Antibody expression and purification is a crucial process in the production of recombinant antibodies for various applications in therapy, diagnostics, and research. The general workflow involves the following steps:

Antibody Expression

Recombinant Antibody Expression

   Antibodies are typically expressed in bacterial (e.g. E. coli) or mammalian cell lines (e.g. CHO, HEK293) using recombinant DNA technology.

   Codon optimization and other techniques are often used to enhance protein production.

   The expressed antibodies can be in the form of full-length IgG, antibody fragments (e.g. Fab, scFv), or engineered formats like bispecific antibodies.

A. Selection of Antibody Format

Monoclonal Antibodies: Produced by creating a hybridoma cell line that secretes a single type of antibody.

Polyclonal Antibodies: Produced by immunizing an animal and collecting antibodies from its serum, which are a mixture of various antibodies.

B. Cloning the Antibody Gene

Gene Identification: Identify the gene encoding the antibody of interest, usually from a hybridoma or immune cells.

Construct Creation: Clone the antibody gene into an expression vector, which is a plasmid or virus designed for high-level protein expression.

C. Cell Line Selection

Expression Host: Choose a suitable cell line for expression. Common hosts include:

Bacteria: E. coli, suitable for simple antibodies but often requires refolding of proteins.

Yeast: Pichia pastoris, which can perform some post-translational modifications.

Mammalian Cells: CHO cells, and HEK293 cells, which can perform complex post-translational modifications similar to those in humans.

D. Transformation and Expression

Transformation: Introduce the expression vector into the chosen host cells.

Culture Conditions: Grow the cells under conditions that induce high-level antibody production.

Antibody Purification

   The cells expressing the recombinant antibodies are lysed, often using a French press or homogenizer, to release the antibodies.

   Initial purification steps like centrifugation and filtration are performed to remove cell debris and other impurities.

Purification Methods

Affinity Chromatography: The most common method, using a column with a ligand (e.g., Protein A or Protein G) that specifically binds to the Fc region of antibodies. This allows for the selective capture of antibodies from the mixture.

Ion Exchange Chromatography: Separates proteins based on their charge.

Size Exclusion Chromatography: Separates proteins based on their size and shape.

Protein A/G/Affinity Resins: Specific for antibodies, simplifying the purification process.

Monitoring and Quality Control

Purity Analysis: Use SDS-PAGE, Western blotting, or other assays to confirm the purity of the antibody.

Activity Testing: Verify that the antibody retains its biological activity, often through ELISA or other functional assays.

Concentration Determination: Measure antibody concentration using techniques like UV absorbance or BCA assay.

Removal of Tags and Buffers Exchange

   If the antibody was expressed with an affinity tag (e.g. His-tag, Fc-tag), it may be necessary to remove the tag using a protease cleavage site.

   Dialysis, desalting, or diafiltration are used to exchange the antibody into the desired buffer for the intended application.

The choice of expression system, purification strategy, and specific techniques employed will depend on the antibody format, the desired purity, and the intended use of the purified antibody. Careful optimization of each step is crucial to obtain a high yield of pure, functional recombinant antibodies.