Christopher Ziegenfuss is Vice President, at BioPharmaSpec, Inc

Christopher Ziegenfuss 

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Biopharmaceutical product development is burgeoning with developments in treatment approaches, chemistries, and vectors giving companies new strategies for targeting disease. Biosimilars continue to build as a sector of this market with an estimated 2025 value in the region of US$42 billion (1). Many other products are also under development with innovative designs in the areas of fusion proteins, antibody drug conjugates (ADCs) and related species, peptides, and oligonucleotides.

No matter the nature of the product under development, one of the single most important factors during that development phase is the analytical characterization of the product. The need to characterize goes beyond the obvious requirement for demonstration of correct structure of the API itself but also demonstrates control of the manufacturing process, assesses impurity profiles, and ultimately feeds back to changes and improvements in the manufacturing process to ensure the best quality product. Characterization data also builds in quality to the product by having that deep knowledge of its structure and compositional profile. A detailed and thorough characterization is required to be presented to regulatory authorities as part of any submission for regulatory approval and marketing authorization.

The expectations for structural analysis are clearly laid out in a guideline published a number of years ago by the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH). ICH Q6B provides a clear framework for expectations regarding the structural characterization of biopharmaceutical products (2). While the document itself is a quarter of a century old at this point, it is laid out in such a way that relevancy is maintained. It uses terms like “to the extent possible”, thus allowing for methodological developments pushing the ability to detect structural details in ever greater and more precise ways. That being said, it is recognized that an update is due to acknowledge and bring into focus current analytical practices and to align it with other ICH guidelines. This guideline forms the key document referenced by regulatory authority documents that detail structural characterization requirements of biopharmaceuticals. This covers new biotechnologically derived protein, glycoprotein, and peptide products as well as biosimilars (3, 4). So why is ICH Q6B so important, and what does it outline?

Biopharmaceuticals, which are produced either in cellular systems or created through chemical synthesis, are molecules with incredible complexity. They can be orders of magnitude larger than what we may consider basic pharmaceuticals to be and with this increased size comes not only increased complexity but increased ways in which this complexity manifests. One must consider not only the basic linear structure of the molecule but also the three-dimensional (3D) shape, modifications (either wanted or unwanted) that occur on the molecule, and interactions that may occur within and between molecules themselves (e.g., disulfide bridges and aggregation). Also, consideration must be given to impurities that may arise as a result of the manufacturing or purification processes. All these must be investigated to give as a full a structural characterization of the product as possible and also to use the data as a tool to hone the manufacturing process itself to produce the best quality drug possible. ICH Q6B details the aspects of molecular structure for which characterization is expected.

Firstly, and perhaps most importantly, ICH Q6B requires that the primary amino acid sequence of the product (protein or polypeptide) is determined. This is crucial because a recombinant protein or synthesized peptide must have a known and specific amino acid sequence to fulfill its function. No precise methodology is given for how this should be performed, but modern methods of sequencing rely heavily on state-of-the-art mass spectrometry to provide the most complete and accurate data. Depending on the instrument type, mass spectrometers (e.g., quadrupole time-of-flight [Q-TOF] geometry and similar instruments) can generate mass values with very high mass accuracy (low ppm). This high mass accuracy in combination with this type of instruments’ ability to fragment peptides generates precise sequence information.

Proteins are proteolytically digested to peptides using strategies tailored to the protein under investigation. Peptides are separated and analyzed by on-line liquid chromatography–mass spectrometry (LC-MS), and fragmentation data generated are then used to determine peptide and ultimately protein sequence. This is not to say that MS will be able to generate 100% of the sequence coverage. There may be regions of the protein where peptide generation is difficult or fragment ions are weak or ambiguous. In these instances, gas phase sequencing of purified peptides (also known as Edman sequencing) can be performed to sequentially identify amino acids based on their chromatographic elution positions following specific, sequential chemical labeling.

ICH Q6B also requires that the overall amino acid composition of the product is investigated. To achieve this, the relative molar amounts of each amino acid in the product need to be determined. This is typically carried out using relative quantitation of amino acids following acid hydrolysis of the product and chromatographic separation of the released and derivatized amino acids. Comparison to known amounts of standard amino acids allows for accurate quantitation of each amino acid. It is recognized that no single acid hydrolysis condition will be suitable for all amino acids, so this must be taken into account in terms of recoveries of amino acids and if necessary alternate hydrolytic approaches taken.

Both the N- and C-termini of the product are required to be assessed to demonstrate the degree of homogeneity in each case. Post-translational processing may result in changes to the termini (e.g., ragged ends or N-terminal pyroglutamination), and these must be investigated. N-terminal analysis can be performed by gas phase sequencing if there is a free amine group at the N-terminus (e.g., no pyroglutamination or other blocking group), but there is no equivalent analysis for the C-terminus. Therefore, these terminal investigations are often performed as part of the peptide mapping study.

Peptide mapping is one of the most data-rich analyses that ICH Q6B requires. This investigation is, in some ways, associated with the procedure for primary amino acid sequencing discussed previously. However, peptide mapping focuses on mass analysis of proteolytically released peptides and comparison to the expected masses. The use of high-end mass spectrometers that can generate real-time fragmentation data allows a further confirmation of the identity of peptides detected. Peptide mapping generates a wealth of data, because the combination of specific proteolytic digest and on-line LC-MS analysis of the digest products allows not only accurate mass identification of the peptides themselves but also the identification of any post translational modifications (

). These may either be specific to the production process itself (such as pyroglutamination of the N-terminus as is frequently seen in monoclonal antibodies (mAbs) or may be the result of purification processes (such as amino acid oxidation or deamidation events) and may indicate that process change is required. Peptide mapping also generates data that are required for other areas of ICH Q6B, specifically terminal amino acid sequence identification and disulfide bridge analysis. It also identifies glycosylation sites and can be used to assess the degree of glycosylation site occupancy for glycoproteins. It thus fits well into a multi-attribute monitoring workflow.

Figure 1: Examples of the most commonly found post-translational

modifications on proteins. These can be identified and investigated through

peptide mapping studies with detailed glycan characterization taking place

through specific studies. (Figure courtesy of the authors)

Disulfide bridges are links formed across a protein chain, or between protein chains, via the side chain thiol groups of the amino acid cysteine. These bridges serve to maintain the correct 3D shape of the molecule and are very precisely formed, such that the same bridges will be formed from the same pair of cysteine residues in each copy of the protein. The disulfide bridges in a protein are required to be identified to confirm that first they are present and secondly that they are correctly formed. The presence of incorrect disulfide bridges is termed “scrambling” and indicates the presence of a misfolded protein population. This population is likely to result in inactive material but could at worst lead to an immunogenic response in the patient or product aggregation. Disulfide bridges can be identified through targeted proteolytic and/or chemical digestion strategies and MS-based analyses and can be seen as akin to peptide mapping, albeit focused specifically on the bridges (

). It is also important to assess for any unbridged cysteine residues.

Figure 2: Disulfide bridge analysis. Bridged peptides are identified by mass following proteolytic digestion. Fragment ions or a reduction step are then used to confirm the bridge identification. (Figure courtesy of the authors)

Many of the biopharmaceutical products on the market today are glycoproteins with mAbs being the most widespread example. For glycosylated products, it is a requirement that the sugar structures of the molecule (known collectively as glycosylation) are thoroughly characterized. This requires a quantitative analysis of the individual monosaccharides in the glycans (which can be performed by gas chromatography [GC]-MS or LC with fluorescence detection for sialic acids), but most significantly the overall glycan profile must be investigated. This usually involves release of the glycans, and either fluorescent labeling and analysis by on-line LC-MS with fluorescence detection (

) or MS analysis of released glycans following chemical derivatization, depending on the type of glycan being analyzed. This process allows the relative abundances of each glycan to be assessed along with the identification of each glycan’s composition through mass values detected.

Figure 3: Fluorescence profile of released N-glycans from a monoclonal antibody following fluorescent labelling and liquid chromatography-mass spectrometry (LC-MS) analysis. MS provides mass information allowing identification of N-glycan compositions. (Figure courtesy of the authors)

To be able to fully define their structures, the linkages between the monosaccharides must also be characterized. Linkage analysis can be performed through a series of elegant chemical steps culminating in GC-MS analysis of the end products. The elution positions and fragmentation patterns identify the original linkage and type of each monosaccharide. Knowing the biosynthetic pathways of glycosylation along with the data generated, it is then possible to define the population of glycans on the product to the extent possible. ICH Q6B also requires that, for glycoproteins with more than one glycosylation site, the glycan profile at each site must also be characterized. Glycosylation is not a templated post-translational modification (PTM) and can be highly heterogenous, depending on the glycoprotein. A detailed knowledge of glycan biosynthesis is needed to fully interpret the data and define the glycan structures indicated by the data.

Modern MS instruments have very high mass accuracy and resolution, allowing an accurate assessment of the molecular weight of the intact product and associated heterogeneity (low ppm). It not only provides data related to the size of the molecule but also may provide orthogonal data supporting other primary assessments of key molecular characteristics including post-translational modifications. Analysis under various conditions (e.g., native [

], reduced, and/or following deglycosylation) allows for an assessment of the overall product mass as well as individual protein chains in multimeric species and protein chains in the absence of glycan heterogeneity.

Figure 4: Intact molecular weight of a sample of Herceptin (trastuzumab). On-line liquid chromatography-mass spectrometry (LC-MS) of the sample was performed followed by transformation of the raw mass data to generate the intact mass values. The mass differences observed between the major signals are consistent with differing levels of galactose on the N-glycans. Minor signals indicate that other sources of heterogeneity exist in the sample,and these can be further investigated. (Figure courtesy of the authors)

The MS data should be used alongside data from more traditional methods such as sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (or its more modern capillary-based equivalent of capillary electrophoresis-SDS) and size-exclusion chromatography (SEC) when compiling a robust characterization package, to generate orthogonal data sets. With their technical and methodological improvements, advanced techniques provide valuable information on overall product mass profiles. With properly designed experiments, these analyses provide data that will be indicative of the quality of the manufacturing process and the product itself.

Charge isoform analysis provides a profile of species within the sample based on separation of the various charge states. There are many structural attributes within a protein that can affect the charge profile, the most frequently encountered being the post-translational modification deamidation, the presence of sialic acid in glycoproteins, or in the case of monoclonal antibodies (mAbs) the presence of pyroglutamate or heavy chain C-terminal lysine.

ICH Q6B notes the isoform pattern can be assessed using isoelectric focusing or other appropriate techniques. Frequently the capillary-based technique of image capillary isoelectric focusing (icIEF) is used due to its speed, resolution, reproducibility, and reliability of UV and fluorescence-based detection for relative quantitation. Data generated gives charge profile information, which can be further investigated using specific sample treatments prior to reanalysis. This is another area where collection of orthogonal data builds a thorough understanding of the product. For example, charge-based PTMs detected during peptide mapping or glycan analysis experiments may give rise to unique isoform profiles. Along with isoelectric focusing, ion-exchange chromatography can be used to assess isoforms as well as chromatographic patterns.

It is vital to be sure of the amount of product being administered to ensure patient safety and for product bioassay data generation. A common UV absorption reading can be used to measure protein content. When this strategy is applied, it is critical to determine the extinction coefficient. This determination can be achieved using a combination of ultraviolet-visible spectroscopy spectrophotometry and optimized amino acid analysis that has been assessed specifically on the product to demonstrate its suitability. Very often this encompasses a qualification or validation of the method to ICH Q2(R2) specifications (5,6).

Some aspects of structural characterization requirements specified by ICH Q6B overlap with one another. In the case of electrophoretic patterns for example, the same icIEF and CE-SDS experiments applied under the headings of size and/or isoform patterns can be used to provide the required data on identity, homogeneity, and purity for the characterization dossier that this analytical section covers.

The identity, homogeneity, and purity of biologic products needs to be evaluated, and this can be carried out using a variety of chromatographic techniques. The most commonly employed are reverse-phase, SEC, and ion exchange chromatography, each evaluating a different physicochemical characteristic of the molecule. As with other characterization assays, additional or substitute methods such as HIC or HILIC (hydrophilic interaction chromatography), can be used to enhance knowledge and understanding of the product depending on its structural characteristics. Several chromatographic techniques are expected to be applied to separate out components based on different physical properties such as charge, hydrophobicity, and mass. This gives the most comprehensive investigation into the overall composition of the sample because any coeluting species in one technique will likely separate in others.

Spectroscopic techniques are applied during characterization to assess the higher order (secondary and tertiary) structure. The 3D shape of a biological product plays a critical role in its function and therefore must be characterized to the extent possible. The higher order structures of molecules can be composed of different types of structural features, depending on the nature of the primary amino acid sequence and how it interacts with itself in its solution environment to produce the final 3D shape. At the secondary structural level, ordered structural features such as alpha helices or beta sheet structures can be produced, depending on the amino acid sequence, but regions of more random-type structure can also be found. How these secondary structural units interact with one another and assemble in three dimensions gives rise to the tertiary level of molecular structure. Various techniques should be applied during characterization, each of which has its own relative strengths and will thus probe the structure in different and orthogonal ways. The techniques of circular dichroism (CD), Fourier transform infrared spectroscopy (FT-IR), fluorescence analysis, microfluidic modulation spectroscopy (MMS) and nuclear magnetic resonance (NMR, both 1D and 2D) are all appropriate for higher order structural (HOS) analysis and provide a meaningful orthogonal dataset (7).

An assessment of aggregation is important as part of an investigation into product related impurities, which is also covered by ICH Q6B. Orthogonality is again important here and the application of two techniques such as SEC-multi-angle light scattering (MALS) (a column-based method, 

) and SV-AUC (a non-column-based method), which work on different physical principles, gives meaningful data.

Figure 5: Size exclusion chromatography-multi-angle light scattering (SEC-MALS) analysis of a protein showing the presence of mostly monomer (peak eluting at approx. 6 mins) but other higher mass species are also detected such as dimer, and trimer eluting at approx. 5.5 mins and 5.0 mins respectively. Lower levels of other higher oligomeric states are also present. The UV profile is shown with the MALS mass data given a discreet lines across peaks in the upper portion of the image. (Figure courtesy of the authors)

The ICH Q6B guidance document has been adopted by regulatory agencies due to its comprehensive coverage of structural requirements both for the product itself but also for impurities found within it. These in-depth characterization studies must be performed during product development and following significant process changes and are expected to be present in any drug application document.

The nature of biologically produced biopharmaceuticals inevitably results in some level of heterogeneity that must be assessed and for which techniques must be used that can provide clear and detailed analytical data. Researchers who follow the suggested guidelines outlined in ICH Q6B, using state-of-the-art analytical equipment and techniques, will be well-positioned to move their product through the regulatory processes and also for critically assessing the production process itself.

Mordor Intelligence. Biosimilars Market Size | Industry Growth & Forecast Report. Mordorintelligence.com (accessed May 5, 2025).

ICH. Specifications. Q6B. Test Procedures and Acceptance Criteria for Biotechnological/Biological Products (ICH, March 1999).

FDA. Development of Therapeutic Protein Biosimilars: Comparative Analytical Assessment and Other Quality-Related Considerations, Draft Guidance for Industry (CDER, CBER, 2019).

EMA. Guideline on Similar Biological Medicinal Products Containing Biotechnology-derived Proteins as Active Substances: Quality Issues (revision 1) (May 22, 2014).

ICH. Q2(R2). Analytical Validation (ICH, 2023).

Easton, R.L. Structural Characterization Methods for Biosimilars: Fit-for-Purpose, Qualified, or Validated. GaBI J., 2022 11, 41-44

Greer, F and Easton, R.L. Biosimilars–Increasing Regulatory Focus on Orthogonal Analytical Characterization. International Biopharmaceutical Industry, 2021 4, 10-13.

Richard Easton, PhD, is Technical Director–Structural Analysis, BioPharmaSpec Ltd, and Christopher Ziegenfuss is Vice President, at BioPharmaSpec, Inc.

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Volume 49, No. 5
June 2025
Page: 22–28

When referring to this article, please cite it as Easton, R. and Ziengenfuss, R. ICH Q6B for Analytics. Pharmaceutical Technology 2025 49 (5).

Articles

ICH Q6B provides expectations and a clear framework for the structural characterization of biopharmaceutical products. 