Custom Antibody Production

Production of partially humanized antibody

Technology overview

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). 

(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

6x10⁶ exponentially 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 bank was 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).