After production the monoclonal antibody is purified from the media of cultured hybridomas or from the ascites fluid .

The contaminants in the cell culture media consist primarily of media components such as growth factors, hormones, and transferrins. In contrast, the in vivo (ascites ) sample is likely to have host antibodies, proteases, nucleases, nucleic acids, and viruses. In both cases, other secretions by the hybridomas such as cytokines may be present. There may also be bacterial contamination and, as a result, endotoxins that are secreted by the bacteria. Depending on the complexity of the media required in cell culture, and thus the contaminants in question, one method (in vivo or in vitro) may be preferable to the other.

The sample is first conditioned, or prepared for purification. Cells, cell debris, lipids, and clotted material are first removed, typically by filtration with a 0.45 µm filter. The method using Protein A/G affinity chromatography is the fastest way to purify the antibodies in a single step.

The antibodies selectively binds to Protein A/G, so a high level of purity (generally >80%) is obtained. However, this method may be problematic for antibodies that are easily damaged, as harsh conditions are generally used. A low pH can break the bonds to remove the antibody from the column. In addition to possibly affecting the product, low pH can cause Protein A/G itself to leak off the column and appear in the eluted sample. Gentle elution buffer systems that employ high salt concentrations are also available to avoid exposing sensitive antibodies to low pH. Cost is also an important consideration with this method because immobilized Protein A/G is a more expensive resin.

To achieve maximum purity in a single step, affinity purification can be performed, using the antigen to provide exquisite specificity for the antibody. In this method, the antigen used to generate the antibody is covalently attached to an agarose support. If the antigen is a peptide, it is commonly synthesized with a terminal cysteine, which allows selective attachment to a carrier protein, such as KLH during development and to the support for purification. The antibody-containing media is then incubated with the immobilized antigen, either in batch or as the antibody is passed through a column, where it selectively binds and can be retained while impurities are washed away. An elution with a low pH buffer or a more gentle, high salt elution buffer is then used to recover purified antibody from the support.

To further select for antibodies, the antibodies can be precipitated out using sodium sulfate or ammonium sulfate. Antibodies precipitate at low concentrations of the salt, while most other proteins precipitate at higher concentrations. The appropriate level of salt is added in order to achieve the best separation. Excess salt must then be removed by a desalting method such as dialysis.

The final purity can be analyzed using a chromatogram. Any impurities will produce peaks, and the volume under the peak indicates the amount of the impurity. Alternatively, gel electrophoresis and capillary electrophoresis can be carried out. Impurities will produce bands of varying intensity, depending on how much of the impurity is present

The other methods involving the priciples of ion-excahnge and gel chromatography can also be used to purify the antibodies, but are generally not preferred as it involves a lot of steps and it invovles several steps. But if the antibodies are really sensitive and cannot tolerate the harsh conditions that are used in protein A/G affinity chromatography, then purification using ion-exchange columns is a better choice.