Home » Antibody Characterization Methods You Should Know

Antibody Characterization Methods You Should Know

by Nathan Zachary

The world would be dull and repetitive if everyone looked the same. This applies to the immune system as well. Every individual human, or any animal, has an independent immune profile, which means they produce antibodies to fight off bacteria and viruses differently from others. 

The characterization of antibodies is a must for understanding their function, the procedures that should be used for their purification and analysis, and the other necessary steps for a successful application. However, antibody characterization methods are probably the least understood technique in antibody research. 

This post puts together a list of common antibody characterization methods in order of usefulness. 

Characterization Via Production And Development

The rationale for defining a mAb varies depending on the stage of development and production. It is vital to understand what you are looking for and why. The former is used to confirm that you have the correct mAb and to identify possibly essential quality characteristics of the antibody. To some extent, the latter is determined by these characteristics. “They will indicate which product-related contaminants you will need to consider during process development and then throughout manufacture,” adds Malmquist.  

Three analysis methods determine the custom antibody production: process control and lot release, stability indicators, and characterization. Characterization ensures that the drug’s properties stay the same and that the levels of product-related impurities are acceptable. When carried out correctly, analytical approaches allow producers to detect high-molecular-weight aggregates resulting from mAbs sticking together throughout the manufacturing process. Similarly, analytical methods are utilized to identify contaminants such as host cell proteins and leached Protein A. 

Common Factory And Lab Characterization 

Another defining trend is that pharmaceutical companies are bringing laboratory-based analytical procedures into manufacturing. The technique involves corporations deploying analytical instruments near manufacturing lines, which “will boost data frequency, minimize reaction times, and enhance system control,” according to Malmquist. According to Malmquist, real-time release testing may be achievable in the future, which would dramatically cut release time by maintaining quality targets satisfied during the production process. 

Western Western 

Simple Western assays can characterize single- and double-phosphorylated isoforms at their expected pIs. You will also be able to quantify biological reactions with size and charge methodologies. What’s more, the best part? You only need a few nanoliters! 


Therapeutic monoclonal antibodies require precise charge heterogeneity characterization. iCE systems provide high resolution, rapid analysis, and general methodologies for quick and easy charge variant characterization. Free solution IEF in a capillary column (cIEF) allows for the detection of targeted protein zones in less than 10 minutes, and high-resolution leads to simple, reliable quantification of protein charge variations, making iCE excellent for the investigation of monoclonal antibodies. 

Affinity Chromatography

A common technique makes use of affinity chromatography. Here, the crude sample is run through a column containing a resinous stationary phase that contains protein A. Protein A has a high sensitivity for the Fc (fragment, crystallizable) sections of antibodies, which is how it collects the antibody. Affinity chromatography is a rapid and easy process, although it has the disadvantage of being more expensive than other approaches.  

Protein A resin has a shorter lifespan than other stationary phases, which accounts for a large portion of this expense. The crude material is initially run through an affinity column and then polished using ion exchange chromatography in a multistep process, including affinity chromatography (IEX). 

Cation Exchange Chromatography 

Cation exchange chromatography is a common way to clean up a monoclonal antibody that has already been isolated. In this step, the antibody binds the solid phase, and impurities are permitted to pass through or wash off the protein. Anion exchange chromatography, a supplementary approach, may also be used, in which contaminants are caught on the anion exchange column while the antibody passes through. The total elimination of pollutants associated with cells frequently requires two steps of ion-exchange chromatography. 

Size-exclusion chromatography (SEC)

Due to volume restrictions, size-exclusion chromatography (SEC), a less common last step for eliminating remaining proteins and impurities, frequently uses a size-based filter rather than a column. Another common technique is tagged affinity chromatography. In this procedure, a fusion protein carrying a terminal affinity tag, such as a polyhistidine tag, is used to create a recombinant antibody. Molecules in the chromatography column have been functionalized to bind the tag with high affinity. Examples of these molecules include tris-NTA, a nickel complex that holds histidine residues securely but also reversibly.  

This method enables capturing and purifying antibodies in a single step through immobilized metal ion affinity chromatography. A drawback of this method is the creation of antibodies fitted with a tag, which may prevent the antibody from adequately attaching to its target antigen. As a result, removing this tag necessitates additional steps in the procedure. 


There you have it, a cornucopia of antibody characterization methods that should help you meet almost any challenge. Each has advantages and disadvantages, so before choosing one method, try and find out more. Choose the antibody characterization method best for your application to ensure optimal results. The new antibody characterization school brings a host of new terms and techniques. Don’t be afraid to try variations on each method to see what works best for you and your antibodies.

Related Posts

Techcrams logo file

TechCrams is an online webpage that provides business news, tech, telecom, digital marketing, auto news, and website reviews around World.

Contact us: info@techcrams.com

@2022 – TechCrams. All Right Reserved. Designed by Techager Team