA Assay Kit (Sigma-Aldrich). Samples were separated by electrophoresis in 7.5% denaturing polyacrylamide gel, sample load was 10 μg per well for cell lysates and the equivalent of 0.5 ml of conditioned medium. Gel slabs were stained by colloidal Coomassie stain (Fermentas) according to manufacturer's procedure or used for blotting. Transfer to the membrane, blocking, hybridization and staining were performed as described previously .
Purification of FVIII
Purification of FVIII from the cell culture supernatant was carried out as described in  with changes.
All solutions used for the purification, except the final composition, contained 0.02% Tween-80. To the conditioned culture medium or cell suspension, NaCl was added to 0.3 M and CaCl2 to 27 mM. The resulting suspension was incubated for 30 min with stirring at 37 °C, clarified by centrifugation, supplemented with 10 mM of L-histidine pH 7.0 and 0.02% of Tween-80 and applied in 100 ml portions to the 1 ml HiTrap Capto MMC column (GE Healthcare), equilibrated with a solution 1-A (0.3 M NaCl, 10 mM CaCl2, 10 mM L-histidine, pH 7.0), flow rate 1 ml/min.
Column was washed by 10 volumes of solutions 1-B, 1-C and 1-D at 2 ml/min. Solution 1-B contained 1 M NaCl, 50 mM CaCl2, 50 mM L- histidine, pH 6.5. Solution 1-C contained 0.1 M NaCl, 50 mM CaCl2, 50 mM L-histidine, pH 6.5. Solution 1-D contained 0.3 M NaCl, 10 mM CaCl2, 10 mM L-histidine, 0.25 M L-arginine-HCl, 10% ethylene glycol, pH 6.5. FVIII was eluted at 1 ml/min by the solution 1-E (0.3 M NaCl, 20 mM CaCl2, 20 mM L- histidine, 0.8 M L- arginine-HCl, 50% ethylene glycol, pH 6.5).
Eluted FVIII solution was diluted 8-fold with a solution 2-A (10 mM NaCl, 10 mM L-histidine, 10 mM CaCl2, pH 6.5) and applied to the 1 ml Tricorn 5/50 column (GE Healthcare) with the SP Sepharose FF (GE Healthcare), equilibrated with a solution 2-B (0.15 M NaCl, 10 mM L- histidine, 10 mM CaCl2, pH 6.5), flow rate 2 ml/min. Column was washed with 20 ml of the solution 2-B and FVIII was eluted by the solution 2-C (0.34 M NaCl, 35 mM CaCl2, 45 mM L-arginine-HCl, 0.2 M sorbitol, 10 mM L-histidine, pH 6.5). Resulting solution of the semi-purified FVIII was immediately frozen in liquid N2 and stored in the −80 °C freezer.
Thawed FVIII solution was applied on the 1 ml Tricorn 5/50 column, filled with the immunoaffinity resin VIII Select (GE Healthcare) at 0.2 ml/min. Prior to the application of the sample, the column was equilibrated with the solution 3-A (0.3 M NaCl, 20 mM CaCl2, 20 mM L- Histidine, pH 6.5). The column was washed sequentially at a rate of 2 ml/min by 10 ml of the solution 3-A and 3 ml of the solution 3-B (1 M NaCl, 20 mM CaCl2, 20 mM L-Histidine, pH 6.5). FVIII was eluted with the solution 3-C (1.5 M NaCl, 20 mM CaCl2, 20 mM L- Histidine, 50% ethylene glycol, pH 6.5).
Eluate was diluted 15-fold with the solution 4-A (10 mM NaCl, 10 mM L- histidine, 10 mM CaCl2, pH 6.5) and applied at 2 ml/min to the 1 ml HiTrap Capto Q column (GE Healthcare), equilibrated with the solution 4-B (0.1 M NaCl, 0.02 M L-histidine, 20 mM CaCl2, pH 6.5). Column was washed with 20 ml of the 4-C solution (0.3 M NaCl, 20 mM L-histidine, 20 mM CaCl2, pH 6.5). Elution was performed by the 4-D solution (0.4 M NaCl, 0.02 M CaCl2, 20 mM L-histidine, pH 6.5).
FVIII solution was applied in 2 ml portions at 0.4 ml/min flow rate to the size exclusion column Tricorn 10/300 Superdex 200 (GE Healthcare), equilibrated with the 5-A solution(9 g/L NaCl, 0.25 g/L CaCl2, 1.5 g/L L-histidine, 0.01% Tween-80, 3 g/L sucrose, pH 7.0) at the flow rate 1 ml/min. Monomer of the FVIII in the long-term storage solution was collected in the final volume of 5–8 ml, divided into small aliquots, frozen in liquid N2 and stored frozen for further analysis.
Coomassie-stained bands of FVIII chains were cut from the polyacrylamide gel, washed twice (to remove the dye) with 100 μl of 40% acetonitrile in 0.1 M NH4HCO3 for 20 min at 37 °C, dehydrated by 100 μl of acetonitrile and completely dried in vacuo. Four μl of 15 μg/ml modified trypsin solution (Promega) in 50 mM NH4HCO3 added to each 3–4 mm3 gel piece. Hydrolysis was carried out for 5 h at 37 °C, then 7 μl of 0.5% trifluoroacetic acid (TFA) in 10% aqueous acetonitrile solution was added and mixed thoroughly. The upper layer of the solution above the gel was used for MALDI-TOF analysis.
2 μl aliquots of the peptide mixtures were mixed with 0.5 μl of 2.5-dihydroxybenzoic acid solution (Aldrich, 20 mg/ml in 20% aqueous acetonitrile, 0.5% TFA), applied to target spots and air dried.
Mass spectra were obtained on a MALDI-TOF instrument UltrafleXtreme (BrukerDaltonics, Germany) in the positive ion mode with the reflectron. For each sample analyzed the accuracy of the monoisotopic mass measurements was maintained as at least 0.003% (30 rrm) by the calibration on trypsin autolysis peaks. Spectra were obtained in the mass range 700–4500 m/z. Peptide mapping was conducted using the GPMaw program (Lighthouse data, Denmark).