Monoclonal antibodies (mAbs) are a preferred class of protein therapeutics used for the treatment of various diseases because of their ability to target specific tissues for drug delivery or the modulation of cellular activities.


Cellular production and downstream manufacturing processes commonly introduce heterogeneity to mAb structures by way of post-translational or chemical modifications that can have potential effects on product efficacy, safety, and stability. As such, thorough characterization of mAbs is required to fulfill regulatory requirements and bring new therapeutics to market.

The Delights of Diversity

A Multitude of Separation Techniques Combined with Mass Spectrometry for Characterization in Biopharmaceutical Development

Charge variant analysis using ion-exchange columns

Charge variant analysis uses ion-exchange chromatography (IEX) to separate proteins based on differences in charge, making it an ideal method for both qualitative and quantitative evaluation of charge heterogeneity.


Anion-exchange columns contain positively-charged surface ligands that interact with negatively-charged analytes, while cation-exchange columns have negatively-charged surface ligands that interact with positively-charged analytes. Early ion-exchange chromatography methods used salt gradients to disrupt ionic interactions of the proteins with the stationary phase and elute the analyte. More recent methods employ a pH gradient to provide impeccable separation results on columns as short as 50 mm in length, allowing for a five-fold increase in productivity as traditional salt gradient IEX must be ran on 250 mm long columns to maintain resolution.

Thermo Scientific offers both anion- and cation-exchange columns in the MAbPac and ProPac column families described on Charge Variant Analysis HPLC Columns. Among these, our ProPac 3R SCX and ProPac 3R SAX Columns offer the most advanced column technology for charge-variant analysis in our portfolio. Using a novel monodispersed polymeric resin, ProPac 3R IEX Columns deliver the resolution power, lot-to-lot reproducibility, and robustness needed for reliable charge-variant analysis workflows.


Thermo Scientific CX-1 pH Gradient Buffer Solutions can be used to generate highly reproducible, linear pH gradients using cation-exchange chromatography, as described in our CX-1 gradient buffers product specification sheet, Simple method development for charge variant characterization.

BioPharma workflow solutions

Chromatography columns and consumables

Get an easy start on your biopharmaceutical analysis with the right application specific consumables. Learn how our portfolio of complete biopharma workflow solutions can simplify your methods in our biopharma workflow brochure.


Conformational variance analysis

Proteins therapy products can be subjected to a variety of biochemical modifications during processing, delivery, and storage, which have been shown to affect the safety and efficacy of these therapeutics.


Oxidation of amino acid residues can alter the hydrophobic nature of a therapeutic protein either by increasing the polarity of the amino acid’s oxidized form or by causing a conformational change of the therapeutic protein.


Although conformational variance analysis can be accomplished through charge variance analysis, hydrophobicity-based HPLC methods such as reversed phase liquid chromatography and hydrophobic interaction liquid chromatography (HIC) have proven to be the most suitable solution for separating protein variants with little difference in hydrophobicity, such as oxidation variants.


Oxidation that causes the conformational variance can come from interactions with surfactants in the production process or from other points in the process. As a result, there is an incentive to monitor the surfactants used throughout production to discover the root cause of any non-conformities related to drug stability. A high-resolution surfactant analysis of polysorbate, a commonly-used surfactant in biopharmaceutical manufacturing, by charged aerosol detection (CAD) can be found in the application note Polysorbate 80 profiling by HPLC with mass and charged aerosol detection.


Thermo Scientific MAbPac HIC-20 Columns are high-resolution, silica-based HIC columns with a unique proprietary stationary phase that provides high resolution and rugged stability and is particularly good for antibody (AB) oxidation and analysis of disulphide protein variants. Separation of mAb oxidation variants using a MAbPac HIC-20 column is further described in the application note Separation of Monoclonal Antibody (mAb) Oxidation Variants on a High-Resolution HIC Column.

Antibody-drug conjugate (ADC) analysis

Antibody-drug conjugates (ADCs) are a class of biopharmaceutical drugs designed as a targeted therapy for treating cancer and typically composed of a mAb covalently attached to a cytotoxic drug via a chemical linker. ADCs are a hot topic in research and development of anticancer drugs as they combine the advantages of highly specific targeting abilities with highly potent killing effect to achieve accurate and efficient elimination of cancer cells.


The conjugation of drugs often results in ADC molecules which are heterogeneous with respect to both the distribution and loading of cytotoxic drugs on the mAb. Unconjugated mAbs have significantly lower potency, and the ADCs with high drug loads are subject to rapid renal clearance.


As the number of drugs attached to the mAb has been shown to directly affect the safety and efficacy of the drug, it is critical to fully characterize and monitor the heterogeneity of ADCs during development and production.

Site-specific labeling of NISTmAb to synthesize ADCs with DIBO-Angiotensin II, DIBO-Biotin, and DIBO-AF488.

The attachment of a cytotoxin alters the hydrophobicity of the antibody so that, during HPLC, the least hydrophobic unconjugated antibody will elute first, and elution time increases as the number of drugs attached increases. HIC is, therefore, considered to be the method of choice for characterizing the distribution of ADC molecules with different drug-to-antibody ratios (DARs) – more information on this topic can be found in the application note High-Resolution Separation of Cysteine-Linked Antibody-Drug Conjugate Mimics Using Hydrophobic Interaction Chromatography. Thermo Scientific MAbPac HIC-Butyl Columns are packed with C4-bonded polymeric particles and the hydrophilic nature of polymer particles and optimal density of butyl functional groups lead to excellent biocompatibility, low carryover, and high resolution.


Mass spectrometry (MS) is also becoming a popular method of ADC characterization as MS provides a more in-depth understanding of ADC biomolecules and their various species. For MS applications, it is important to use MS-suitable solvents, which rules out the use of HIC columns and their high salt content. An alternative column that offers MS-compatibility is the Thermo Scientific MAbPac Reversed Phase (MAbPac RP) Column, which contains a polymer particle with a significantly large pore size (1500 Å) that allows proteins to diffuse very efficiently. The polymer sorbent is an effective interaction source for hydrophobicity that also makes the column very robust, allowing for the use of stronger organic solvents, effective column cleaning, and reduced carryover. The paper Development of NISTmAb-derived Antibody-drug Conjugate (ADC) Standards provides an example of a reversed phase, MS-based, NISTmAb-derived ADC standard application protocol.

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Aggregate analysis

Aggregates are accumulated protein monomers of biopharmaceuticals, which stick together to form dimers, trimers, or larger order structures of antibody molecules. They are typically formed during fermentation, downstream product purification, storage, or mishandling of the drug prior to patient administration. Protein aggregation can cause adverse immunological reactions that result in serious safety and efficacy issues, and thus aggregate formation must be monitored throughout the production process and during storage of the formulated biotherapeutic.


Size-exclusion chromatography (SEC) is the standard method for this aggregate analysis as biopharmaceutical monomers can be differentiated from aggregates and mAb fragments by their size.


As SEC is one of the few chromatography methods that exhibits no ‘on-column’ focusing, the pre-column dispersion on the HPLC system used is extremely important, especially at reduced flow rates on smaller internal diameter (i.d) columns, as there will be no focusing of broad peak volumes at the head of the column.


If HPLC systems with more dispersive volumes are used with more modern SEC columns (e.g., larger and longer tubing, larger flow cells, and, in general, greater dead volumes), dispersion can easily lead to an up to 50% increase in peak widths. The low dispersion Thermo Scientific Vanquish Flex Quaternary UHPLC system was used to perform SEC protein aggregate analysis in order to control and study the effects of dispersion, as described in the application note The importance of correct UHPLC instrument setup for protein aggregate analysis by size-exclusion chromatography.


Thermo Scientific MAbPac SEC-1 Columns are silica-based UHPLC columns with a pore size of 300 Å, applicable for the molecular weight range of the monomers and dimers of a typical 150 kDa mAb. These columns utilize a proprietary covalently-bonded diol hydrophilic layer to prevent secondary interactions and peak tailing, in contrast to traditional SEC columns on the market where biopharmaceuticals still can show non-specific binding. Protein aggregation analysis of biotherapeutic mAbs run on a MAbPac SEC-1 column on a low dispersion UHPLC system displays the high-resolution capabilities of the column, as described in the application note A universal chromatography method for aggregate analysis of monoclonal antibodies.

A Universal Eluent System for Method Scouting and Separation of Biotherapeutic Proteins by Ion-Exchange, Size-Exclusion, and Hydrophobic Interaction Chromatography | Analytical Chemistry

For biopharmaceutical research and development labs, it may be beneficial to have a full setup for easy scouting of all biotherapeutic protein characterization and quality control methods to assure correct characterization. This article provides an automated method scouting approach along with a selection of different methods that require minimal manual intervention.

Glycan analysis using HILIC HPLC columns

Glycosylation – the attachment of sugar moieties to proteins – is done as part of the post-translational modification (PTM) process to optimize a biotherapeutic’s efficacy. The PTM is characterized by various glycosidic linkages, including N-, O-, and C-linked glycosylation, glypiation (GPI anchor attachment), and phosphor-glycosylation.


Glycosylation is one of the key critical quality attributes (CQAs) of mAb-based biotherapeutics, as any structural changes can impact a biological drug’s safety, efficacy, clearance, and immunogenicity.


Accurate and precise quantification of mAb-released N-glycans is best performed on a hydrophilic interaction liquid chromatography (HILIC) column due to the polarity of the glycans. Thermo Scientific Accucore 150 Amide HILIC UHPLC Columns have a unique proprietary selectivity bound to solid core particles, which increases overall efficiency of the column without substantially increasing backpressure. For more in depth explanation of solid core technology, visit our Accucore HPLC and UHPLC Columns webpage. Accucore 150 Amide HILIC columns have a larger pore size of 150 Å that is more compatible with peptides, allowing for improved peak shape and increased separation accuracy. The application note Accurate and precise quantification of mAb-released N-glycans with an amide HILIC column describes the effectiveness of the quantification of human IgG-released N-glycans when using an Accucore 150 Amide HILIC column.

Peptide mapping with UHPLC

Due to the inherent complexity of biotherapeutics, regulatory agencies require the comprehensive characterization of these drug product to ensure product quality, safety, and efficacy. Primary sequence verification and the identification and relative quantitation of post-translational modifications (PTMs) of proteins are important characterization steps for therapeutic proteins that are frequently performed using peptide mapping methods.


Peptide mapping is used to measure several critical quality attributes (CQAs) required for the characterization of any biotherapeutic proteins and involves the treatment of proteins with a protease (e.g., trypsin) to produce a series of peptides which are separated, detected, and analyzed by liquid chromatography mass spectrometry (LC-MS). Liquid chromatography-mass spectrometry and the related multi-attribute method (MAM) analysis enable characterization and monitoring of a wide range of CQAs simultaneously as described in the article Optimized Multi-Attribute Method Workflow Addressing Missed Cleavages and Chromatographic Tailing/Carry-Over of Hydrophobic Peptides.


Host cell proteins (HCPs), which are process-related impurities derived from Chinese hamster ovary (CHO) cell host organisms during biotherapeutic manufacturing, can also be analyzed through peptide mapping with NISTmAb verification of common CHO peptides using an UHPLC-MS application setup.


Various Thermo Scientific columns can be used for peptide mapping. Thermo Scientific Acclaim VANQUISH C18 UHPLC Columns are highly retentive and contain 2.2 µm silica particles with a pore size of 120 Å. These columns also have a surface area of 300 m²/g and 18% carbon content, ensuring maximum retention of complicated samples such as for peptide mapping.


Thermo Scientific Hypersil GOLD C18 Columns are also excellent for peptide mapping analysis as they are packed with ultrapure silica particles bonded to short butyl chains for weak hydrophobic retention. The quality of the silica minimizes secondary interactions that could cause undesired retention such as peak tailing. Hypersil GOLD C18 has a pore size of 175 Å, which allows for continuous diffusion and less resistance to mass transfer and is highly beneficial for the peak shape of the peptide.


Regardless of which column you choose, Thermo Scientific SMART Digest Trypsin Kits provide a significant advance in sample preparation for biopharmaceutical protein research. The kits provide a method for fast and simple protein digestion with high reproducibility, high sensitivity, and high levels of data quality in a format that is compatible with automation, reducing the time spent on a digest to as little as 45 minutes. Visit our Protein Digestion for HPLC and MS Peptide Mapping webpage for more information.

Easy, fast and reproducible analysis of host cell protein (HCP) in monoclonal antibody preparations.

Comparability study for the determination of post-translational modifications by automated high-throughput peptide mapping analysis.

Development of a robust multi-attribute method suitable for implementation in a QC environment.