While extracting antibodies from the monoclones maintained either in vitro or in vivo methods, certain contaminants such as growth factors, hormones, transferrins etc. of culture media, or host antibodies, proteases, nucleases, nucleic acids etc. also may be extracted.

Hence the sample is first conditioned, or prepared for purification. Cells, cell debris, lipids, and clotted material are first removed, typically by filtration with a 0.45pm filter and condensed by ultra filtration or dialysis.

Since most of the charged impurities are usually anions such as nucleic acids and endotoxins, they are often separated by ion exchange chromatography.

Salvage Pathway:

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A salvage pathway is a pathway in which nucleotides (purines and pyrimidines) are synthesized from intermediates in the degradative pathway for nucleotides. They are used to recover bases and nucleosides that are formed during degradation of RNA and DNA.

This is important in some organs because some tissues cannot undergo de novo synthesis. The salvaged bases and nucleosides can then be converted back into nucleotides.

De Novo Synthesis

It refers to the synthesis of complex molecules from simple molecules such as sugars or amino acids, as opposed to their being recycled after partial degradation.

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For example, nucleotides are not needed in the diet as they can be constructed from small precursor molecules such as formate and aspartate

Polyethylene Glycol:

Polyethylene Glycol is a family of long chain polymers made up of ethylene glycol subunits. It allows a slowed clearance of the carried protein from the blood.

This makes for a longer acting medicinal effect and reduces toxicity, and it allows longer dosing intervals.

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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 keyhole limpet haemocyanin (KLH) during development and helps in purification. The antibody-containing media is then incubated with the immobilized antigen, either in batch or 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 gentler, high salt elution buffer is then used to recover purified antibody from the support.

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.