Production of partially humanized
QMCF Technology for protein and antibody expression comprises of (1) genetically modified eukaryotic cell lines and (2) expression plasmids that stably
replicate in these cells.
The QMCF cell line used for protein production are a modified suspension CHO (CHOEBNALT85) cells which express besides a target protein two additional proteins for providing replication initiation and effective maintenance of the expression vectors. The replication initiation function is provided by mouse polyomavirus Large T antigen. The stable maintenance function for the vector is provided by the Epstein-Barr virus` (EBV) EBNA1 protein, which by acting on the family of repeats (FR) element provides chromatin attachment and segregation/partitioning function to the expression vector in mitosis and cell division.
QMCF expression plasmids consist of
two viral-originated DNA elements for replication and maintenance of the
plasmids in dividing cells. The replication of the plasmid is assured by Py
minimal replication origin. For maintenance of the QMCF plasmids FR of EBV is
used which consists of multiple binding sequences for EBV EBNA-1. DNA binding
domains of EBV EBNA-1 interact specifically with appropriate binding sequences
in plasmids and non-specifically with chromatin DNA using second DNA binding
domains. These interactions assure stable maintenance and partitioning of the
expression plasmid during mitosis.
cDNA isolation and expression plasmid construction
cDNA of the antibody variable regions was isolated from antibody-expressing mouse hybridoma culture using methodology described in Orlandi et al. 1989; PNAS. Variable regions-encoding cDNA-s were generated and cloned into the pUC19 vector. 10 subclones were analyzed by sequencing and sequence alignment to avoid mutations generated during DNA amplification.
To construct expression plasmid of
partially humanized antibody the variable-regions encoding cDNA-s were inserted
into the antibody expression vector fusing variable regions in frame with
constant regions of human IgG1 heavy- and light chains without adding or
changing amino acid sequences (Figure 1).
Figure 1. (A) Schematic representation of pQMCF antibody expression vector. Maintenance sequence (Epstein-Barr Virus Family of Repeats (FR)); PyV core origin– murine polyomavirus minimal replication origin; SV40 pr – SV 40 promoter controlling expression of Neo resistance gene; Neo/Km – Neomycin/Kanamycin resistance marker; Two different expression cassettes for either antibody heavy and light chain expression. PolyA – polyadenylation sequence; RSV-LTR – Rous sarcoma virus promoter with long terminal repeat. CMV – humancytomegalovirus promoter; Human IgG1 LC – humanIgG1 light chain constant region-encoding cDNA; Human IgG1 HC – humanIgG1 heavy chain constant region-encoding cDNA.
(B) Schematic representation of antibody heavy and light chain constant regions containing expression cassettes. AarI and Eco31I are restriction sites for insertion of the antibody variable regions.
(C) AarI and Eco31I restriction sites` dislocation from recognition sites enables insertion of the antibody variable regions in frame without adding or changing amino acid sequence.
Transfection, antibiotic selection and protein expression
growing CHOEBNALT85 cells (viability 96%) were transfected with 1 µg of
antibody-expression plasmid (BioRad Gene Pulser) with efficiency approximately
85%. The details of cell culture growth and viability are represented in Figure 2.
Figure 2. Growth of the antibody expressing CHOEBNALT85 cell culture. 48h after transfection G418 was
added. 72h after transfection expression analysis was performed. 10 days after
transfection expression cell bank was generated. Days 13 to 23 the
production phase was carried on with temperature shift to 30°C and adding nutrients to the medium.
The viability steadied over 90% in 19 days after transfection. While the viability started to drop, the supernatant was clarified by centrifugation and filtered through 0.45 µm filter and stored at -20°C prior to purification.
To select plasmid containing cell
population, G418 was added 48 hours after transfection. The final volume of the cell culture has been expanded to 400 ml
by repeated dilutions. On the 11th day after transfection the production cell
generated (5 vials 1x107 cells/vial). Thereafter the culture was placed at 30°C and fed additionally. On the 11th days after the temperature shift (22 days after transfection) the culture was harvested, the supernatant was filtrated and stored at -20°C prior to purification.
During the process the antibody expression was tested by Western blot analysis after 72 hours after transfection and at the end of the process by Coomassie-stained SDS-PAGE analysis, respectively Figure 3 and Figure 4.
Figure 3. Western Blot analysis of chimeric antibody early-stage expression. Line 1. Protein ladder (PageRuler Prestained protein ladder, Fermentas). Line 2. Cell lysate of antibody expressing CHOEBNALT85; Line 3. Supernatant (10µl) of antibody expressing CHOEBNALT85
Figure 4. Coomassie-stained SDS-PAGE postproduction analysis of chimeric antibody. Line 1. Protein ladder (PageRuler Prestained protein ladder, Fermentas). Line 2. Supernatant (10µl) of antibody expressing CHOEBNALT85.
Purification and analysis
For the purification of chimeric antibody protein G affinity chromatography followed by gel-filtration (Superdex 200) was used. After purification the antibody concentration was measured spectrophotometrically (Nanodrop, A280). Calculated productivity of the antibody was 200 mg/L. Purified antibody was analyzed in Coomassie-stained SDS-PAGE (Figure 5) and after that preparative gel filtration was performed to analyze protein prep for presence of possible aggregates (Figure 6).
Figure 5. SDS-PAGE analysis of chimeric antibody purified from the supernatant of CHOEBNALT85 cell culture. Line 1. Marker (PageRuler Prestained Protein Ladder (Fermentas, #SM0671). Line 2. 8 µg of purified antibody.
Figure 6. Preparative gel-filtration analysis of chimeric antibody. Vo designates void volume, IgG designates IgG1 standard. Blue line represents pAB1 antibody, red line represents standards (IgG, BSA, Ovalbumine and myoglobine).
Antibody production starting from expression cell bank
One vial from expression cell bank of chimeric
antibody was thawed in 25 ml fresh medium to achieve culture density 2x106
cell/ml. 24h after thawing G418 was added and in 7 days the culture was expanded to
4,0 liters final volume. 8 days after thawing the production phase was started while the temperature was lowered
to 30°C and the culture was repeatedly fed.
13 days after beginning of production phase the culture was harvested, cell
culture supernatant was filtrated and stored at -20°C prior to purification.
The antibody was purified by Protein G affinity chromatography and analyzed by Coomassie-stained SDS-PAGE in non-reduced and reduced conditions (Figure 7). Concentration of purified antibody was measured spectrophotometrically (Nanodrop, A280). Calculated productivity of the antibody was similar to the production measured before cell bank generation (200 mg/L).
Figure 7. SDS-PAGE analysis of chimeric antibody purified from the supernatant of CHOEBNALT85 cell culture. Line 1. 10 µg of purified antibody (non-reduced conditions); Line 2. Marker (PageRuler Prestained Protein Ladder (Fermentas, #SM0671). Line 3. 10 µg of purified antibody (reduced conditions).