MAb screening & characterizationReaders familiar with our monoclonal antibody (MAb) characterization solutions know that we communicate with our customers via webinars featuring industry experts, talks, and posters presentations. During the course of these events, customers present us with many questions about their work, and here are some interesting and valuable questions posed by our customers on MAb screening & characterization!

I also wanted to share a couple of interesting reads that readers might find useful. First, is a list of therapeutic MAbs approved or in review in the European Union or United States. And, the second, an insightful read on the monoclonal antibody development business in China. (Both were shared on one of the MAb groups on LinkedIn.)

Here goes with the FAQs.

How long does it take to perform the Protein A, size exclusion chromatography (SEC) , and Ion Exchange (IEX) steps on your fully automated MAb analysis platform?

Using our current biomolecules liquid chromatography analysis platform (Thermo Scientific Dionex UltiMate 3000 Biocompatible Analytical LC System) and our new 3 μm monoclonal antibody analysis column (Thermo Scientific Dionex MAbPac SCX-10 column), we can get the total run time for all three analyses down to 60 min. For a detailed description of the method and results, view this poster, Automated Monoclonal Antibody 2-Dimensional Workflow: from Harvest Cell Culture to Variant Analysis (downloadable PDF).

Do the peaks in your pH-gradient ion-exchange chromatography (pH-IEC) method correlate to the peaks in cIEF method? Can I use pH-IEC to collect impurities seen on the cIEF method?

We are not sure as we have not done any experiments using the cIEF method but think it may be similar because the mechanism of separation is same.

Has you tried packing a sub-2 micron particle resin ion-exchange column to further increase the resolution and reduce analysis time?

The lowest particle size we have used is 3 μm. We have not attempted using sub-2 micron particle resin because the tests with the 1.7 μm columns were not satisfactory. Our newly released 3 μm small-particle columns (referred to in the previous answer) are ideal for charge variant analysis and provide excellent high-resolution results with very fast analysis. These columns are an excellent compliment to our industry gold-standard column for the high-resolution separation of proteins and MAbs (Thermo Scientific ProPac WCX-10 column). (In case you are interested in the ProPac columns, here is a short tips and tricks guide for improving monoclonal antibody variant analysis and characterization for these columns.)

What is the advantage of online protein characterization?

The key advantages of online protein characterization are savings of time and labor and the automation of routine analysis. We have developed an automated on-line MAb platform (Thermo Scientific Dionex UltiMate 3000 Titanium solution) for high-throughput routine analysis featured in this poster, Development of an Automated Method for Monoclonal Antibody Purification and Analysis (downloadable PDF).

The poster also highlights the capabilities of our autosampler (Thermo Scientific Dionex WPS-3000T bioinert autosampler) and Chromeleon Chromatography Data Software (CDS). This method includes the ability to automate high-flow fraction collection and reinjection for high-throughput analysis of biopharmaceuticals.

Is it sufficient to just implement a platform method or is there more to do?

It depends on your needs. For example, if you need to look into the charge-variant analysis in-depth, then you can set up another automated 2-D workflow using the same system. The first dimension will be ion-exchange and the second dimension will be reverse phase. Samples collected from the ion-exchange can be pre-concentrated onto a trap column before they are injected onto the reverse-phase column.

Can you use dynamic light scattering (DLS) to characterize proteins?

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How many samples do you typically run in parallel when doing glycan analysis?

For any analysis, you will need multiple runs and samples.

Which method is best for quantitation of charged isoforms, HPLC-CEX or cIEF?

We recommend and use CEX ourselves.

Which percentage of aggregates is acceptable for monoclonal antibodies?

This depends on your customer’s sample and their product level of acceptance.

What about the ability to generate enough material to look at PK or other studies using this approach? Genentech and others recently have adapted displacement chromatography to the cation exchange analysis. Have you done this?


The reverse-phase method is used for detecting LC and HC of MAbs currently, but is not good for intact, native form. What do you think about it?

The solvents used in the reverse-phase method denature the proteins.

What are the recommended analytical methods for glycan structural characterization?

We reccomend HPLC-MS/MS for the separation and characterization of glycans present on MAbs.


If you have questions you would like answered on this topic, don’t forget to add them to the Comments box below. Our experts will be pleased to provide answers.