How are recombinant antibodies made?

Recombinant antibodies are produced by genetically engineering cells to express and secrete specific antibody proteins. This process allows for the creation of antibodies with defined characteristics and consistent production.

Here's a breakdown of the process:

1. Identifying and Cloning Antibody Genes:

   • The first step involves identifying the genes that encode the heavy and light chains of the desired antibody. This can be achieved through several methods:
     • Hybridoma Technology (Starting Point): If an antibody-producing hybridoma cell line exists (created by fusing antibody-producing B cells with myeloma cells), the genes can be amplified from the hybridoma's DNA or RNA using PCR (Polymerase Chain Reaction).
     • Phage Display/Yeast Display/Ribosome Display: These are in vitro selection techniques where a library of antibody variable regions is displayed on the surface of bacteriophages, yeast cells, or ribosomes, respectively. Antibodies with the desired binding specificity are selected by affinity purification. Once selected, the DNA encoding these antibodies is easily recovered.
     • Single B Cell Cloning: Individual B cells from an immunized animal or patient can be screened for antibody production. The genes encoding the heavy and light chains are then amplified from the selected B cells.

2. Designing the Expression Vector:

   • The cloned antibody genes are then inserted into a specially designed expression vector. This vector is a DNA molecule that contains all the necessary elements for gene expression in the chosen host cell. Key elements include:
     • Promoter: A DNA sequence that initiates transcription of the antibody genes. Strong promoters ensure high levels of antibody production.
     • Signal Sequence: A DNA sequence that directs the newly synthesized antibody protein to the endoplasmic reticulum (ER) for proper folding and secretion.
     • Selection Marker: A gene that allows for the selection of host cells that have successfully taken up the expression vector (e.g., antibiotic resistance gene).
     • Terminator: A DNA sequence that signals the end of transcription.

3. Introducing the Expression Vector into Host Cells (Transformation/Transfection):

   • The expression vector containing the antibody genes is introduced into host cells. Common host cells include:
     • Bacteria (e.g., E. coli): Relatively inexpensive and easy to culture, making them suitable for large-scale antibody production, especially for antibody fragments (e.g., scFv, Fab). However, bacteria lack the glycosylation machinery found in mammalian cells, so bacterial-produced antibodies are not glycosylated.
     • Mammalian Cells (e.g., CHO, HEK293): Capable of proper protein folding, assembly, and glycosylation, which is crucial for the function of many therapeutic antibodies. Culturing mammalian cells is more complex and expensive than culturing bacteria.
     • Yeast (e.g., Pichia pastoris): Offer a balance between the ease of bacterial culture and the protein processing capabilities of mammalian cells.
   • The process of introducing the vector into host cells is called transformation (for bacteria) or transfection (for mammalian cells).

4. Culturing the Host Cells and Antibody Production:

   • The transformed/transfected host cells are cultured under optimal conditions to promote cell growth and antibody production. This usually involves providing the cells with the necessary nutrients, temperature, and pH. Mammalian cell culture often requires more sophisticated bioreactors.
   • The cells then express the antibody genes, producing the recombinant antibody protein.

5. Purification of Recombinant Antibodies:

   • The recombinant antibodies are then purified from the cell culture medium or from the cells themselves. Common purification methods include:
     • Protein A/G Chromatography: These proteins bind specifically to the Fc region of antibodies, allowing for efficient capture and purification.
     • Affinity Chromatography: Using an antigen that binds specifically to the antibody of interest.
     • Ion Exchange Chromatography: Separates proteins based on their charge.
     • Size Exclusion Chromatography: Separates proteins based on their size.

6. Characterization and Quality Control:

   • The purified antibodies are then characterized to ensure their purity, binding affinity, and functionality. This may involve techniques like ELISA, SDS-PAGE, mass spectrometry, and cell-based assays.

Recombinant antibody technology offers several advantages over traditional hybridoma technology, including the ability to produce antibodies with defined specificities, engineer antibody fragments, and scale up production more efficiently.