Production of partially humanized antibody
QMCF Technology comprises of genetically modified eukaryotic cell line and expression plasmids that stably replicate and maintain in these cells.
QMCF cell lines are stable suspension or adherent cells (for instance CHOEBNALT85) into which two genes have been introduced in order to provide replication initiation and effective maintenance functions to appropriate QMCF expression vectors. The replication initiation function is provided by mouse polyomavirus Large T antigen, and the stable maintenance function for the vector is provided by the Epstein-Barr virus EBNA-1 protein, which by acting on the 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. There is a large set of QMCF plasmids that in addition to the regulatory maintenance and replication elements contain different expression cassettes and allow to design protein production experiment in most proper manner.
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 a QMCF 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 – SV40
promoter controlling expression of Neo resistance gene; Neo/Km –
Neomycin/Kanamycin resistance marker; two different expression cassettes for heavy and light chain expression containing RSV-LTR and CMV promoters; PolyA – polyadenylation sequence; Human IgG1 LC – human IgG1 light chain constant region-encoding cDNA; Human IgG1 HC – human IgG1 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
6x10^6 exponentially growing CHOEBNALT85 cells (viability 96%) were transfected with 1 µg of antibody-expression plasmid (Bio-Rad 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 post transfection G418 was added.
72h post transfection expression analysis was performed. 10 days post transfection expression cell bank was generated. Days 13 to 23 the production phase at 30°C was performed, additional nutrients were added to the medium. The viability was kept > 90% during 19 days post the transfection. If viability declined, the supernatant was clarified by centrifugation and filtration and stored at -20°C prior to purification.
To select plasmid containing cell population, G418 was added 48 h after transfection. The final volume of the cell culture has been expanded to 400 ml by repeated dilutions. On the day 11th after the transfection, a production cell bank was generated (5 vials 1x10^7 cells/vial). Thereafter the culture was placed at 30°C and fed additionally. 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 h 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 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 antibody purified
from CHOEBNALT85 supernatant. 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 the cell bank of chimeric antibody was thawed in 25 ml fresh medium to achieve culture density 2x10^6 cell/ml. 24 h after thawing G418 was added and in 7 days the culture was expanded to 4,0 litres final volume. 8 days after thawing the production phase was started and 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
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).