The characterization of product-related variants in monoclonal antibodies involves identifying and quantifying the size and charge of variants that can impact the activity, efficacy, and safety of the antibodies. These variants represent distinct molecular forms that may arise from processes such as fragmentation, dimerization, aggregation, or post-translational modifications. The characterization of variants typically involves isolating the relevant species using a semi-preparative scale HPLC and analyzing them using various analytical techniques and biological assays. The commonly used analytical techniques include size-exclusion and ion-exchange chromatography, light scattering, mass spectrometry, capillary isoelectric focusing, and capillary electrophoresis-sodium dodecyl sulfate with ultraviolet or laser-induced fluorescence detection, among others. Additionally, functional assessments are performed using cell-based assays and binding assays to assess the biological activities of the variants. Identifying product-related variants through characterization enables the recognition of impurities that compromise the quality and safety of the drug.
Recombinant monoclonal antibodies (mAbs) are biotherapeutics known for their high selectivity in binding to target antigens and inducing an immune response. Multiple structural variants may arise in mAbs due to post-translational modifications (PTMs) or processes like fragmentation, dimerization, or aggregation (1). The common modifications in mAbs include N-linked glycosylation, oxidation, deamidation, isomerization, glycation, cysteinylation, C-terminal lysine cleavage, among others, leading to increased heterogeneity and diverse charge variants (Table I) (1–5). These product-related variants may form at any stage of the antibody manufacturing process, including cell culture, downstream recovery, or storage. These variants can impact quality attributes like stability, potency, and serum half-life, thereby limiting the product shelf-life (6).
According to the ICH Q6B Guidelines (7), product-related variants comparable to the desired product in terms of activity, efficacy, and safety are deemed product-related substances, while those deviating in these properties are labeled as product-related impurities. For instance, C-terminal lysine or N-terminal pyroglutamate variants are not expected to affect safety or efficacy, as these regions are highly exposed and not part of any ligand binding sites. In contrast, variants with deamidation and isomerization in the complementary determining region (CDR) can reduce antigen binding affinity and potency, categorizing them as product-related impurities (8–10). Similarly, oxidation in the Fc region may affect neonatal receptor (FcRn) binding, potentially influencing the drug’s half-life in serum (10–12). Generally, modifications in the Fc region do not significantly affect Fab function, however, modifications in the Fab region, especially in the CDR region, are more likely to affect antigen binding and potency (13).
To ensure product quality, it is essential to comprehensively characterize, quantify, and closely monitor product-related variants throughout the product lifecycle. The characterization of variants typically involves isolating the relevant species and extensively analyzing them using various techniques, which are discussed below.
Analytical assays, such as Size Exclusion and Cation Exchange separations with High-Performance Liquid Chromatography (SE-HPLC and CEX-HPLC, respectively), are used to monitor variants contributing to heterogeneity in Drug Substances (DS) and Drug Products (DP). Figure 1 shows typical chromatograms of an IgG1 mAb product.
It is critical to understand the properties of product-related impurities and substances observed in SE-HPLC and CEX-HPLC assays, and their differences from the desired drug product. This is primarily because species like truncated forms, aggregates, and degradation products can impact activity, efficacy, and safety. Therefore, thorough product characterization helps establish individual and collective acceptance criteria for product-related substances and impurities. Additionally, it is a regulatory expectation that all peaks from chromatography release tests are identified (14).
To perform a comprehensive characterization of the variants, isolation of each variant with high purity (>80% enriched) and suitable quantities–often milligram levels–is required. This is challenging for biologic products in which the variant species are less than 1% of the total content in the product. Coupling SE- or CEX-HPLC, which use volatile salts, directly to native mass spectrometry is an emerging and promising technique for variant characterization (15). Despite this advancement, isolation of product-related variants through offline fraction collection remains necessary to obtain material for bioactivity assays, in order to determine the structure-function relationship. A semi-preparative scale HPLC is often used to isolate size, charge, and hydrophobic variants in mAbs.
Characterizing variants observed in SE- and CEX-HPLC involves transferring these analytical HPLC methods to a semi-preparative scale for the collection of a sufficient quantity of each fraction. Method transfer, however, can be challenging due to differences in column dimensions, particle sizes, and flow rates. Moreover, variations in tubing lengths and components may contribute to band broadening caused by extra column volume, which may impact peak resolution. Therefore, successful scale up requires careful optimization and adjustments at the semi-preparative scale to ensure a close match of the chromatographic profile to the analytical scale. These adjustments may involve modifying the gradient in analytical HPLC to facilitate variants’ proper elution and separation with the large volumes of the semi-preparative column. Scaling up also requires larger volumes of solvents and additional consumables, highlighting the importance of balancing efficiency to manage costs effectively.
In addition, maintaining reproducibility across injections is critical when collecting fractions with semi-preparative HPLC, particularly when pooling fractions from multiple injections is required to obtain sufficient quantities of low-abundant variants. Once semi-preparative HPLC method is optimized for reproducibility, fractions containing desired variants are collected, but it is crucial to collect individual peaks instead of grouping low-abundant peaks. However, a single peak does not necessarily indicate a single variant but may contain one or more species. The success of variant characterization depends on the purity of the fractions. Fraction purity is assessed by analyzing each fraction alongside the unfractionated starting material using analytical HPLC. The chromatographic profiles are overlaid with the unfractionated samples, and their elution order is confirmed. In instances where co-fractionated species are present and might interfere with accurate characterization of the variants, re fractionation of the collected fractions may be necessary to ensure the isolated fractions are pure and can be reliably characterized. Figure 2 shows a semi-preparative HPLC fractionation workflow commonly used for variant characterization.
The main peak, which is the desired drug product and elutes as a major peak, is collected, and analyzed alongside the variant fractions as a control. The main peak usually lacks C-terminal lysine in the heavy chains but is glycosylated with neutral oligosaccharides at the conserved asparagine residue in the Fc region. Figure 3 shows an example of overlaid SEC chromatograms depicting the unfractionated material and four fractions of a mAb product, where each fraction appears highly enriched compared to the original unfractionated sample. It is important to control the artifacts introduced during fraction collection and sample preparation. Modifications can occur during concentration, buffer exchange, pH variations, and freeze/thaw cycles. The side-by-side characterization of the main peak with each variant is recommended as an assay control (13). Modifications can also be lost during sample preparation. Succinimide is unstable under typical denaturation, reduction, alkylation, and enzymatic digestion conditions used for generating peptides for LC-MS analysis (13).
Although fraction collection is the initial step toward thorough characterization, determining the contribution of sialic acid to acidic species and C-terminal lysine to the basic species requires enzymatic removal under native conditions before fraction collection. Sialic acid can be removed using sialidase, and C-terminal lysine can be removed by carboxypeptidase B (CPB) without affecting antibody structures. In certain cases, treatment with CPB or sialidase may be necessary before fraction collection to enrich other variants overlapping with C-terminal lysine or sialic acid variant peaks. Furthermore, the collection of Fab and Fc fragment fractions obtained from enzymatic digestion can help localize the acidic or basic species associated with Fab or Fc.
Following successful fraction collection, various analytical techniques are used for characterizing the variants. Orthogonal methods, as outlined in Table II, are used to provide unambiguous characterization of the variants.
In-depth characterization of variants in mAbs DS and DP can be challenging due to their extremely low abundance. This challenge can be addressed by exposing DS and DP to various stress conditions to enrich these low-abundant species prior to isolation. The selection of stress conditions is based on an extensive understanding of the therapeutic protein’s biophysical properties, degradation pathways, and the likelihood of exposure to those conditions during processing, packaging, shipping, and handling. Commonly applied forced degradation conditions include high temperature, freeze-thaw cycles, agitation, extremes of pH, light exposure, and oxidation. The variants most frequently observed in these forced degraded samples include aggregate, fragment, oxidized, and deamidated species. Each stress condition tends to generate specific types of variants in abundance. For instance, incubation with hydrogen peroxide generates oxidized species. A major degradation pathway resulting from heat stress is the formation of aggregates, comprising both insoluble (precipitates and particles) and soluble aggregates of both covalent and non-covalent nature (14). Forced degradation conditions should be carefully selected to mainly modify the same sites that are present in those identified in the separated acidic and basic species. When modifications at other sites cannot be avoided, the impact of off-target modifications should be considered (16). In addition to stressed samples, various in-process samples, such as those from mixed mode chromatography (MMC) fractions, serve as a valuable source for enriched variants. Therefore, along with in-process samples, forced degradation studies designed to induce and identify product-related variants enhance our ability to comprehensively characterize and understand the diverse variants present in DS and DP.
Size variants represent distinct molecular forms of therapeutic proteins that may result from processes like fragmentation, dimerization, or aggregation during manufacturing or storage. Size variants are one of the CQAs for which specifications must be set for batch release.
SE-HPLC is a widely used technique for monitoring size variants. It separates proteins based on their hydrodynamic radius, where smaller molecules permeate column matrix pores and elute later than larger molecules that pass through more quickly, resulting in distinct chromatographic peaks. SEC primarily separates the monomeric IgG from dimers, trimers, or higher order aggregates (high molecular weight or HMW species) and fragments (low molecular weight or LMW species). This allows for the quantification and characterization of different size variants. Although SEC theoretically involves no analyte interaction with a stationary phase, secondary interactions based on charge or hydrophobicity are possible. To minimize these interactions, additives such as arginine or isopropyl alcohol are commonly used. Figure 1(a) provides a representative UV chromatogram example of size variants in a mAb monitored by SE-HPLC (17).
Mass spectrometry (MS) is a powerful and accurate method for determining the molecular mass of size variants. The use of enzymes such as PNGaseF for deglycosylation removes N-linked glycans, simplifying the MS peak profile. This facilitates the identification of truncated variants at the hinge region and localization of the cleavage site. Cleavage in the hinge region is a major degradation pathway for mAbs. In IgG1 mAbs, if all inter-chain disulfide linkages are conserved, cleavage generates Fc-Fab, Fab, and Fc fragments (Figure 4). The Fc-Fab fragment is an intact mAb lacking one Fab arm, potentially impacting potency if interaction with the target receptor requires both Fab arms. The Fab fragment lacks Fc-mediated effector function and exhibits a reduced circulation half-time in serum. In the presence of a reducing agent, the Fc-Fab, Fc, and Fab transform into HC, LC, 1/2Fc, and Fd species, with Fd representing the piece of the heavy chain included in the Fab (Figure 4c) (18). Additionally, deglycosylation of mAbs facilitates the identification of glycation variants wherein reducing sugars covalently link to amines of lysine residues. To locate the glycation site, a bottom-up peptide mapping approach is commonly used (19).
Size exclusion chromatography with multi-angle light scattering and refractive index detectors (SEC-MALS-RI) is another powerful analytical technique for characterizing size variants under non-denaturing conditions. In SEC-based separation, the elution position relies not only on the protein’s molecular weight but also on its shape. Additionally, interactions with the column matrix can alter the elution position. However, the benefit of coupling MALS to SE-HPLC is that SEC-MALS is able to provide absolute molar mass irrespective of elution time, column calibration standards, molecular conformations, and non-ideal column interactions. SEC-MALS helps in identifying and determining the molecular weight of monomers, dimers, oligomers, and aggregate species, as well as truncated fragments at the hinge region.
Capillary electrophoresis with sodium dodecyl sulfate and ultraviolet or laser-induced fluorescence detection (CE-SDSUV or CE-SDS-LIF, respectively) are orthogonal methods for the quantitative estimation of size variants under denaturing conditions, with or without reducing disulfide linkages. According to the monograph 129 of the United States Pharmacopeia (USP) (20), SEC-HPLC is considered a robust method for measuring monomer and HMW species, while CE-SDS provides reliable quantitation of LMW species of mAbs. CE-SDS under non-reducing conditions is used to monitor the purity of denatured intact antibodies, while CE-SDS under reduced conditions is used to monitor intact light chains, heavy chains, and non glycosylated heavy chains (21). Figure 5 provides representative electropherograms of size variants in a mAb monitored by non-reduced and reduced CE-SDS. Together with SEC-MALS and RPLC-MS analysis, CE-SDS provides additional insights into identifying size variants and determining whether aggregates and dimers consist of covalent or non-covalent bonds.
Size variant fractions are analyzed using CEX-HPLC to evaluate their charge properties and gain a better understanding of product heterogeneity. While charge-based methods are effective for assessing dimers composed of several distinct species, they may not be as suitable for monitoring fragmentation or aggregation due to their sensitivity to chemical and structural modifications (1,2). In fact, it can be challenging to determine which peaks are a result of changes in size. In more complex scenarios, such as characterizing highly heterogeneous aggregate species, the CEX-HPLC method may show a broad and difficult-to-quantify peak in the basic region of the chromatogram. Low molecular weight fragments can produce acidic and basic variants (22).
In addition to these analytical techniques, functional assessments of variants are performed using cell-based assays and various binding assays (Table II) to assess the impact on bioactivity including the Fab- and Fc-mediated functions. The target and Fc receptor binding assays that are used include ELISA, alphaLISA, and Surface Plasmon Resonance (SPR). Cell-based assays such as anti-proliferation assays and ADCC cytotoxicity assays are important to assess the product potency and effector functions. Figure 6 shows the potency assessment result of the aggregate, dimer, and LMWS1 fractions collected from a thermally stressed mAb product (Figure 3) using a cell-based proliferation inhibition assay, indicating that the isolated dimer species is as potent as the unfractionated mAb reference standard, whereas the aggregate and the low molecular species (fragments) have either completely lost their potency or are much less potent.
Charge variants in mAbs primarily result from PTMs that modify their isoelectric point (pI) or charge distribution profile. Table I outlines key modifications contributing to acidic and basic variants commonly found in mAbs. Acidic species have a lower apparent pI, while basic species have a higher apparent pI when analyzed using isoelectric focusing (IEF)-based methods. In ion exchange chromatography-based methods, the retention times relative to the main peak define acidic and basic species. Acidic species elute before the main peak in CEX-HPLC or after the main peak in anion exchange-HPLC (AEX-HPLC). Given the basic pI of most human IgGs, CEX-HPLC is typically used to characterize the charge variants.
In ion-exchange HPLC, electrostatic interactions between the ionic groups of the stationary phase and those on the mAb surface form the basis of the separation. For example, in CEX-HPLC, separation is based on the interaction between positively charged analytes and a stationary phase with negatively charged functional groups. The antibody is subsequently eluted from the column by a salt gradient, pH gradient, or a combination of both. Basic variants with a net positive charge interact more strongly with the negatively charged stationary phase and elute later in the chromatogram. Conversely, acidic variants with a net negative charge experience weaker interactions and elute earlier. Figure 1(b) provides a representative example of a UV chromatogram of charge variants in a mAb monitored by CEX-HPLC.
The acidic and basic charge variants are characterized by multiple analytical methods outlined in Table II. High resolution MS is a powerful technique for identifying PTMs. The identification and quantification of modifications in the acidic and basic charge variant fractions often use bottom-up approaches, which involve enzymatic digestion and reversed-phase liquid chromatography-tandem mass spectrometry (RPLC-MS/MS) analysis. Tandem MS enables the determination of the position of the modifications within the peptide. Tandem MS also facilitates the identification of glycopeptides, the determination of glycosylation sites, and the quantification of various glycoforms (23). RPLC-MS/MS has been demonstrated to complement the HILIC-FLD (hydrophilic interaction chromatography with fluorescence detection) method for glycan analysis (23). Figure 7 shows the major PTMs in the charge variants of a mAb product identified by RPLC-MS/MS analysis of fractions collected from CEX-HPLC. In addition, the middle-down approach by RPLC-MS could also be used for the rapid assessment of oxidation in mAbs (24). This involves enzymatic cleavage below the hinge region followed by the reduction of inter-chain disulfide bonds, allowing the separation of the LC, Fd, and 1/2Fc fragments (Figure 8).
Imaged capillary isoelectric focusing (icIEF) is a high-resolution technique that separates variants based on their pI or net charge. In isoelectric focusing, a continuous pH gradient is established in the capillary by ampholytes upon the application of high voltage. A protein migrates along the gradient to the point at which the overall charge is neutral. The pI markers spiked in the sample help establish the pI of unknown proteins through interpolation. icIEF has become the industry standard technology for charge variant analysis due to its fast run time, high resolution, and compatibility with quality control processes (25).
As discussed earlier, functional assessments using cell-based assays and various binding assays are also important in assessing the impact of PTMs on the charge variants to the bioactivities. For instance, in a study by Harris RJ et al, the isolated acidic fractions containing highly enriched deamidation in CDR, and basic fractions containing highly enriched isomerization in CDR, showed reduced potency compared to the main peak fraction collected from a mAb product (9).
Monoclonal antibodies commonly exhibit multiple product-related variants with differences in charge, molecular weight, or other properties. While the chemical nature of the main species is usually well-understood, characterizing the variants is integral for understanding their effect on safety, efficacy, and potency. There are many factors to consider in the characterization of the product-related variants, including variant isolation technique, starting material design and selection, and the availability of extended characterization methods. A comprehensive understanding of product-related variants in monoclonal antibodies not only enhances product development but also ensures regulatory compliance and supports the continuous improvement of manufacturing processes. Ultimately, it contributes to the production of safe and effective biopharmaceuticals.
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Navin Rauniyar and Xuemei Han are with Tanvex Biopharma USA, Inc in San Diego, California. Direct correspondence to: nrauniyar@tanvex.com
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