The desired antibodies should be extracted from a media sample of cultured hybridomas or a sample of ascites fluid. The contaminants in the cell culture sample consists of growth factors, hormones, and transferrins. The in vivo sample however, contains host antibodies, proteases, nucleases, nucleic acids, and viruses. Other secretions by the hybridomas (like cytokines) may be present in both the cases. Endotoxins may also be present in case of bacterial contamination as they are secreted by the bacteria.

For purification, the sample is conditioned by removing cells, cell debris, lipids, and clotted materials through centrifugation and then filtration through a 0.45μm filter. Membrane fouling can be caused by the large particles in the further steps of purification. Moreover, the product concentration in the sample might not be sufficient, particularly in cases where the desired antibody is produced by a low-secreting cell line. Therefore, the sample is subjected to ultrafiltration or dialysis for condensation.

The charged impurities are mostly anions like nucleic acids and endotoxins, and are separated by ion exchange chromatography. At sufficiently low pH, cation exchange chromatography is conducted so that the desired antibody binds to the column and the anions flow through. While at sufficiently high pH, anion exchange chromatography is: conducted so that the desired antibody flows through column and the anions bind to it.

Based on their isoelectric point (pI), many proteins and anions can be separated. For example, pI of albumin (4.8) is lower than the pI of most monoclonal antibodies (6.1). So, the average charge of albumin molecules at a certain pH is more negative. Size exclusion chromatography is used for removing transferrin.

Affinity purification is conducted to obtain maximum purity in a single step as the antigen used delivers antibody specificity. In this technique, the antibody-generating antigen is covalently bonded to agarose support. If the antigen is a peptide, it is synthesised with a terminal cysteine that allows selective attachment to a carrier protein (Keyhole Limpet Hemocyanin, KLH) during development and to promote purification.

The media, containing antibody is incubated with the immobilised antigen in batch. It can also be incubated as the antibody is passed through a column, at which it binds selectively and the impurities are rinsed. The purified antibody is recovered from the support by an elution using a low pH buffer or a gentle high salt elution buffer.

Sodium or ammonium sulphate is used to precipitate out the antibodies for their further selection. Antibodies precipitate at low salt concentrations, while other proteins precipitate at higher concentrations. To obtain best separation, salt should be added in sufficient amount, and the excess salt can be removed by desalting method like dialysis.

Chromatogram is used for the analysis of final purity. Presence of any impurities can be detected by the formation of peaks and the amount of impurity is indicated by the volume under the peaks. Gel electrophoresis and capillary electrophoresis can also be performed for the analysis of final purity. Presence of any impurities can be detected by the formation of bands of varying intensity.

Applications of MAbs

The application of monoclonal antibodies are:

1. Diagnostic applications,
2. Therapeutic applications,
3. Protein purification, and
4. Miscellaneous applications.