Robust Analytical Characterization:
Accurate, Reliable Quantitation for Biotherapeutics

Biopharmaceuticals have revolutionized the treatment of a number of diseases. With this success, and continued advances in biotherapeutic options, consistently reliable manufacturing and quality control processes are required. Accurate and robust analytical testing equipment and methodologies are critical.

Biotherapeutics such as monoclonal antibodies (mAbs) are typically produced using recombinant methodologies and bioprocessing technology, which can result in the generation of impurities, post-translational modifications (PTMs), and protein aggregates that can affect drug safety and efficacy. Identifying and analyzing critical quality attributes (CQAs) of complex biotherapeutic molecules is therefore a fundamental process throughout each development stage of biopharmaceutical protein production.

In this resource, explore some of the chromatographic applications for CQA monitoring within the most common bioanalysis workflows. See how Agilent Biopharma and AdvanceBio HPLC columns and technology can help ensure reliable analytical results and effective biotherapeutics.

Titer Determination and Purification

Monoclonal antibodies have become one of the most important and fastest-growing classes of biopharmaceutical products in recent years. Since the approval of the first therapeutic antibodies in 1986, not only has genetic engineering enabled increased antibody specificity, but the manufacturing processes used for mAb production have evolved significantly. A pivotal improvement has been the ability to accurately measure antibody concentration (or titer). This has enabled researchers to select the most effective, high-yielding transfected cells, but also to monitor mAb concentration throughout the development process.

Affinity chromatography is ideally suited for mAb titer determination, using antibody-antigen interactions to separate mAbs from a complex mixture. The use of monolithic Protein A or Protein G affinity chromatography columns, such as the Agilent Bio-Monolith Protein A and Protein G LC columns, can provide the most effective separations. These columns provide analytical separation of all immunoglobulin (IgG) subclasses and the capture of mAb from complex matrices. These Agilent columns are compatible with HPLC and UHPLC systems.

Agilent Bio-Monolith Protein A and Protein G LC affinity columns

Agilent Bio-Monolith Protein A and Protein G LC affinity columns

“Great result and compatible with HPLC & UHPLC.”
"It provides quick and accurate mAb titer, we can also use it to purify small amounts of mAb for some characterization experiments."

Kok Looi
Pfizer Inc

Protein Identification and Impurity Profiling

Biotherapeutic proteins are often heterogeneous, with fermentation products containing a mixture of fragments and other product-related impurities. For the assurance of protein identity, intact mass analysis using large-pore columns provides a fast and precise method of target protein and impurity characterization.

As well as intact mass analysis, biotherapeutics may be analyzed at their subunit level after digestion or reduction to improve sensitivity and provide sequence confirmation. Reversed-phase LC/MS is commonly used to analyze mAbs at the intact or subunit level, with highly optimized columns for mAb separations using HPLC and UHPLC now available.

LC/MS of intact therapeutic monoclonal antibodies using Agilent AdvanceBio RP-mAb

Agilent AdvanceBio RP-mAb columns are based on Poroshell technology and have been specifically developed for the unique challenges faced during mAb characterization to deliver rapid results with improved accuracy. Superficially porous technology allows for sub-two micron sensitivities while maintaining pressure profiles similar to those of large particle separations.

In this application note, AdvanceBio RP-mAb columns with C4 and Diphenyl chemistries, 3.5 µm superficially porous particles, and 450 Å pore size were coupled to an accurate-mass Agilent 6530 LC/Q-TOF to deliver fast and high-resolution analysis of mAbs and antibody-drug conjugates.

Separation of deamidated peptides with an Agilent AdvanceBio Peptide Plus column

While difficult to detect by mass spectrometry, the deamidation of glutamine and asparagine is one of the most common protein degradation reactions, and this analysis forms an important factor in biopharmaceutical development.

In this application note, improved resolution of deamidated peptide variants using LC/MS is achieved using a charged-surface C18 column under formic acid conditions. Featuring a hybrid, endcapped C18 stationary phase on a 100 Å pore size, 2.7 µm particle modified with a positively charged surface, the Agilent AdvanceBio Peptide Plus columns show greater selectivity for deamidated protein variants over unmodified forms, compared to a standard C18 column.

Agilent AdvanceBio Peptide Mapping

With a 120 Å pore size and superficially porous 2.7 μm particles, the AdvanceBio Peptide Mapping columns enable reliable peptide mapping performance while being two to three times faster than fully porous HPLC columns.

“The 2.7 μm particles provide an excellent bridge between HPLC and UHPLC, compatible with most 600 bar instruments, but able to withstand much higher pressures.”

April Rachamim
Global Product Manager - Biocolumns & Bioconsumables at Agilent Technologies

Agilent AdvanceBio Peptide Plus

AdvanceBio Peptide Plus columns are reversed-phase, superficially porous particle LC columns optimized for the separation of target peptides, impurities, and post translational modifications. These columns feature a hybrid endcapped C18 stationary phase on a 100 Å pore size, 2.7 μm particle modified to have a charged surface.

AdvanceBio Peptide Plus columns enable you to identify multiple critical quality attributes confidently by LC/MS. They provide the speed, sensitivity, and efficiency you need to quickly confirm and identify target proteins and peptides.

Agilent AdvanceBio RP-mAb

AdvanceBio RP-mAb columns are designed to optimize the performance of intact and reduced mAb analysis when analyzing monoclonal antibodies for biopharmaceutical discovery, development, and QA/QC applications.

AdvanceBio RP-mAb columns deliver higher resolution and faster run times to provide accurate, reproducible results. Available in a range of chemistries: SB-C8, C4, and diphenyl.

Intact level analysis banner Analysis of mAb application note Peptide level analysis banner

Glycan Analysis

As with oxidation and deamidation, glycosylation is another important PTM that plays a key role in biotherapeutic function and efficacy. Analysis of the distribution and composition of N-gylcans therefore represents a critical attribute of increasing importance for biopharmaceutical manufacturing and regulatory authorities.

Determining the glycan fingerprint can be a complex, time-consuming process, often using enzymatic methods involving PNGase F to cleave intact N-linked glycans for labeling with a suitable fluorophore, separation through chromatographic techniques such as hydrophilic interaction chromatography (HILIC), and downstream detection by fluorescence or mass spectrometry.

Agilent AdvanceBio Gly-X sample preparation, along with AdvanceBio Glycan Mapping columns, deliver rapid, high-resolution, and reproducible glycan identification by providing streamlined workflows for N-glycan release, labeling, cleanup, and separation.

Sialic Acid video tile

The Agilent AdvanceBio Sialic Acid Profiling and Quantitation kit represents a sensitive, high-throughput approach to both sialic acid profiling and quantitation. the workflow can be completed in around 5 hours.

Charge Variant Analysis

Biomolecules such as mAbs are large, heterogenous macromolecules that can exist as multiple charged species, with different levels of amino acid PTMs, such as oxidation, deamidation, and glycosylation forming the net charge of a molecule. In conjunction, these attributes impact biomolecular function and ultimately efficacy and toxicity, and in the case of mAb-based biotherapeutics, affect antigen binding. These charge variants represent a core critical quality attribute and require strict monitoring during the biopharmaceutical manufacturing process.

Significant optimization is often required for each analyte of interest. Crucial to the process is the determination of the molecule isoelectric point (pI), and therefore the pH of mobile phase conditions, to aid in column retention. While strong cation exchange columns can provide improved ease of use, weaker cation exchange columns are often required for mAbs to enable improved peak shape and resolution through ion exchange chromatography.

How shallow can you go?

This application note details the analysis of charge variants of trastuzumab and NISTmAb reference standards with the Agilent 1290 Infinity II Bio LC system, specifically designed for use in biochromatography, and Bio mAb HPLC columns for high-resolution charge-based separations of mAbs. An iron-free flow path and high-performance binary pump deliver accurate and precise solvent mixing and enable the creation of shallow solvent gradients, best suited for mAb separation.

Aggregate Analysis

During the biopharmaceutical manufacturing process, biomolecules are subject to multiple stress conditions, including changes in temperature, pH, concentration, and even mechanical shear stresses – all of which can make the molecule susceptible to aggregation. This can affect the quality and performance of the final product, making aggregate analysis a CQA for biopharmaceutical production and is typically monitored using size exclusion chromatography (SEC).

While SEC is an effective technique for the separation of protein monomers from their aggregates, in biopharmaceutical production, the process can often be made more complex due to the presence of hydrophobic cytotoxic drugs and secondary interactions. The use of bioinert, robust columns optimized for the accurate quantitation of aggregation and fragment analysis is therefore a key factor to consider.

Agilent 1290 Infinity II Bio LC system

“This is the best bioinert UHPLC system available.”
"This new line of Bioinert UHPLC will blow the other vendors away. It truly is the best bioinert system out on the market."

Justin Jeong
Genentech, Inc

Elevate your mAb aggregate analysis

This application note details the use of SEC using minimized system dead volumes on the Agilent 1290 Infinity II Bio LC system and the unique hydrophilic bonding chemistry of the Agilent AdvanceBio SEC column. Using a protein standard mixture and mAbs, this methodology can separate proteins and their aggregates and provide the highest resolutions while minimizing maintenance costs.

With a unique hydrophilic coating, the Agilent AdvanceBio SEC column speeds up analysis while maintaining an accurate level of resolution between critical peaks – making it ideal for high-resolution, high-throughput separations.

*Certain images and/or photos on this page are the copyrighted property of, its contributors or its licensed partners and are being used with permission under the relevant license. These images and/or photos may not be copied or downloaded without permission from Other images courtesy of Agilent Technologies.