Pharmaceutical Analysis

Three Methods for Critical Quality Attribute Determination of Monoclonal Antibodies

In this article, from Issue 12 of the Analytix Reporter, produced by Merck, three step-by-step protocols to monitor critical quality attributes (CQAs) of monoclonal antibodies were developed using adalimumab as an example.

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Three_Methods_AR12Monoclonal antibodies are glycoproteins used as drugs in the treatment of a variety of cancers and immune system disorders. These large biomolecules have complex structures making thorough characterization essential to ensure the safety and efficacy of drugs. This article provides an overview of the different workflows that can be used for an accurate characterization of monoclonal antibodies' critical quality attributes (CQAs).


Increasing importance of monoclonal antibody (mAb) therapeutics has fundamentally changed the pharmaceutical market in recent years. These macromolecules are used in cancer therapies and in combating autoimmune and other diseases. 

A reliable characterization of mAbs is essential to ensure the safety and efficacy of both the innovator and biosimilar drugs. Therefore, the critical quality attributes (CQAs) are defined for each protein along with their acceptable ranges.

Common analytical techniques employed to verify CQAs being within the set limits include capillary zone electrophoresis, isoelectric focusing, ion exchange, size exclusion (SEC), reversed phase (RP) or hydrophilic interaction liquid chromatography (HILIC), coupled with ultraviolet (UV), fluorescence, or mass spectrometric (MS) detection. Strategies used to aid the analysis include chemical or enzymatic fragmentation of the protein into subunits, removal and analysis of glycans, and proteolytic digestion of the protein into smaller peptides followed by their sequencing. A combination of these techniques can also be applied. For the analysis of mAbs from cell culture supernatants, an affinity purification step is often used to prepare samples prior to their analysis.

Recently, step-by-step workflows for three approaches commonly used in mAb characterization were prepared.

These approaches determine:

  • the mass of the intact molecule and of the heavy and light chains (intact and middle-up mass analysis),
  • the amino acid sequence (peptide mapping), and
  • the glycosylation state (glycan analysis).

Download the full article to read more about these three approaches and how they satisfy the requirements of regulatory authorities to ensure efficacy and safety of these drugs.

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*The life science business of Merck operates as MilliporeSigma in the U.S. and Canada.

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