Detection and quantification of single chain rFVII impurity in final drug products by SE-UPLC and CE-SDS as an alternative to SDS-PAGE.

Recombinant factor VII, produced in recombinant BHK cell line, is secreted as a single chain zymogen form (rFVII, non-activated) in cell culture supernatant and subsequently converts to its active form during anion exchange chromatography step in the downstream purification process, with the aid of calcium ion. Single chain rFVII impurity (non-activated form) in final drug products should not exceed more than 3.0 % of total rFVIIa content. Therefore, one of the most essential quality control tests in pharmaceutical companies is to precisely quantify and report this impurity. SDS-PAGE, as a traditional method in quality control laboratories to quantify single chain rFVII, is a laborious, time-consuming, low output, and semi-quantitative method for quantification of non-activated form impurity which utilizes a densitometer to scan the gel and calculate the non-activated form band density. In this work, we developed two novel instrumental-based techniques (SE-UPLC and CE-SDS) with superior precision, accuracy, sensitivity, and efficiency that overcome SDS-PAGE shortcomings. The results of both methods were comparable to SDS-PAGE and showed an even higher correlation with expected values. Finally, we concluded that these two methods could be used as a high throughput routine method in quality control laboratories as an alternative choice to manual SDS-PAGE.

Characterization of mAb size heterogeneity originating from a cysteine to tyrosine substitution using denaturing and native LC-MS.

Upon assessing the comparability between a biosimilar mAb and its reference product by non-reducing CE-SDS, increased levels of a heavy-heavy-light chain (HHL) variant, present as a low molecular weight (LMW) peak, were observed. RPLC-MS applied at top, middle-up and bottom-up level revealed the existence of Cys-to-Tyr substitutions, predominantly at position HC226 involved in connecting LC and HC, explaining the abundant HHL levels. Antigen binding was not impacted by the presence of this size variant suggesting a non-covalent association of Tyr substituted HHL and LC. The latter complex is not maintained in the denaturing conditions associated with CE-SDS and RPLC-MS. Its existence could, nevertheless, be confirmed by native SEC-MS which preserves non-covalent protein interactions during separation and electrospray ionization. Amino acid analysis furthermore demonstrated a depletion of Cys during the fed-batch process indicating that the observed size/sequence variant is not of genetic but rather of metabolic origin. Native SEC-MS showed that supplementing the cell culture medium with Cys halts misincorporation of Tyr and promotes the formation of the desired mAb structure. To the best of our knowledge, Cys-to-Tyr substitutions preventing interchain disulfide bridge formation have not been described earlier. This observation adds to the impressive structural heterogeneity reported to date for mAbs.

Using enhanced development tools offered by analytical Quality by Design to support switching of a quality control method.

Quality by Design (QbD) principles play an increasingly important role in the pharmaceutical industry. Here, we used an analytical QbD (AQbD) approach to develop a capillary electrophoresis sodium dodecyl sulfate under reducing conditions (rCE-SDS), with the aim of replacing SDS-polyacrylamide gel electrophoresis (SDS-PAGE) as release and stability test method for a commercialized monoclonal antibody product. Method development started with defining analytical method performance requirements as part of an analytical target profile, followed by a systematic risk assessment of method input parameters and their relation to defined method outputs. Based on this, design of experiments studies were performed to identify a method operable design region (MODR). The MODR could be leveraged to improve method robustness. In a bridging study, it was demonstrated that the rCE-SDS method is more sensitive than the legacy SDS-PAGE method, and a conversion factor could be established to compensate for an off-set due to the higher sensitivity, without losing the correlation to the historical data acquired with the former method. Overall, systematic application of analytical Quality by Design principles for designing and developing a new analytical method helped to elucidate the complex dependency of method outputs on its input parameters. The link of the method to product quality attributes and the definition of method performance requirements were found to be most relevant for derisking the analytical method switch, regarding impact on the control strategy.

Chromatography assisted in-vitro refolding and purification of recombinant peptibody: Recombinant Romiplostim a case study.

In-vitro protein refolding is one of the key rate-limiting unit operations in manufacturing of fusion proteins such as peptibodies expressed using E. coli. Dilution-assisted refolding is the most commonly used industrial practice to achieve the soluble, native functional form of the recombinant protein from the inclusion bodies. This study is focused on developing a chromatography-assisted in-vitro refolding platform to produce the biologically active, native form of recombinant peptibody. Recombinant Romiplostim was selected as a model protein for the study. A plug flow tubular reactor was connected in series with capture step affinity chromatography to achieve simultaneous in-vitro refolding and capture step purification of recombinant Romiplostim. Effect of various critical process parameters like fold dilution, temperature, residence time, and Cysteine: DTT ratio was studied using a central composite based design of experiment strategy to achieve a maximum refolding yield of selected peptibody. Under optimum refolding conditions, the maximum refolding yield of 57.0 ± 1.5 % and a purity of over 79.73 ± 3.4 % were achieved at 25-fold dilution, 15 °C temperature, 6 h residence time with 6 mM and 10 mM of cysteine and DTT, respectively. The formation of native peptibody structure was examined using various orthogonal analytical tools to study the protein's primary, secondary, and tertiary structure. The amino acid sequence for the disulfide-linked peptide was mapped using collision-induced dissociation (CID) to confirm the formation of interchain disulfide bonds between Cys7-Cys7 and Cys10-Cys10 similarly for intra-chain disulfide bonds between Cys42-Cys102, and Cys148-Cys206. The developed protocol here is a valuable tool to identify high-yield scalable refolding conditions for multi-domain proteins involving inter-domain disulfide bonds.

End-to-end approach for the characterization and control of product-related impurities in T cell bispecific antibody preparations.

Antibody-based T cell-activating biologics are promising therapeutic medicines being developed for a number of indications, mainly in the oncology field. Among those, T cell bispecific antibodies are designed to bind one tumor-specific antigen and the T cell receptor at the same time, leading to a robust T cell response against the tumor. Although their unique format and the versatility of the CrossMab technology allows for the generation of safer molecules in an efficient manner, product-related variants cannot be completely avoided. Therefore, it is of extreme importance that both a manufacturing process that limits or depletes product-related impurities, as well as a thorough analytical characterization are in place, starting from the development of the manufacturing cell line until the assessment of potential toxicities. Here, we describe such an end-to-end approach to minimize, quantify and control impurities and -upon their functional characterization- derive specifications that allow for the release of clinical material.

Characterization of light chain c-terminal extension sequence variant in one bispecific antibody.

Protein modifications such as post-translational modifications (PTMs) and sequence variants (SVs) occur frequently during protein biosynthesis and have received great attention by biopharma industry and regulatory agencies. In this study, an aberrant peak near light chain (LC) was observed in the non-reduced capillary electrophoresis sodium dodecyl sulfate (nrCE-SDS) electrophoretogram during cell line development of one bispecific antibody (BsAb) product, and the detected mass was about 944 Da higher than LC. The corresponding peak was then enriched by denaturing size-exclusion chromatography (SEC-HPLC) and further characterized by nrCE-SDS and peptide mapping analyses. De novo mass spectra/mass spectra (MS/MS) analysis revealed that the aberrant peak was LC related sequence variant, with the truncated C-terminal sequence "SFNR" ("GEC"deleted) linked with downstream SV40 promotor sequence "EAEAASASELFQ". The unusual sequence was further confirmed by comparing with the direct synthetic peptide "SFNREAEAASASELFQ". It was demonstrated by mRNA sequencing of the cell pool that the sequence variant was caused by aberrant splicing at the transcription step. The prepared product containing this extension variant maintained well-folded structure and good functional properties though the LC/Heavy chain (HC) inter-chain disulfide was not formed. Several control strategies to mitigate the risk of this LC related sequence variant were also proposed.

Recent advances in capillary gel electrophoresis for the analysis of proteins.

The purpose of this review is to highlight noteworthy advancements in the field of capillary gel electrophoresis for the separation and analysis of proteins from the period of 2015-2021. This review will provide an overview of the historical perspective and principles of the technique, introduce the challenges and limitations commonly faced, and highlight the advancements made to overcome these issues and broaden our knowledge of the method. Finally, applications of capillary gel electrophoresis and future directions for the technique will be presented.

Capillary electrophoresis Western blot using inkjet transfer to membrane.

Traditional Western blots are commonly used to separate and assay proteins; however, they have limitations including a long, cumbersome process and large sample requirements. Here, we describe a system for Western blotting where capillary gel electrophoresis is used to separate sodium dodecyl sulfate-protein complexes. The capillary outlet is threaded into a piezoelectric inkjetting head that deposits the separated proteins in a quasi-continuous stream of <100 pL droplets onto a moving membrane. Through separations at 400 V/cm and protein capture on a membrane moving at 2 mm/min, we are able to detect actin with a limit of detection at 8 pM, or an estimated 5 fg injected. Separation and membrane capture of sample containing 10 proteins ranging in molecular weights from 11 - 250 kDa was achieved in 15 min. The system was demonstrated with Western blots for actin, ß-tubulin, ERK1/2, and STAT3 in human A431 epidermoid carcinoma cell lysate.

Identification of a CE-SDS shoulder peak as disulfide-linked fragments from common CH2 cleavages in IgGs and IgG-like bispecific antibodies.

Fragmentation is a well-characterized degradation pathway of therapeutic antibodies and is usually monitored by capillary electrophoresis-sodium dodecyl sulfate (CE-SDS). Although fragments due to cleavage in CH2 domains linked by intrachain disulfide bonds are common and can be detected by reduced reversed-phase - liquid chromatography mass spectrometry (RP-LCMS) and reduced CE-SDS methods, their separation in nonreduced CE-SDS (nrCE-SDS) has not been reported but speculated as comigrating with intact IgG. A shoulder peak in nrCE-SDS was observed in the stability samples of an IgG-like bispecific antibody and was determined to be mainly caused by fragments from clipping at the C-terminus of leucine (L)306 or L309 (EU numbering) in the CH2 domain of both heavy chains (HCs) and, to a lesser degree, at the C-terminus of L182 in the CH1 domain of the knob HC. Subunit LCMS analysis verified that the crystallizable fragment contained variants with one or multiple mass additions of ~18 Da due to clipping. Further investigation revealed that CH2 clippings at L306 and L309 were largely due to proteolytic activity, and cleavages were present at various levels in all in-house IgG1 and IgG4 molecules studied. Our study shows that CH2 domain cleavages, with complementary fragments still linked by intrachain disulfide, can be electrophoretically resolved as a front shoulder of the main peak in nrCE-SDS. Given the high occurrence of CH2 cleavages in antibodies, these findings will have broad applicability and could help manufacturers of therapeutic antibodies in process improvement, product characterization, investigations, formulation stability, and stability comparability studies.

Interlaboratory method validation of capillary electrophoresis sodium dodecyl sulfate (CE-SDS) methodology for analysis of mAbs.

Capillary electrophoresis sodium dodecyl sulfate (CE-SDS) is an analytical method to assess the purity of proteins, commonly applied to monoclonal antibodies (mAbs) in the biopharmaceutical industry. To address the need to standardize the CE-SDS method in the pharmaceutical industry and to enhance the confidence in method transfer between laboratories operating different commercial capillary electrophoresis (CE) instrument platforms, an interlaboratory CE-SDS method validation was organized involving 13 laboratories in 13 companies on four different types of commercial capillary electrophoresis instruments. In the validation, a commercial mAb therapeutic was used as the sample. The validation process followed the analytical guidelines set by the ICH guidelines (International Conference for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use). The method's precision, accuracy, linearity and range, and limit of quantitation (LOQ) were validated in the study. Variations of all the parameters validated in the study passed the pre-set criteria defined at the beginning of the study. The definition was based on previously published works and the intended application purpose of the CE-SDS method for mAbs. The study proved that the CE-SDS method fits its intended application purpose as a size impurity assay and size heterogeneity characterization assay for mAb therapeutic products. This study is the first time a CE-SDS method is validated by multiple laboratories using different commercial CE instrument platforms and on a commercial mAb therapeutic. Its results will enhance the confidence of the biopharmaceutical industry to develop CE-SDS methods and transfer CE-SDS methods between different laboratories.

A comparative study of CE-SDS, SDS-PAGE, and Simple Western-Precision, repeatability, and apparent molecular mass shifts by glycosylation.

SDS gel electrophoresis is a commonly used approach for monitoring purity and apparent molecular mass (Mr) of proteins, especially in the field of quality control of biopharmaceutical proteins. The technological installation of CE-SDS as the replacement of the slab gel technique (SDS-PAGE) is still in progress, leading to a continuous improvement of CE-SDS instruments. Various CE-SDS instruments, namely Maurice (CE-SDS/CE-SDS PLUS) and Wes by ProteinSimple as well as the microchip gel electrophoresis system LabChip® GXII Touch™ HT by PerkinElmer were tested for precision and repeatability compared to SDS-PAGE (Bio-Rad). For assessing these quality control parameters, standard model proteins with minor post-translational modifications were used. Overall, it can be concluded that the CE-SDS-based methods are similar to SDS-PAGE with respect to these parameters. Quality characteristics of test systems gain more significance by testing proteins that do not behave like model proteins. Therefore, glycosylated proteins were analyzed to comparatively investigate the influence of glycosylation on Mr determination in the different instruments. In some cases, high deviations were found both among the methods and with regard to reference values. This article provides possible explanations for these findings.

Online top-down mass spectrometric identification of CE(SDS)-separated antibody fragments by two-dimensional capillary electrophoresis.

Size heterogeneity analysis by capillary sieving electrophoresis utilizing sodium dodecyl sulfate (CE(SDS)) with optical detection is a major method applied for release and stability testing of monoclonal antibodies (mAbs) in biopharmaceutical applications. Identification of mAb-fragments and impurities observed with CE(SDS) is of outstanding importance for the assessment of critical quality attributes and development of the analytical control system. Mass spectrometric (MS) detection is a powerful tool for protein identification and characterization. Unfortunately, CE(SDS) is incompatible with online MS-hyphenation due to strong ionization suppression of SDS and other separation buffer components. Here, we present a comprehensive platform for full characterization of individual CE(SDS)-separated peaks by CE(SDS)-capillary zone electrophoresis-top-down-MS. The peak of interest is transferred from the first to the second dimension via an 8-port valve to remove MS-incompatible components. Full characterization of mAb byproducts is performed by intact mass determination and fragmentation by electron transfer dissociation, higher-energy collisional dissociation, and ultraviolet photodissociation. This enables online determination of intact mass as well as sequence verification of individual CE(SDS)-separated peaks simultaneously. A more substantiated characterization of unknown CE(SDS) peaks by exact localization of modifications without prior digestion is facilitated. High sensitivity is demonstrated by successful mass and sequence verification of low abundant, unknown CE(SDS) peaks from two stressed mAb samples. Good fragmentation coverages are obtained by MS2, enabling unequivocal identification of these mAb-fragments. Also, the differentiation of reduced/non-reduced intra-protein disulfide bonds is demonstrated. In summary, a reliable and unambiguous online MS2 identification of unknown compounds of low-abundant individual CE(SDS) peaks is enabled.

A comparative study of CE-SDS, SDS-PAGE, and Simple Western: Influences of sample preparation on molecular weight determination of proteins.

The development of capillary electrophoresis, especially CE-SDS devices, has led CE-SDS to become an established tool in a wide range of applications in the analysis of biopharmaceuticals and is increasingly replacing its method of origin, SDS-PAGE. The goal of this study was to evaluate the comparability of molecular weight (MW) determination especially by CE-SDS and SDS-PAGE. For ensuring comparability, model proteins that have little or no posttranslational modifications and an IgG antibody were used. Only a minor influence of sample preparation conditions, including sample buffer, temperature conditions, and different reducing agents on the MW determination were found. In contrast, the selection of the MW marker plays a decisive role in determining the accurate apparent MW of a protein. When using different MW markers, the deviation in MW determination can exceed 10%. Interestingly, CE-SDS and 10% SDS-PAGE hardly differ in their trueness of MW determination. The trueness in relation to the reference MW for each protein was calculated. Although the trueness values for the model proteins considered range between 1.00 and 1.11 using CE-SDS, they range between 0.93 and 1.03 on SDS-PAGE, depending on the experimental conditions chosen.

Capillary Electrophoresis-Sodium Dodecyl Sulfate with Laser-Induced Fluorescence Detection as a Highly Sensitive and Quality Control-Friendly Method for Monitoring Adeno-Associated Virus Capsid Protein Purity.

The capsid protein purity of adeno-associated virus (AAV) is considered a critical quality attribute of AAV-based gene therapy products. However, the analytical methods currently available to monitor the viral capsid proteins, which are present in extremely low concentrations, have limited sensitivity and robustness, thus limiting their general applicability. As a result, there is an urgent need to develop robust separation methods with highly sensitive detection. In this article, we describe the first denaturation and fluorescence labeling procedure for AAV capsid proteins using the pyrylium dye Chromeo™ P503, enabling the establishment of the first capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) method combined with laser-induced fluorescence (LIF) detection for AAV. Upon optimization using a quality-by-design approach, the newly developed method features a simple and robust one-step sample preparation workflow resulting in consistently labeled and denatured viral protein samples, which can subsequently be separated and quantified by CE-LIF. The method has been validated to be accurate and precise with a linear range of 50-150% of the nominal concentration of 2.0 × 1011 vector genomes per mL (vg/mL). The detection limit and quantitation limit were established to be 8.0 × 107 vg/mL (∼0.8 ng/mL) and 4.2 × 108 vg/mL (∼4 ng/mL), respectively, representing the highest sensitivity achieved for AAV capsid protein quantitation reported to date and a linear dynamic range of 8.0 × 107-3.0 × 1011 vg/mL. A comparison of the CE-SDS LIF method with existing methods, such as CE-SDS ultraviolet and sodium dodecyl sulfate-polyacrylamide gel electrophoresis with SYPRO Ruby stain, indicated that the new method has superior resolution and a significant increase in signal intensity. Capsid protein purity analysis of multiple AAV serotypes, including AAV5, scAAVrh10, AAV2, and AAV6, has been demonstrated for the first time using the same method, indicating the newly developed AAV labeling procedure and CE-LIF analysis could serve as a Quality Control-friendly platform and best-in-class analytical method for the control of AAV capsid protein purity.

Four-step approach to efficiently develop capillary gel electrophoresis methods for viral vaccine protein analysis.

Vaccines against infectious diseases are urgently needed. Therefore, modern analytical method development should be as efficient as possible to speed up vaccine development. The objectives of the study were to identify critical method parameters (CMPs) and to establish a set of steps to efficiently develop and validate a CE-SDS method for vaccine protein analysis based on a commercially available gel buffer. The CMPs were obtained from reviewing the literature and testing the effects of gel buffer dilution. A four-step approach, including two multivariate DoE (design of experiments) steps, was proposed, based on CMPs and was verified by CE-SDS method development for: (i) the determination of influenza group 1 mini-hemagglutinin glycoprotein; and (ii) the determination of polio virus particle proteins from an inactivated polio vaccine (IPV). The CMPs for sample preparation were incubation temperature(s) and time(s), pH, and reagent(s) concentration(s), and the detection wavelength. The effects of gel buffer dilution revealed the CMPs for CE-SDS separation to be the effective length, the gel buffer concentration, and the capillary temperature. The four-step approach based on the CMPs was efficient for the development of the two CE methods. A four-step approach to efficiently develop capillary gel electrophoresis methods for viral vaccine protein analysis was successfully established.

Improved CE(SDS)-CZE-MS method utilizing an 8-port nanoliter valve.

Capillary sieving electrophoresis utilizing SDS (CE(SDS)) is one of the most applied methods for the analysis of antibody (mAb) size heterogeneity in the biopharmaceutical industry. Inadequate peak identification of observed protein fragments is still a major issue. In a recent publication, we introduced an electrophoretic 2D system, enabling online mass spectrometric detection of generic CE(SDS) separated peaks and identification of several mAb fragments. However, an improvement regarding system stability and handling of the approach was desired. Here, we introduce a novel 8-port valve in conjunction with an optimized decomplexation strategy. The valve contains four sample loops with increased distances between the separation dimensions. Thus, successively coinjection of solvent and cationic surfactant without any additional detector in the second dimension is enabled, simplifying the decomplexation strategy. Removal efficiency was optimized by testing different volumes of solvents as presample and cationic surfactant as postsample zone. 2D measurements of the light and heavy chain of the reduced NIST mAb with the 8-port valve and the optimized decomplexation strategy demonstrates the increased robustness of the system. The presented novel set-up is a step toward routine application of CE(SDS)-CZE-MS for impurity characterization of proteins in the biopharmaceutical field.

Formation of an Unprecedented Impurity during CE-SDS Analysis of a Recombinant Protein.

PURPOSES: The main purposes of this article are to describe an unprecedented phenomenon in which significant amount of a shoulder peak impurity was observed during normal non-reducing capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) analysis of a recombinant fusion protein X, and to evaluate the root cause for this phenomenon. METHODS: A series of experiments were conducted to study the nature of this degradation. Effects of iodoacetamide (IAM), heating temperature, duration, and SDS on the formation of this specific impurity were evaluated using a variety of characterization techniques. RESULTS: The formation of the impurity as observed in CE-SDS was actually due to alkylation of lysine and serine residues with IAM, as confirmed by peptide mapping and LC-MS/MS, which increased the molecular weight and therefore decreased the electrophoretic mobility. The amount of impurity was also strongly dependent on sample preparation conditions including the presence or absence of SDS. CONCLUSIONS: Our study clearly suggested that even though IAM has been used extensively as an alkylation reagent in the traditional non-reducing CE-SDS analysis of monoclonal antibodies and other proteins, alkylation with IAM could potentially lead to additional impurity peak, and therefore complicating analysis. Therefore, before performing CE-SDS and other analyses, the effects of sample preparation procedures on analytical results must be evaluated. For protein X, IAM should be excluded for CE-SDS analysis.

Characterization and elimination of artificial non-covalent light Chain dimers in reduced CE-SDS analysis of pertuzumab.

Capillary electrophoresis sodium dodecyl sulfate (CE-SDS), either in reduced (rCE-SDS) or non-reduced (nrCE-SDS) form, is widely used for purity evaluation and impurity analysis of monoclonal antibody (mAb) drugs. The accuracy of the method may be interfered by artificial species resulted from sample preparation or electrophoresis operation if it is not well optimized. In a routine analysis of pertuzumab for both innovator Perjeta® and biosimilar HLX11 samples, a cluster of unknown peaks located between light chain (LC) and heavy chain (HC) were observed in rCE-SDS and making the purity of (LC + HC)% unacceptable. They can hardly be reduced by regular method optimization such as changing buffer pH, denaturing temperature or incubation time to achieve the (LC + HC)% expectation. Here, the peaks are first characterized and determined to be non-covalently formed LC-LC dimers by multiple techniques including reversed-phase liquid chromatography-mass spectrometry (LC-MS). These artifacts are then eliminated through enhancing capillary separation temperature to 60 °C and decreasing the separation voltage to 9.5 kV, an unusual CE-SDS operation setting. Finally, a developed rCE-SDS method is presented for successful evaluation of pertuzumab purity and impurities, which is further confirmed by an alternative reduced microchip-based gel electrophoresis. In summary, the developed method provided an accurate and reliable purity evaluation and size variant profiling for batch releasing, stability testing and quality study of reduced pertuzumab samples.

Using differential scanning calorimetry for the development of non-reduced capillary electrophoresis sodium dodecyl sulfate methods for monoclonal antibodies.

Analysis of non-reduced and reduced monoclonal antibodies (mAbs) by capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) is routinely used to detect product size variants and process-related impurities. Levels of high molecular weight (HMW) forms obtained from this method usually trend comparably to those obtained by orthogonal methods such as size-exclusion ultra-high performance liquid chromatography (SE-UHPLC). However, in the presented case study, comparison of CE-SDS data for three IgG1 mAbs (trastuzumab, mAb1, and mAb2) showed a discrepancy between amounts of observed HMW forms in mAb2 compared with its native forms determined by SE-UHPLC (~17% vs. ~0.5%, respectively). SDS chemical denaturation, as measured by differential scanning calorimetry, demonstrated that the high thermal stability of mAb2 caused an unidentified HMW peak observed by non-reduced (NR)-CE-SDS, which was the result of improper denaturing, resulting in a partially folded species. More so, this strategy enabled the rapid identification of optimal SDS concentration and temperature conditions needed for suitable denaturation for mAb2. This case study presents an alternative option for quick optimization of NR-CE-SDS methods when characterizing mAbs or other thermally stable proteins. Also, this strategy can be used to understand basic biophysical mechanisms of protein unfolding and investigate the higher-order structure imparted by specific sequences and understand how these sequences might affect the results of an analytical method such as CE-SDS.

Determination of size variants by CE-SDS for approved therapeutic antibodies: Key implications of subclasses and light chain specificities.

In the present work, a generic non-reducing capillary electrophoresis sodium dodecyl sulphate (nrCE-SDS) method was tested for a wide range of 26 FDA and EMA approved monoclonal antibodies (mAbs) and 2 antibody drug conjugates (ADCs) as well as for the NISTmab, in a QC environment (e.g. testing quality requirements for batch manufacturing or batch release). This method allows obtaining rapidly and accurately the amount of size variants in drug products within about 40 min and may be used for batch release and consistency as well as for stability and shelf-life. First, the method repeatability was found to be excellent in terms of relative migration times and relative proportions of fragments (average RSD values of 0.3 and 0.2 %, on relative migration times and relative percentages of fragments, respectively), thanks to the addition of an internal standard. A panel of chimeric, humanized and human mAbs were tested, belonging to different subclasses (heavy chain gamma 1, 2, 2/4 and 4) and light chain types (κ or λ) and produced in different cell lines (CHO, NS0 and SP2/0). For all these biopharmaceutical products, the amount of H2L2 species was comprised between 90.9 % and 97.7 %, except for the two mAbs belonging to the IgG1λ subclass, namely avelumab and belimumab, which were prone to partial reduction during the sample preparation at 70 °C. Based on the CE-SDS results obtained for a diverse panel of therapeutic antibodies investigated in this study, and covering a wide range of structural and physico-chemical properties, a specification on the intact antibody content (H2L2) greater than 90 % can be achieved.