In recent years, methods for purifying monoclonal antibodies have advanced significantly. Generally, each step is designed to remove specific impurities from the clarified harvest. The choice of a particular chromatography step depends on the physicochemical properties of the target antibody. Due to variations in antibody characteristics, a chromatography method effective for one monoclonal antibody may not work as well for others.

I. Columns involved in the purification process

II. Properties of various affinity proteins used for antibody purification

 III. Ion exchange chromatography

All monoclonal antibodies have a basic isoelectric point (pI) within the range of 7.3–8.9. The pI of an antibody determines its elution behavior in ion-exchange chromatography. If the pH is maintained below the antibody's pI, the antibody carries a net positive charge. Conversely, when the pH is kept above the antibody's pI, the antibody acquires a net negative charge.Cation-exchange or anion-exchange chromatography can be incorporated into the purification scheme based on the target impurities to be removed. Cation-exchange chromatography is primarily used for product-related impurities, such as host cell proteins, while anion-exchange chromatography is employed to remove host cell DNA and host cell proteins. Additionally, anion-exchange chromatography serves as a critical column for demonstrating viral clearance during the purification process.Anion-exchange chromatography operates in a flow-through mode, where all negatively charged impurities bind to the column, while the positively charged antibody is eluted as it passes through.

IV. Experimental procedures for monoclonal antibody purification by affinity chromatography

1. Equilibrate the HiTrap protein A column (1 mL) with 10 mL of binding buffer suitable for the IgG subclass. This column is intended for use with single mouse ascites (5-15 mg immunoglobulin/single mouse ascites).

2. Dilute the ascites with an equal volume of binding buffer and load onto the column at a flow rate of 0.5 mL/min. Retain the flow-through to avoid losing unbound antibodies.

3. Wash the column with 10 mL of binding buffer at a flow rate of 1 mL/min.

4. Elute the bound protein using 2-5 column volumes of elution buffer suitable for the IgG subclass, and collect 0.5 mL fractions using a fraction collector. Measure A280 to identify the eluted protein. Pool the protein fractions and neutralize with 1 M Tris–HCl buffer (pH 9.0) (check with pH paper).

5. Dialyze the eluted protein against 2 L of PBS at 4°C overnight.

6. Aliquot into 1 mL/tube and store at -80°C.

V. Other purification methods

After the Protein A step, trace amounts of process-related impurities (such as host cell proteins (HCPs), DNA, leached Protein A, endotoxins, and some cell culture media additives) as well as product-related impurities (high-molecular-weight aggregates and low-molecular-weight degradation products) typically remain.  Subsequent chromatography steps are often described as polishing steps, as they reduce these trace impurities to levels that ensure product safety.

Various chromatography modes, including cation exchange (CEX), anion exchange (AEX), hydrophobic interaction (HIC), and hydroxyapatite (HA), have been employed as polishing steps in antibody purification processes.  Although less common, the use of immobilized metal affinity chromatography (IMAC) and size exclusion chromatography (SEC) has also been reported.

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