Size exclusion chromatography of biopharmaceutical products: From current practices for proteins to emerging trends for viral vectors, nucleic acids and lipid nanoparticles.

The 21st century has been particularly productive for the biopharmaceutical industry, with the introduction of several classes of innovative therapeutics, such as monoclonal antibodies and related compounds, gene therapy products, and RNA-based modalities. All these new molecules are susceptible to aggregation and fragmentation, which necessitates a size variant analysis for their comprehensive characterization. Size exclusion chromatography (SEC) is one of the reference techniques that can be applied. The analytical techniques for mAbs are now well established and some of them are now emerging for the newer modalities. In this context, the objective of this review article is: i) to provide a short historical background on SEC, ii) to suggest some clear guidelines on the selection of packing material and mobile phase for successful method development in modern SEC; and iii) to highlight recent advances in SEC, such as the use of narrow-bore and micro-bore columns, ultra-wide pore columns, and low-adsorption column hardware. Some important innovations, such as recycling SEC, the coupling of SEC with mass spectrometry, and the use of alternative detectors such as charge detection mass spectrometry and mass photometry are also described. In addition, this review discusses the use of SEC in multidimensional setups and shows some of the most recent advances at the preparative scale. In the third part of the article, the possibility of SEC for the characterization of new modalities is also reviewed. The final objective of this review is to provide a clear summary of opportunities and limitations of SEC for the analysis of different biopharmaceutical products.

Enhancing Selectivity of Protein Biopharmaceuticals in Ion Exchange Chromatography through Addition of Organic Modifiers.

Charge heterogeneity among therapeutic monoclonal antibodies (mAbs) is considered an important critical quality attribute and requires careful characterization to ensure safe and efficacious drug products. The charge heterogeneity among mAbs is the result of chemical and enzymatic post-translational modifications and leads to the formation of acidic and basic variants that can be characterized using cation exchange chromatography (CEX). Recently, the use of mass spectrometry-compatible salt-mediated pH gradients has gained increased attention to elute the proteins from the charged stationary phase material. However, with the increasing antibody product complexity, more and more selectivity is required. Therefore, in this study, we set out to improve the selectivity by using a solvent-enriched mobile phase composition for the analysis of a variety of mAbs and bispecific antibody products. It was found that the addition of the solvents to the mobile phase appeared to modify the hydrate shell surrounding the protein and alter the retention behavior of the studied proteins. Therefore, this work demonstrates that the use of solvent-enriched mobile phase composition could be an attractive additional method parameter during method development in CEX.

Why is there no biosimilar of Erbitux®?

Monoclonal antibody (mAb)-based therapies have been a major advance in oncology patient care, even though they represent a significant healthcare cost. Biosimilars, launched in Europe in 2004 are an economically attractive alternative to expensive originator biological drugs. They also increase the competitiveness of pharmaceutical development. This article focuses on the case of Erbitux® (cetuximab). This anti-EGFR (Epidermal Growth Factor Receptor) monoclonal antibody is indicated for metastatic colorectal cancer (2004) and squamous cell carcinoma of the head and neck (2006). However, despite the expiration of the patent in Europe in 2014 and estimated annual sales of 1.681 million US dollars in 2022, Erbitux® has not yet faced any approved biosimilar challenges in the United States or in Europe. Here, we outline the unique structural complexity of this antibody highlighted by advanced orthogonal analytical characterization strategies resulting in risks to demonstrate biosimilarity, which may explain the lack of Erbitux® biosimilars in the European and US markets to date. The development of Erbitux® biobetters are also discussed as alternative strategies to biosimilars. These biologics offer expected additional safety and potency benefits over the reference product but require a full pharmaceutical and clinical development as for New Molecular Entities.

High-Throughput Chromatographic Separation of Oligonucleotides: A Proof of Concept Using Ultra-Short Columns.

Ion-pairing reversed-phase liquid chromatography (IP-RPLC) is the reference separation technique for characterizing oligonucleotides (ONs) and their related impurities. The aim of this study was to better understand the retention mechanism of ONs, evaluate the applicability of the linear solvent strength (LSS) retention model, and explore the potential of ultra-short columns having a length of only 5 mm for the separation of model ONs. First, the validity of the LSS model was evaluated for ONs having sizes comprised between 3 and 30 kDa, and the accuracy of retention time predictions was assessed. It was found that ONs in IP-RPLC conditions follow an "on-off" elution behavior, despite a molecular weight lower than that of proteins. For most linear gradient separation conditions, a column length between 5 and 35 mm was found to be appropriate. Ultra-short columns of only 5 mm were therefore explored to speed up separations by considering the impact of the instrumentation on the efficiency. Interestingly, the impacts of injection volume and post-column connection tubing on peak capacity were found to be negligible. Finally, it was demonstrated that longer columns would not improve selectivity or separation efficiency, but baseline separation of three model ONs mixtures was enabled in as little as 30 s on the 5 mm column. This proof-of-concept work paves the way for future investigations using more complex therapeutic ONs and their related impurities.

Boosting the Separation of Adeno-Associated Virus Capsid Proteins by Liquid Chromatography and Capillary Electrophoresis Approaches.

The purity of the three capsid proteins that make up recombinant adeno-associated virus (rAAV) is considered a critical quality attribute of gene therapy products. As such, there is a clear need to develop separation methods capable of rapidly characterizing these three viral proteins (VPs). In this study, the potential benefits and limitations of different electrophoretic and chromatographic methods were evaluated, including capillary electrophoresis-sodium dodecyl sulfate (CE-SDS), reversed phase liquid chromatography (RPLC), hydrophilic interaction chromatography (HILIC), and hydrophobic interaction chromatography (HIC), for the analysis of VPs obtained from different serotypes (i.e., AAV2, AAV5, AAV8, and AAV9). CE-SDS is considered to be the reference method and provides a suitable separation of VP1-3 proteins using generic conditions and laser induced fluorescence detection. However, the characterization of post-translational modifications (i.e., phosphorylation, oxidation) remains difficult, and species identification is almost impossible due to the lack of compatibility between CE-SDS and mass spectrometry (MS). In contrast, RPLC and HILIC were found to be less generic than CE-SDS and require tedious optimization of the gradient conditions for each AAV serotype. However, these two chromatographic approaches are inherently compatible with MS, and were shown to be particularly sensitive in detecting capsid protein variants resulting from different post-translational modifications. Finally, despite being non-denaturing, HIC offers disappointing performance for viral capsid proteins characterization.

Tackling Issues Observed during the Development of a Liquid Chromatography Method for Small Molecule Quantification in Antibody-Chelator Conjugate.

In the context of targeted radionuclide therapy, antibody-chelator conjugates (ACCs) are an evolving class of antibody-related drugs with promising applications as tumor-targeted pharmaceuticals. Generally, a typical ACC consists of a recombinant monoclonal antibody (mAb) coupled to radionuclide via a chelating agent. Characterizing the ACC structure represents an analytical challenge since various impurities must be constantly monitored in the presence of formulation components during the quality control (QC) process. In this contribution, a reliable method devoted to the monitoring of an ACC sample, and its small molecule-related synthesis impurities, has been developed via liquid chromatography (LC). A problem-solving approach of common analytical issues was used to highlight some major issues encountered during method development. This included separation of poorly retained impurities (issue #1); interferences from the formulation components (issue #2); analysis of impurities in presence of ACC at high concentration (issue #3); and recovery of impurities during the whole analytical procedure (issue #4). To the best of our knowledge, this is the first time that a chromatographic method for the analysis of ACC synthesis impurities is presented. In addition, the developed approach has the potential to be more widely applied to the characterization of similar ACCs and other antibody-related drugs.

Interlaboratory Evaluation of a User-Friendly Benchtop Mass Spectrometer for Multiple-Attribute Monitoring Studies of a Monoclonal Antibody.

In the quest to market increasingly safer and more potent biotherapeutic proteins, the concept of the multi-attribute method (MAM) has emerged from biopharmaceutical companies to boost the quality-by-design process development. MAM strategies rely on state-of-the-art analytical workflows based on liquid chromatography coupled to mass spectrometry (LC-MS) to identify and quantify a selected series of critical quality attributes (CQA) in a single assay. Here, we aimed at evaluating the repeatability and robustness of a benchtop LC-MS platform along with bioinformatics data treatment pipelines for peptide mapping-based MAM studies using standardized LC-MS methods, with the objective to benchmark MAM methods across laboratories, taking nivolumab as a case study. Our results evidence strong interlaboratory consistency across LC-MS platforms for all CQAs (i.e., deamidation, oxidation, lysine clipping and glycosylation). In addition, our work uniquely highlights the crucial role of bioinformatics postprocessing in MAM studies, especially for low-abundant species quantification. Altogether, we believe that MAM has fostered the development of routine, robust, easy-to-use LC-MS platforms for high-throughput determination of major CQAs in a regulated environment.

Comprehensive evaluation of zwitterionic hydrophilic liquid chromatography stationary phases for oligonucleotide characterization.

Hydrophilic interaction chromatography (HILIC) has been proposed as a valuable alternative to ion-pairing reversed-phase chromatography (IP-RPLC) for oligonucleotide (ON) analysis. In this context, the potential of seven zwitterionic HILIC columns has been evaluated against amide- and poly-hydroxy fructan-functionalized HILIC columns and a C18 column operated under IP-RPLC mode. Based on the retention characteristics of key small molecule pairs, each zwitterionic HILIC column showed a unique radar-shaped profile, suggesting different selectivities for distinct structural differences. Unmodified DNA and RNA samples were then evaluated, and the columns classified based on their retentivity. Two zwitterionic columns were particularly promising in terms of overall resolution, especially for the largest ONs (> 40-mer). Finally, separations between a chemically modified drug-like ON and its closely related impurities were performed. Although the ZIC-cHILIC column showed similar selectivity values as compared to the reference IP-RPLC technique, all columns demonstrated a general decrease in selectivity due to the minor structural differences present in the highly complex samples. This work highlights the utility of zwitterionic HILIC mode for ON analysis and it reveals the importance of understanding columns characteristics - in terms of retention and selectivity - when selecting a stationary phase for specific ON applications.

Ultra-Fast Middle-Up Reversed Phase Liquid Chromatography Analysis of Complex Bispecific Antibodies Obtained in Less Than One Minute.

This work illustrates the benefits and limitations of using ultra-short reversed phase liquid chromatography (RPLC) columns for the characterization of various complex bispecific antibodies after prolonged thermal stress at the middle-up level of analysis. First, we have demonstrated that alternative organic modifiers, such as isopropanol, can be used in RPLC mode without generating excessive pressure, thanks to the prototype 10 × 2.1 mm, 2.7 µm particle column. However, compared to acetonitrile, the selectivity was not improved, at least for the selected biopharmaceutical products. Importantly, very fast separations (sub-1 min) of high quality were systematically obtained for the different samples when using a spectroscopic detector, but a severe loss of performance was observed with mass spectrometry (MS) detection due to dispersion effects. Based on these results, there is a clear need to improve the interfacing between LC and MS (shorter/thinner tubing) to mitigate band broadening.

Anion-Exchange Chromatography at the Service of Gene Therapy: Baseline Separation of Full/Empty Adeno-Associated Virus Capsids by Screening of Conditions and Step Gradient Elution Mode.

Gene therapy is opening unprecedented opportunities for novel therapeutic approaches. Based on the concept of rescuing function mutations by co-expressing the correct gene to allow biological functions to be restored, it requires the use of viral vectors to ensure the proper delivery of therapeutic genes. In this context, recombinant adeno-associated viruses (rAAV) are the most widely used vectors. Their biomanufacturing process requires the insertion of the therapeutic gene into the rAAV (full capsids). However, a percentage of rAAV that do not contain the desired gene (empty capsids), as well as partly filled capsids, might also be produced, potentially impacting the efficiency of the therapy. Therefore, the determination of the rAAV capsids' full/empty ratio needs to be monitored to ensure consistent product quality and efficacy. Anion-exchange chromatography (AEX) can serve this need. In this contribution, thorough AEX method development, including a mobile phase, a stationary phase and gradient conditions, has highlighted its potential in supporting gene therapy. Taking advantage of the fact that viral capsids follow an "on/off" retention behavior, the application of a step gradient approach to the rAAV serotype 8 (rAAV8) allowed the unprecedented separation of rAAV8 full/empty capsids, with a resolution gain of 3.7 as compared to the resolution obtained with a fully optimized linear gradient. Finally, the developed analytical approach allowed a precise and accurate baseline separation and quantification of full and empty rAAV8 capsids, with the potential to be applied as a high-throughput quality control (QC) method.

The impact of low adsorption surfaces for the analysis of DNA and RNA oligonucleotides.

As interest in oligonucleotide (ON) therapeutics is increasing, there is a need to develop suitable analytical methods able to properly analyze those molecules. However, an issue exists in the adsorption of ONs on different parts of the instrumentation during their analysis. The goal of the present paper was to comprehensively evaluate various types of bioinert materials used in ion-pairing reversed-phase (IP-RPLC) and hydrophilic interaction chromatography (HILIC) to mitigate this issue for 15- to 100-mer DNA and RNA oligonucleotides. The whole sample flow path was considered under both conditions, including chromatographic columns, ultra-high-performance liquid chromatography (UHPLC) system, and ultraviolet (UV) flow cell. It was found that a negligible amount of non-specific adsorption might be attributable to the chromatographic instrumentation. However, the flow cell of a detector should be carefully subjected to sample-based conditioning, as the material used in the UV flow cell was found to significantly impact the peak shapes of the largest ONs (60- to 100-mer). Most importantly, we found that the choice of column hardware had the most significant impact on the extent of non-specific adsorption. Depending on the material used for the column walls and frits, adsorption can be more or less pronounced. It was proved that any type of bioinert RPLC/HILIC column hardware offered some clear benefits in terms of adsorption in comparison to their stainless-steel counterparts. Finally, the evaluation of a large set of ONs was performed, including a DNA duplex and DNA or RNA ONs having different base composition, furanose sugar, and modifications occurring at the phosphate linkage or at the sugar moiety. This work represents an important advance in understanding the overall ON adsorption, and it helps to define the best combination of materials when analyzing a wide range of unmodified and modified 20-mer DNA and RNA ONs.

Direct coupling of size exclusion chromatography and mass spectrometry for the characterization of complex monoclonal antibody products.

The present study describes the possibilities offered by an innovative bioinert size exclusion chromatography column for size variant characterization of complex monoclonal antibody products. This size exclusion chromatography column includes a novel column hardware surface. The column was prepared from metallic hardware components that were treated to have prototype hydrophilically modified hybrid organic-inorganic silica surfaces called hybrid surface technology. This provides a significant reduction in nondesired hydrophobic and electrostatic interactions that can occur between column and analyte when performing size exclusion chromatography analysis with volatile mobile phase. Compared to a reference stainless-steel column packed with the same batch of packing material, peak tailing, band broadening, and above all recovery of high molecular weight species were distinctly improved for all types of monoclonal antibody products. Based on our observations, we found that 50 mM ammonium acetate in water was a suitable mobile phase offering good compromise in terms of liquid chromatography performance and mass spectrometry sensitivity. In addition, method repeatability (intra- and interday relative standard deviations) on elution times and high molecular weight species peak areas were found to be excellent. By using this innovative size exclusion chromatography material, the low and high molecular weight species contained in various stressed and nonstressed monoclonal antibody products were successfully characterized with mass spectrometry detection.

Bispecific antibody characterization by a combination of intact and site-specific/chain-specific LC/MS techniques.

Bispecific antibodies (bsAbs) are considered as an important class of biopharmaceutical drugs, with about 160 products in clinical trials. From an analytical point of view, the correct chain-association is one of the most critical challenge to monitor during bsAbs development and production. In the present study, a full analytical workflow has been developed based on the use of various chromatographic modes: size exclusion chromatography (SEC), ion exchange chromatography (IEX), reversed phase liquid chromatography (RPLC), and hydrophilic interaction chromatography (HILIC), all combined with high resolution mass spectrometry (MS). This analytical strategy was applied to Hemlibra® (emicizumab), which is certainly the most successful commercial bsAb to date. Using this strategy, it was possible to monitor the presence of mispaired bsAb species and detect and identify additional post-translational modifications (PTMs).

Quantitative N-Glycan Profiling of Therapeutic Monoclonal Antibodies Performed by Middle-Up Level HILIC-HRMS Analysis.

The identification and accurate quantitation of the various glycoforms contained in therapeutic monoclonal antibodies (mAbs) is one of the main analytical needs in the biopharmaceutical industry, and glycosylation represents a crucial critical quality attribute (CQA) that needs to be addressed. Currently, the reference method for performing such identification/quantitation consists of the release of the N-glycan moieties from the mAb, their labelling with a specific dye (e.g., 2-AB or RFMS) and their analysis by HILIC-FLD or HILIC-MS. In this contribution, the potential of a new cost- and time-effective analytical approach performed at the protein subunit level (middle-up) was investigated for quantitative purposes and compared with the reference methods. The robustness of the approach was first demonstrated by performing the relative quantification of the glycoforms related to a well characterized mAb, namely adalimumab. Then, the workflow was applied to various glyco-engineered mAb products (i.e., obinutuzumab, benralizumab and atezolizumab). Finally, the glycosylation pattern of infliximab (Remicade®) was assessed and compared to two of its commercially available biosimilars (Remsima® and Inflectra®). The middle-up analysis proved to provide accurate quantitation results and has the added potential to be used as multi-attribute monitoring method.

The importance of being metal-free: The critical choice of column hardware for size exclusion chromatography coupled to high resolution mass spectrometry.

The goal of the study was to evaluate the possibilities offered by a new generation of metal-free SEC column to perform direct SEC-MS of protein biopharmaceuticals using ammonium acetate as the main mobile phase additive. The prototype metal-free SEC column hardware used in this work was a polyether ether ketone (PEEK) infused stainless steel tube including PEEK frits. This PEEK-lined column provides a fully bioinert and metal-free fluidic path, while maintaining the stability of the metal hardware, and could be a good solution to limit possible undesired interactions between proteins and column wall/frits. This prototype metal-free SEC column was systematically compared with a conventional stainless-steel SEC column hardware packed with the same stationary phase material. Four different mAb products, namely trastuzumab, palivizumab, bevacizumab and NISTmAb, and one antibody drug conjugate (ADC), trastuzumab emtansine, were selected as test samples. It appears that peak symmetry, separation of low molecular weight species (LMWS), and the recovery of high molecular weight species (HMWS) were significantly improved for the different biopharmaceutical products on the metal-free SEC column. It has also been demonstrated that the largest differences between standard and metal-free SEC columns were observed for the most basic mAbs (high pI), which confirms that electrostatic interactions between the mAb and the metallic parts of the column (frits and inlet tube) could be responsible for the issues observed when performing SEC analysis with volatile mobile phase. Finally, it was feasible to perform SEC-MS analysis for a wide range of biopharmaceutical products using volatile mobile phase. Our results also highlight that an inappropriate column could bias the quantification of size variants when using MS-compatible mobile phases. Therefore, metal-free column, such as the PEEK-lined column, should be preferentially selected for SEC-MS analysis.

Towards a simple on-line coupling of ion exchange chromatography and native mass spectrometry for the detailed characterization of monoclonal antibodies.

This work describes the direct hyphenation of cation exchange chromatography (CEX) with a compact, easy-to-use benchtop Time of Flight mass spectrometer (ToF/MS) for the analytical characterization of monoclonal antibodies (mAbs). For this purpose, a wide range of commercial mAb products (including expired samples and mAb biosimilars) were selected to draw reliable conclusions. From a chromatographic point of view, various buffers and column dimensions were tested. When considering pH response, buffer stability over time and MS compatibility, the best compromise is represented by the following recipe: 50 mM ammonium acetate, titrated to pH 5.0 (mobile phase A) and 160 mM ammonium acetate, titrated to pH 8.5 (mobile phase B). Despite the broader peaks observed with the 2.1 mm i.d. CEX column, this was preferentially selected for CEX-MS operation, since the efficiency loss (caused by extra-column dispersion) was still acceptable while MS compatibility was strongly enhanced (thanks to low flow rate). In terms of MS, it was important to avoid the use of glass-bottled mobile phases, laboratory glassware and glass vials to minimize loss of MS resolution, sensitivity, and mass accuracy due to metal contaminants. With this new CEX-MS setup, straightforward and rapid analysis (in less than 10 min) of charge variants was possible, allowing the separation and identification of several charge variants.

State-of-the-Art Native Mass Spectrometry and Ion Mobility Methods to Monitor Homogeneous Site-Specific Antibody-Drug Conjugates Synthesis.

Antibody-drug conjugates (ADCs) are biotherapeutics consisting of a tumor-targeting monoclonal antibody (mAb) linked covalently to a cytotoxic drug. Early generation ADCs were predominantly obtained through non-selective conjugation methods based on lysine and cysteine residues, resulting in heterogeneous populations with varying drug-to-antibody ratios (DAR). Site-specific conjugation is one of the current challenges in ADC development, allowing for controlled conjugation and production of homogeneous ADCs. We report here the characterization of a site-specific DAR2 ADC generated with the GlyCLICK three-step process, which involves glycan-based enzymatic remodeling and click chemistry, using state-of-the-art native mass spectrometry (nMS) methods. The conjugation process was monitored with size exclusion chromatography coupled to nMS (SEC-nMS), which offered a straightforward identification and quantification of all reaction products, providing a direct snapshot of the ADC homogeneity. Benefits of SEC-nMS were further demonstrated for forced degradation studies, for which fragments generated upon thermal stress were clearly identified, with no deconjugation of the drug linker observed for the T-GlyGLICK-DM1 ADC. Lastly, innovative ion mobility-based collision-induced unfolding (CIU) approaches were used to assess the gas-phase behavior of compounds along the conjugation process, highlighting an increased resistance of the mAb against gas-phase unfolding upon drug conjugation. Altogether, these state-of-the-art nMS methods represent innovative approaches to investigate drug loading and distribution of last generation ADCs, their evolution during the bioconjugation process and their impact on gas-phase stabilities. We envision nMS and CIU methods to improve the conformational characterization of next generation-empowered mAb-derived products such as engineered nanobodies, bispecific ADCs or immunocytokines.

Characterization of Glycosylated Proteins at Subunit Level by HILIC/MS.

Hydrophilic interaction chromatography (HILIC) coupled to mass spectrometry (MS) is considered as the reference analytical technique for glycans profiling, especially for the characterization of glycosylated protein therapeutics such as monoclonal antibodies (mAbs) and mAbs-related products. Although HILIC/MS is mainly known to profile enzymatically released and fluorescently labeled N-glycans, the recent commercialization of new widepore HILIC amide bonded stationary phases packed with sub-2 µm particles has allowed for remarkable separations also at the subunit level. Here, we describe a simple protocol to perform the mAb glycans profiling at subunit level by HILIC/MS.

Alternative mobile phase additives for the characterization of protein biopharmaceuticals in liquid chromatography - Mass spectrometry.

When analyzing large complex protein biopharmaceuticals, ion-pairing agents imparting low pH are widely used as mobile phase additives to improve the chromatographic performance. However, one of the most effective additives in RPLC and HILIC, trifluoroacetic acid (TFA), is known as a strong suppressor of the MS signal and limits its use in hyphenated techniques. In this study, we evaluated a wide range of acidic additives to find alternatives to TFA that provided comparable chromatographic performance and improved MS sensitivity. It was observed that stronger acidic additives were required for intact level analysis compared to subunit level analysis and that the additive nature had a larger impact on the chromatographic performance in HILIC mode compared to RPLC. Therefore, four additives were identified as valuable alternatives to TFA in RPLC mode, namely, difluoroacetic acid (DFA), dichloroacetic acid (DClAA), trichloroacetic acid (TClAA), and methanesulfonic acid (MSA). Only one of these additives provided acceptable performance in HILIC mode, namely, TClAA. After evaluation of the MS performance, TClAA was discarded due to the apparent loss of intensity in both RPLC-MS and HILIC-MS mode. Together, these results demonstrate that for HILIC-MS analysis TFA remains the gold standard additive. However, DFA was found as promising alternative to TFA for RPLC-MS analysis and could play an important role in the development of methods for the characterization of the increasingly complex protein biopharmaceuticals.

Therapeutic Fc-fusion proteins: Current analytical strategies.

Fc-Fusion proteins represent a successful class of biopharmaceutical products, with already 13 drugs approved in the European Union and United States as well as three biosimilar versions of etanercept. Fc-Fusion products combine tailored pharmacological properties of biological ligands, together with multiple functions of the fragment crystallizable domain of immunoglobulins. There is a great diversity in terms of possible biological ligands, including the extracellular domains of natural receptors, functionally active peptides, recombinant enzymes, and genetically engineered binding constructs acting as cytokine traps. Due to their highly diverse structures, the analytical characterization of Fc-Fusion proteins is far more complex than that of monoclonal antibodies and requires the use and development of additional product-specific methods over conventional generic/platform methods. This can be explained, for example, by the presence of numerous sialic acids, leading to high diversity in terms of isoelectric points and complex glycosylation profiles including multiple N- and O-linked glycosylation sites. In this review, we highlight the wide range of analytical strategies used to fully characterize Fc-fusion proteins. We also present case studies on the structural assessment of all commercially available Fc-fusion proteins, based on the features and critical quality attributes of their ligand-binding domains.