Effects of various disaccharide adaptations on recombinant IgA1 production in CHO-K1 suspension cells

Plasmid construction

The DNA sequence of the IgA variable region was obtained from a hybridoma cell line infused with immune cells from dengue-infected volunteers in Thailand was established by SPYMEG technology in a previous work (Setthapramote et al. 2012). The following primers were used to clone the variable region of the IgA: 5′-ATGAAACACCTGTGGTTCTTCCTCCT-3’ (sense primer for amplification of the heavy chain), 5’-TGCACGTGAGGTTCGCTTCTGAACC-3’ (antisense primer for amplification of the heavy chain), 5’-ATGGCCTGGWYYCCTCTCYTYCTS-3’ (sense primer for amplification of the lambda chain), and 5’-TGGCAGCTGTAGCTTCTGTGGGACT-3’ (antisense primer for amplification of the lambda chain). The amplified variable regions were ligated into pGEM®-T Easy vector (Promega, Madison, WI, USA). The IgA1 heavy chain and lambda chain constant region were synthesized by the GeneArt™ gene synthesis service (Thermo Fisher Scientific, Waltham, MA, USA) based on DNA sequence from GenBank Accession IDs AH007035.2 and JF806287.1, respectively. The heavy chains and lambda chains of variable regions and constant regions were inserted in pQCXIP (Takara Bio, Shiga, Japan) and pQCXIH (Takara Bio), respectively, to construct IgA1 expression vectors, pQCXIP heavy chain and pQCXIH light chain.

Construction of IgA1 stably producing CHO-K1 cells

Stable transfection was done by electroporation using the Neon® transfection system (Thermo Fisher Scientific) following the manufacturer’s instructions. In brief, cells were seeded in a 6-well plate 24 h before electroporation. During the transfection day, cell density was calculated, and 1×106 cells/mL were collected. Cells in the tube were centrifuged at 400×g for 5 min at room temperature and washed with phosphate-buffered saline (PBS) and centrifuged again at 400×g for 5 min at room temperature. Cells were resuspended with resuspension buffer R with a final cell density of 2×107 cells/mL and mixed with 1.5 μg of pQCXIP heavy chain and 1.5 μg of pQCXIH light chain. The mixture was then aspirated and electroporated under the following conditions: voltage 1150 V, width 5 ms, pulses 10. After electroporation, transfected cells were incubated at 37 °C with 5% CO2. After 24 h post-transfection, 100 μg/mL hygromycin B (Fujifilm Wako Pure Chemical, Osaka, Japan) and 1 μg/mL puromycin dihydrochloride dihydrate (Fujifilm Wako Pure Chemical) were added. Medium and antibiotics were changed every 3–4 days for 21 days. Once the cells reached confluence, antibiotics were increased by a twofold concentration until reaching 2 mg/mL hygromycin B and 20 μg/mL puromycin dihydrochloride dihydrate.

Cell lines and cultivation

Serum-free adapted CHO-K1 cells were cultured using BMPro-W medium (Funakoshi, Tokyo, Japan) supplemented with 6 mM L-glutamine (Nacalai Tesque, Kyoto, Japan) and incubated at 37 °C with 5% CO2. The recombinant IgA1 stably producing CHO-K1 cell line was cultured using BMPro-W medium supplemented with 6 mM L-glutamine, 2 mg/mL hygromycin B, and 20 μg/mL puromycin dihydrochloride dihydrate. IgA1 stably producing CHO-K1 cells were adapted to 150 mM of each of different disaccharides: sucrose, maltose, lactose, and trehalose (Fujifilm Wako Pure Chemical). In brief, IgA1 stably producing CHO-K1 cells were adapted to 50 mM of each disaccharide stepwise until the disaccharide concentration reached 150 mM. Cell viability and cell growth were determined visually.

Shake flask batch culture sampling and evaluation.

Approximately 3×105 cells/mL of disaccharide-adapted IgA1 CHO-K1 SF cells were seeded in a 50 mL baffled shaker flask filled with 30 mL of medium supplemented with 150 mM of disaccharide and incubated at 37 °C with 5% CO2 at 105 rpm. Samples were collected every 24 h and evaluated for total cell count and cell viability by trypan blue exclusion assay using TC20™ Automated Cell Counter (Bio-Rad, Hercules, CA, USA). The specific growth rate was calculated using this formula,

$$\ln \left( }}} \right) = \mu t,$$

(1)

where VCD = final viable cell density, VCD0 = initial viable cell density, µ = specific growth rate, and t = time.

In addition, glucose consumption and lactate production were analyzed using a BF-9 multi-use biosensor (Oji Scientific Instruments, Hyogo, Japan). After 9 days, samples from the media were collected to determine IgA-specific productivity as shown below. The remaining media were used to purify recombinant IgA1.

Specific productivity by sandwich enzyme-linked immunosorbent assay (ELISA)

Sandwich ELISA was done to evaluate the specific productivity. In brief, a microplate was coated with 5 μg/mL goat anti-human IgA (MBL, Nagoya, Japan) capture antibody in 50 μL of PBS pH 7.4 and incubated at 4 °C overnight. The coating solution was removed, and the wells were washed with 200 μL wash buffer (0.05% Tween 20 in PBS) three times. Wells were blocked with 1% BSA in wash buffer for 1.5 h at room temperature. After washing with wash buffer three times, IgA standard purified from human serum (Bethyl Laboratories, Montgomery, TX, USA) at different concentrations was introduced and cell-free supernatant containing IgA1 was added, and the plate was incubated for 1.5 h at 37 °C. Samples were discarded and washed under the same conditions as above. Horseradish peroxidase (HRP)-conjugated goat anti-human IgA (MBL) was added and incubated for 1.5 h at room temperature. After washing three times in the same manner as above, 200 μL of Sigmafast o-phenylenediamine dihydrochloride substrate (Sigma-Aldrich, St. Louis, MO, USA) was added and incubated in the dark at room temperature for 12 min. The reaction was stopped with 100 μL of 3 M HCl. The reaction was measured using an iMARK™ microplate reader (Bio-Rad) at 450 nm wavelength. Data were analyzed using microplate reader manager 6 (Bio-rad) using a linear curve. Specific IgA1 productivity (qp) was calculated using the formula below:

First, the integrated viable cell density (IVCD) was calculated using the trapezium rule:

$$IVCD_ = IVCD_ + 0.5 \times \left( + VCD_ } \right) \times \Delta t$$

(2)

Specific IgA1 productivity was calculated between two culture times by plotting IgA1 concentration (P) vs IVCD:

$$q_ = \left( - P_ } \right)/\left( - IVCD_ } \right)$$

(3)

Tests were done in triplicates and standard error of mean (SEM) were used to calculate for the error bar.

Preparation of intracellular and extracellular fractions

The intracellular fractions of nonadapted and maltose-adapted CHO-K1 suspension cells were collected using Cytobuster™ protein extraction reagent (Merck, Darmstadt, Germany) following manufacturer’s instructions. In brief, cells were collected and washed with PBS twice. After which, 1 mL of cytobuster protein extraction reagent were added and incubated at room temperature for 5 min. Then, samples were centrifuge at 15,000 × g for 5 min. Supernatant were collected as the intracellular fraction and used for SDS-PAGE. Seven-day cultured medium was used as the extracellular fraction.

IgA1 purification, western blotting, and silver staining

IgA1 was purified using peptide M agarose (InvivoGen, San Diego, CA, USA) following the manufacturer’s instructions. The purified IgA1s were then separated under 10% polyacrylamide gels. After separation, the IgA1s were transferred to polyvinylidene difluoride membrane (Immobilon-P, Merck, Kenilworth, NJ, USA) at 15 V for 25 min. The membranes were blocked with 1% skim milk for 1 h then washed with PBS with 0.5% Tween 20 three times. The membranes were incubated with HRP-conjugated goat anti-human IgA for 1 h. Silver staining was done using the Silver Stain II Kit (Fujifilm Wako Pure Chemical) following the manufacturer’s instructions.

Recombinant IgA1 aggregation analysis

Purified recombinant IgA1 was concentrated using the Amicon 10 K Spin Column (Merck). The concentrated samples were separated using size exclusion chromatography—high-performance liquid chromatography (SEC-HPLC). In brief, 1 μg of purified recombinant IgA1 was injected into the HPLC system (LaChrom L-7000; Hitachi, Tokyo, Japan) under the following conditions: column TSKgel Ultra SW Aggregate 7.8 mm internal diameter (ID)×30.0 cm (Tosoh Bioscience, Tokyo, Japan) UV wavelength at 280 nm, mobile phase 40 mM, and sodium phosphate buffer pH 6.7 containing 400 mM sodium perchlorate at 0.3 mL/min for 60 min. The column temperature was 25 °C. The results collected were then interpreted as aggregate percentage and monomeric IgA1 percentage.

Purified recombinant IgA1 from maltose-adapted cell line was used to assess the effects of different reducing agents to reduce the aggregation caused by disulfide bond formation. Different concentrations of dithiothreitol (DTT) (Nacalai Tesque), Tris (2-carboethyl) phosphine hydrochloride (TCEP HCl) (Fujifilm Wako Pure Chemical), Glutathione (Nacalai Tesque), Cystamine diHCl (Nacalai Tesque) was mixed with recombinant IgA1 at 37 °C for 1 h and run under non-reducing SDS-PAGE.

Statistical analysis

All quantitative data were collected in triplicates. Statistical analysis was done using Microsoft excel. For comparison of the different groups, one-way ANOVA with post-hoc Tukey’s HSD test was done to compare which groups have significant difference. Values p < 0.05 was considered having significant differences.

留言 (0)

沒有登入
gif