Capillary Electrophoresis-sodium Dodecyl Sulfate Research Articles

Characterization workflow for fragments detected in capillary electrophoresis sodium dodecyl sulfate analysis of therapeutic monoclonal antibodies.

Product-related fragments in monoclonal antibodies (mAbs) can have a significant impact on the efficacy and safety of the product. Capillary electrophoresis sodium dodecyl sulfate (CE-SDS) is a commonly used method for fragment quantification, but it has challenges in peak identification due to the inability to enrich components and the incompatibility of SDS with mass spectrometry (MS). This article presents a workflow for identifying peaks in CE-SDS analysis. The workflow involves comparing the migration time of peaks with that of standards and utilizing MS analysis to identify fragments. By employing this innovative systematic workflow, we successfully identified the CE-SDS impurity peaks of seven antibody products. Among them, four products exhibited characteristic fragments associated with disulfide bonds (light chain [LC], heavy-light [HL] chain, heavy-heavy [HH] chain, and HH-LC) and a glycosylation-related fragment non-glycosylated heavy chain. Additionally, one product showed a fragment formed by the connection of HC_C130 and HC_C130 , which is associated with a thioether bond. Furthermore, two other products displayed amino acid backbone breakage, with one product showing clipping at the HC region of A233 -G285 and the other product showing clipping at the HC regions of A97 -S158 and N342 -T366 . This workflow can be applied in early drug research, process development, or during the biologics license application stage to characterize fragments in therapeutic mAbs analyzed by CE-SDS.

Using capillary electrophoresis sodium dodecyl sulfate (CE-SDS) and liquid chromatograph mass spectrometry (LC-MS) to identify glycosylated heavy chain heterogeneity in the anti-VEGFR-2 monoclonal antibody.

The size variant, which can be measured by capillary electrophoresis sodium dodecyl sulfate (CE-SDS), is a critical quality attribute of monoclonal antibodies (mAbs). The CE-SDS size heterogeneity can hardly be identified by tandem mass spectrometry, which is an intractable obstacle of mAb development and quality control across the industry. We analyzed the purity of an anti-vascular endothelial growth factor receptor 2 (VEGFR-2) mAb, an antagonist of the human VEGFR-2, through reduced CE-SDS and observed glycosylated heavy chain heterogeneity. The heterogeneity has potential impact on safety, efficacy, and stability of drugs for clinical use. Therefore, it should be characterized so as to evaluate its potential risk. In order to identify the heterogeneity, we used mass spectrometry to confirm that the molecular size heterogeneity was not due to peptide bond cleavage in the heavy chain. Subsequently, we employed mass-spectrometry-glycosylation profiling and CE-SDS analysis of various glycosidase-treated samples, in addition to the preparation of mAb references with different glycoforms. Ultimately, we demonstrated that the heavy chain heterogeneity was induced by different levels of galactosylation modifications which will potentially impact the efficacy of antibody drugs (i.e., complement-dependent cytotoxicity). In this study, potential risk caused by heavy chain size heterogeneity was evaluated, which addressed the obstacle of mAb development and quality control. Therefore, this study offers a feasible approach for the investigation and identification of heavy chain heterogeneity in reduced CE-SDS, providing a novel strategy for mAb quality control and evaluation.

Application of capillary electrophoresis-sodium dodecyl sulfate in assessing the purity of monoclonal antibody biopharmaceuticals

Monoclonal antibodies (mAbs) are emerging biopharmaceuticals that have drawn great public attention due to their potential to treat many thorny diseases such as cancers, infections, autoimmune disorders and transplant rejection. However, because their production typically relies on biological means and their biological nature is giant and complex proteins, they are prone to be contaminated by impurities introduced during the production process or generated due to the instability of the products themselves. Both process-related and product-related contaminants need to be strictly monitored throughout the entire process from production to consumption. CE-SDS is a canonical method widely used to determine the purity of biopharmaceuticals like mAbs. Compared with early techniques such as SDS-PAGE, it can separate components of different sizes in the analytes in a quick and precise manner. In this review, we demonstrate the principle underlying the function of CE-SDS and summarize the applications of CE-SDS in assessing major types of impurities. We will also discuss the limitations and advantages of CE-SDS compared with other techniques to point out potential directions for future research.

In-Use Stability of SB12 (Eculizumab, Soliris Biosimilar) Diluted in Saline and Dextrose Infusion Solution after an Extended Storage Period.

SB12 is a biosimilar to eculizumab reference product [SolirisTM (Soliris is a trademark of Alexion Pharmaceuticals, Inc.)] that acts as a C5 complement protein inhibitor. The infusion stability of in-use (diluted) SB12 outside the conditions stated in the reference product's label is unknown. The objective of this study was to assess the stability of SB12 after extended storage in conditions not claimed in the originator label. Infusion stability was assessed in SB12 samples (diluted in 0.9% NaCl, 0.45% NaCl, and 5% dextrose, final concentration of 5mg/mL per clinical trial protocol and the reference product's label) kept at 5±3°C for up to 3months, then 30±2°C/65±5% relative humidity (RH) for 72h. The product was stored in different containers [polyolefin (PO) bags, glass bottles and syringes], and the protocol followed International Conference on Harmonisation (ICH) and European Medicines Agency (EMA) requirements for stability evaluation of biological products. Stability was evaluated using complementary assays, including pH, protein concentration (A280), purity (size exclusion-high-performance liquid chromatography, capillary electrophoresis-sodium dodecyl sulfate, and imaged capillary isoelectric focusing), biological activity (C5 binding and inhibition), and safety (subvisible particles). Except for charge variants in SB12 diluted in 5% dextrose, all results met the stability acceptance criteria. There were no major changes in terms of physicochemical stability, biological activity, and subvisible particles. The infusion stability of SB12 after extended storage (5±3°C for up to 3months, then 30±2°C/65±5% RH for72 h) was demonstrated for longer periods and at higher temperatures than what is stated in the EU and US labels of the reference product. The physicochemical properties, biological activity, and subvisible particles of in-use SB12 diluted in 0.9%NaCl and 0.45%NaCl were maintained under the described conditions and for all tested containers. However, instability was observed for the diluted SB12 in 5%dextrose. These results may reduce the workload of clinical staff and minimize drug waste from treatment delays without any loss in product quality and biological activity.

Efficient and reliable CE-SDS separations by the high-speed turbo CE-SDS® cartridge

Capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) is a modern form of SDS-PAGE that uses capillary electrophoresis to separate proteins by size. The technique is based on the sieving effect of net-like structures in the capillary. In this study, we compared the Maurice Turbo CE-SDS Cartridge™ with the established Maurice CE-SDS PLUS Cartridge, both developed for the Maurice CE system. The Turbo cartridge was found to be up to 4–5 times faster than the PLUS cartridge, while maintaining similar separation efficiency. The limit of quantitation (LOQ) for the Turbo cartridge was estimated to be 0.1 mg/mL with a signal-to-noise ratio (S/N) of 18.55. The linearity of the cartridge was verified in the range of 0.1–4 mg/mL, with correlation coefficient (R2) values of at least 0.99. The separation efficiency remained constant over long series of measurements, and the relative migration time of proteins in a mixture remained stable over 1,000 injections. These results demonstrate that the Turbo CE-SDS Cartridge is a very useful tool for fast and efficient analysis of protein (mixtures). The shorter analysis time can save significant resources by reducing the time and consumables required for analysis, while the high LOQ and linearity ensure that the method is sensitive and accurate.

Multi-channel Deep-UV absorbance measurement setup for multi-capillary electrophoresis with two fiber arrays facing each other

We have developed a simple, compact, and robust eight-channel ultraviolet (UV) absorption detection setup featuring two eight-fiber arrays (an irradiation fiber array and a detection fiber array) whose ends face each other across eight capillaries. UV light of a 220-nm wavelength from a single deuterium lamp is stably and evenly split into eight lights by using a thick homogenizing fiber connected to a fiber bundle with eight thin fibers, which make up the irradiation fiber array. Each capillary is directly irradiated with each UV light emitted from the irradiation fiber array and then each of the UV lights transmitted through the capillaries is collected by each fiber of the detection fiber array without using any other optical devices (e.g., a lens) and is detected by each of the eight photodetectors. This setup achieves simultaneous UV absorption measurement for all eight capillaries with the detection limit of 54 μAU, linear dynamic range of four logs or more, and negligible crosstalk of 0.01% or less. We also implemented an eight-capillary electrophoresis system and simultaneously analyzed eight immunoglobulin G samples, separated by capillary electrophoresis sodium dodecyl sulfate.

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.

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.

Stability Evaluation of the Biosimilar Monoclonal Antibody Using Analytical Techniques.

Determination of the drug substance (DS) and drug product (DP) stability is especially important for biosimilar monoclonal antibodies since it can affect the quality, efficacy, and safety of the drugs. The main objective of this study was to determine the stability of the biosimilar candidate (TUR01) using state-of-the-art (current) analytical techniques. Analytical techniques used in this study were isoelectric focusing on capillary electrophoresis, capillary electrophoresis-sodium dodecyl sulfate, size exclusion chromatography-ultra-high performance liquid chromatography, binding affinity, and physicochemical and microbiological tests. DS was kept in polyethylene terephthalate copolyester, glycol modified (PETG) bottles at ≤-65.0°C and 5.0 ± 3.0°C for 18 months, where the pre-filled syringe stability study was conducted at 5.0 ± 3.0°C for 24 months and 25.0 ± 2.0°C/60% ± 5 relative humidity (RH) for 6 months. The accelerated condition for DS was accepted as 5.0 ± 3.0°C, while it was 25.0 ± 2.0°C for the DP. The results indicated that TUR01 DS was stable when it was stored under long-term storage conditions at ≤-65°C and at 5 ± 3°C at least 18 months. Also, TUR01 DP was stable at 5 ± 3°C for 24 months and at 25 ± 2°C with 60.5% RH for 2 months without any significant changes. State-of-the-art analytical techniques proved to be invaluable tools for evaluate the stability of the TUR01 DS and drug product.

Physicochemical and Biological Stability Assessment of SB11 (Ranibizumab Biosimilar) Under Ambient and In-Use Storage for Intravitreal Administration.

SB11 (Byooviz™) is a ranibizumab biosimilar that acts as a vascular endothelial growth factor (VEGF)-A inhibitor. Stability data for unopened SB11 vials at room temperature are limited and no data are available for SB11 withdrawn into syringes (in-use) for intravitreal administration. SB11 stability was assessed in two different settings: unopened vials stored at 30 ± 2°C/65 ± 5% relative humidity (RH) for 2months, and in-use SB11 withdrawn into syringes stored at 5 ± 3°C for 98days and then 25 ± 2°C/60 ± 5% RH for 24h. The product was stored in the absence of light, and the experimental design followed International Conference on Harmonization and European Medicines Agency requirements for stability evaluation of biological products. Analysis included visual appearance (color, clarity, and presence of visible particles), pH, protein concentration (A280) and purity (size-exclusion high-pressure liquid chromatography, capillary electrophoresis-sodium dodecyl sulfate, imaged capillary isoelectric focusing), biological activity (VEGF binding and neutralization), and safety (sub-visible particulates). Except for charge variants in unopened vials at room temperature after 1month by US standards, all results met the stability acceptance criteria (US and EU) for both unopened vials and for in-use SB11. There were no major changes in terms of physicochemical stability, biological activity and sub-visible particulates. SB11 was stable for longer periods and at higher temperatures than what is stated in the labels of the reference product (Lucentis) and SB11. The physicochemical properties, biological activity, and sub-visible particulates of SB11 in both tested settings (unopened vials at room temperature and in-use product withdrawn into syringes) were maintained under the described storage periods. This information can help to avoid unnecessary delays in patient treatment without any loss in quality and biological activity, lower the workload of health care providers and reduce costs associated with drug waste.

Identification of a monoclonal antibody clipping variant by cross-validation using capillary electrophoresis – sodium dodecyl sulfate, capillary zone electrophoresis – mass spectrometry and capillary isoelectric focusing – mass spectrometry

Critical quality attributes (CQAs) of recombinant monoclonal antibody therapeutics are constantly monitored throughout the life cycle of drug development and manufacturing. In the past few decades, numerous analytical techniques have been developed for the characterization of CQAs. In this regard, non-reduced and reduced capillary electrophoresis – sodium dodecyl sulfate (CE-SDS) methods have been widely adopted by the biopharmaceutical industry for the evaluation of size-related heterogeneities in biologics. In this work we demonstrate that, with recent development of capillary electrophoresis – mass spectrometry (CE-MS) technologies, a clipping variant of bevacizumab may be identified directly by both capillary zone electrophoresis – mass spectrometry (CZE-MS) and capillary isoelectric focusing – mass spectrometry (cIEF-MS) approaches, providing a powerful addition to the traditional CE-SDS analysis workflow. In this novel workflow, linear regression between the mobility and molecular weight first results in an approximate size range of this variant. The intact masses of all species in the bevacizumab are then obtained, after deconvolution of all features identified in the CZE-MS analysis. Subsequent CZE-MS analysis of the subunits of bevacizumab leads to the confirmation of a clipped heavy chain. Furthermore, cIEF-MS of the intact bevacizumab confirms the existence of this clipping variant. The cross-validation between CE-SDS, CZE-MS, and cIEF-MS, creates a comprehensive roadmap for monoclonal antibody size variants profiling. These CE-based analytical techniques are complementary to each other, leading to orthogonal verification for size heterogeneity characterization.

National Control Laboratory Assessment of Quality of Rituximab Biosimilars in India.

In past few years many rituximab (RTX) biosimilars have been launched in India. Biosimilars are products that are similar in terms of quality, safety, and efficacy to its innovator product and are expected to offer improved affordability. The less clinical examination is a significant source of reduction in the cost of development of a biosimilar. However, this clinical relief is predicated on the assumption that there is analytical similarity between the biosimilar and the innovator product. Therefore, the role of National Control Laboratory become very important to ensure the quality of these drugs by carrying out analytical characterization at the point of drug product release level as when referred by National Regulatory Authority for quality evaluation. To assess the similarity between innovator and biosimilars, different physicochemical and biological quality attributes were assessed. A multitude of state-of-the-art analysis of N = 3 RTX biosimilars marketed in India revealed that the impurity profiles of these biosimilars measured by charge variant analysis (cation exchange chromatography-high performance liquid chromatography [HPLC], capillary zone electrophoresis, and capillary isoelectric focusing), aggregates profiling (size exclusion chromatography-HPLC), fragments analysis (capillary electrophoresis-sodium dodecyl sulfate) were found to be significantly varying as compared with the innovator product. There were significant variations in acidic variants (p = 0.023) and basic variants (p = 0.0005), isoelectric point value (p < 0.0001), aggregates (p = 0.0231), and fragments (p < 0.0001) of biosimilars were found as that of innovator product. However, these differences were not affecting the biological activity in the cell-based potency analysis by complement-dependent cytotoxicity (CDC) assay (p = 0.1026), antibody-dependent cell-mediated cytotoxicity (ADCC) (p = 0.3736), and binding assay by flow cytometer fluorescence-activated cell sorting (p = 0.4005) of these biosimilars as compared with the innovator product.

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.

Study of the Stability of Sandoz Rituximab Biosimilar Rixathon®/Riximyo® When Subjected for up to 21 Days to Ambient Storage

AimThe purpose of this study was to evaluate the extended physicochemical and biological stability of Sandoz Rixathon®/Riximyo® (SDZ-RTX) after exposure to out-of-fridge (OOF) conditions.Materials and MethodsThe impact of the short-term temperature excursion on stability parameters of SDZ-RTX was simulated by subsequently exposing the three batches of SDZ-RTX (100 and 500 mg) to OOF conditions, (I) 25 ± 2 °C/60 ± 5% relative humidity (RH) and (II) 30 ± 2 °C/65 ± 5% RH, for up to 21 days after more than the claimed 36-month shelf-life storage in long-term conditions (5 ± 3 °C). Analytical methods used included the cation exchange chromatography (CEX), size exclusion chromatography (SEC), and non-reducing capillary electrophoresis-sodium dodecyl sulfate (nrCE-SDS), as well as biological activity by complement-dependent cytotoxicity (CDC)-bioactivity as well as further methods, for example, related to identity and pharmacopoeia test methods.ResultsNo notable changes were observed across all batches with respect to identity (charge and primary structure), pharmaceutical tests (clarity, visible and subvisible particles analytics, container appearance, degree of coloration, pH, osmolality, extractable volume, and container closure integrity testing), protein content by UV and microbiological parameters (sterility and bacterial endotoxins) under both OOF conditions. Only minor changes were observed for parameters evaluated via SEC, CEX, and nrCE-SDS. For potency (CDC-bioactivity) only one of the batches showed a relevant change. Even for these stability-indicating test methods, all analyzed parameters complied with the shelf-life specifications.ConclusionSDZ-RTX is safe for use even under worst-case conditions, for example, after subjecting it for up to 21 days at OOF conditions (25 ± 2 °C/60 ± 5% RH or 30 ± 2 °C/65 ± 5% RH) after the batches had reached an age that was already beyond the claimed shelf-life.Supplementary InformationThe online version contains supplementary material available at 10.1007/s40268-022-00393-4.

Investigation of an Uncommon Artifact during Reducing Capillary Electrophoresis-Sodium Dodecyl Sulfate Analysis of a Monoclonal Antibody with Dynamic Light Scattering and Reversed Phase High-Performance Liquid Chromatography.

In reducing capillary electrophoresis sodium dodecyl sulfate (CE-SDS) analysis of a monoclonal antibody (mAb-1), the peak area ratio of heavy chain (HC) to light chain (LC) was out of balance, while multiple artifact peaks were observed following the migration of HC. The main purposes of this study were to describe the techniques utilized to eliminate this artifact and clarify the root cause for this interesting phenomenon. We optimized the CE-SDS analysis of mAb-1 by a vairety of techniques including changing the concentration of protein or replacing SDS with a more hydrophobic surfactant (i.e., sodium hexadecyl sulfate (SHS) or sodium tetradecyl sulfate (STS) instead of SDS) in sample and/or the sieving gel buffer. Dynamic light scattering (DLS) and reversed phase high-performance liquid chromatography (RP-HPLC) were used to study the protein-surfactant complex. The artifact could be partially mitigated by reducing the protein concentration and replacing SDS with SHS or STS in the sample and/or the sieving gel buffer solutions. Due to replacing a more hydrophobic surfactant, the HC-surfactant complex formed was more resistant to dissociation, preventing additional hydrophobic HC-HC interaction and aggregation, thus eliminating the artifact problem. DLS and RP-HPLC are powerful supplementary techniques in characterizing the protein-surfactant complex, and hydrophobic surfactants such as SHS and STS could afford more normal electropherograms during the analysis of mAbs.

Physicochemical stability of PF-06439535 (bevacizumab-bvzr; Zirabev®), a bevacizumab biosimilar, under extended in-use conditions.

PF-06439535 (bevacizumab-bvzr; Zirabev®) is a bevacizumab biosimilar. The stability profile and functional activity of PF-06439535 after dilution for intravenous infusion was evaluated following extended storage conditions. PF-06439535 drug product was diluted in 0.9% sodium chloride to produce final concentrations of 1.4 and 16.5 mg/mL of PF-06439535, representing clinically relevant low and high doses for intravenous infusion. Three drug product lots and three infusion bag types (polyolefin, ethylene vinyl acetate, and polyvinyl chloride) were tested. To simulate the potential preparation and administration conditions encountered in a clinical setting, prepared drug solutions were initially stored at 25 ± 5°C for 24 ± 2 h, and then at 5 ± 3°C for up to 6 weeks. Extended storage was followed by storage at 25 ± 5°C for 24 ± 2 h before testing. Physicochemical and biological stability were evaluated according to visual characteristics and pH, protein concentration, particulate content, the proportions of molecular weight variants and charge variants, and relative potency. A wide range of analytical techniques optimized for PF-06439535 assessment were employed, such as size-exclusion chromatography, non-reducing sodium dodecyl sulfate capillary electrophoresis, cation-exchange chromatography, far-UV circular dichroism spectroscopy, differential scanning calorimetry, and an in vitro cell-based bioassay. For all concentrations, drug product lots, infusion bag types, and time points tested, there were no significant changes in protein concentration and no notable differences in visual characteristics (color, clarity, and visible particulates). The abundance of molecular weight variants and charge variants remained stable over the 6-week study period. There were no stability concerns with regard to sub-visible particles. There were no significant changes in primary, secondary, or tertiary structure. Finally, the in vitro relative potency of PF-06439535 was maintained throughout the study period. The stability and biological activity of PF-06439535 was maintained after dilution and storage for up to 6 weeks at 2-8°C, demonstrating the integrity of diluted PF-06439535 under extended in-use conditions.

Structural Characterization of RC28-E, a Recombinant Fusion Protein With Dual Targets on VEGF and FGF2

Vascular endothelial growth factor (VEGF) and fibroblast growthfactor (FGF) play important roles in angiogenesis-related diseases. RC28-E is a soluble fusion protein composed of the human VEGF receptor 1 (VEGFR1) extracellular domain 2 (ECD 2), VEGFR2 ECD 3, FGFR1 ECDs 2 and 3, and the Fc regions of human immunoglobulin G1. By targeting both VEGF and FGF2, RC28-E may represent a useful antiangiogenetic agent, but structural and functional characterizations of this fusion protein are needed. Liquid chromatography–tandem mass spectrometry, size exclusion high-performance liquid chromatography, capillary electrophoresis-sodium dodecyl sulfate, imaged capillary isoelectric focusing, and bio-layer interferometry were used to characterize the properties of RC28-E. The purity of RC28-E was confirmed to be 98% or greater. The glycosylation modification of RC28-E was found to be very complicated, with 11 potential N-linked glycosylation points and 23 types of N-glycans, causing high heterogeneity of the protein. The primary modifications of the amino acid sequence of RC28-E protein included C-terminal K truncation, N-deamidation, and M-oxidation modification. Notably, RC28-E demonstrated a higher affinity for both VEGF and FGF2 than VEGF trap or FGF trap for their respective targets.

Characterization of Grinding-Induced Subvisible Particles and Free Radicals in a Freeze-Dried Monoclonal Antibody Formulation.

The primary objectives of this study were to investigate the degradation mechanisms of freeze-dried monoclonal antibody (mAb) formulations under mechanical grinding, assess the sensitivity and suitability of various particle analysis techniques, analyze the structure of the collected subvisible particles (SbVPs), and analyze the antioxidant mechanism of methionine (Met) under degradation process to gain a thorough understanding of the phenomenon. The freeze-dried mAb-X formulations underwent grinding, and the resultant SbVPs were characterized through visual inspection, flow imaging microscopy, dynamic light scattering, ultraviolet-visible spectroscopy, and size-exclusion high-performance liquid chromatography. We further evaluated the effect of different temperatures and the free radical scavenger Met on SbVP formation. The produced free radicals were detected using electron paramagnetic resonance, and Met S-oxide formation was detected using liquid chromatography-mass spectrometry. In addition, we analyzed the obtained SbVPs using capillary electrophoresis sodium dodecyl sulfate and Fourier transform infrared spectroscopy. Grinding leads to SbVP formation under high temperature and free radical formation. Free radicals produced during grinding require the participation of a macromolecule. Met could then bind to the produced free radicals, thus partially protecting mAb-X from degradation while itself undergoing oxidation to form Met(O). Sensitivity differences between different particle analysis techniques were evaluated, and the obtained SbVPs showed significant changes in secondary structure and the formation of covalent aggregates and fragments. Met plays the role of an antioxidant in protecting macromolecules by quenching the free radicals produced during grinding. To thoroughly characterize SbVPs, multiple and orthogonal particle analysis techniques should be used, and if necessary, SbVPs should be processed by enrichment to accurately analyze primary and high order structures.

Physicochemical stability of PF-05280014 (trastuzumab-qyyp; TrazimeraTM), a trastuzumab biosimilar, under extended in-use conditions.

The stability and functional activity of the trastuzumab biosimilar PF-05280014 (trastuzumab-qyyp; TrazimeraTM), was assessed under extended in-use conditions. PF-05280014 was diluted in 0.9% sodium chloride to final concentrations of 0.2 mg/mL and 4 mg/mL in 3 different types of infusion bags (polyolefin, ethylene vinyl acetate, and polyvinyl chloride). Infusion bags containing diluted PF-05280014 were stored at 25 ± 5° C for 24 h, before storage at 5 ± 3° C for 0, 1, 2, 4, or 6 weeks. Following extended storage, samples of PF-05280014 were removed from the infusion bags and stored at 25 ± 5° C for 24 h before biophysical and functional characterization. In addition to the visual characteristics of each sample at the various time points, the stability of PF-05280014 was assessed using a variety of biophysical techniques, including size-exclusion high-performance liquid chromatography, non-reducing sodium dodecyl sulfate capillary electrophoresis, cation-exchange chromatography, peptide mapping, far-UV circular dichroism spectroscopy, and differential scanning calorimetry. The functional activity of PF-05280014 was evaluated using a cell-based growth inhibition assay. For all PF-05280014 concentrations, time points and infusion bags tested, there were no significant differences in visual characteristics or in protein concentration. The were no significant changes in the relative abundance of molecular weight or charge variants throughout the 6-week study period. Similarly, there were no significant changes in primary structure or in secondary structure content during the study. The relative potency of PF-05280014 was also maintained throughout the 6-week period. The stability and functional activity of PF-05280014 was maintained following dilution in 0.9% sodium chloride and storage for up to 6 weeks at 2-8° C.

A high-resolution multi-attribute method for product characterization, process characterization, and quality control of therapeutic proteins

During the manufacturing of therapeutic proteins, Critical Quality Attributes (CQAs) have been monitored by conventional methods, such as cation exchange chromatography (CEX), reduced capillary electrophoresis-sodium dodecyl sulfate (rCE-SDS), and 1,2-diamino-4,5-methylenedioxybenzene (DMB) labelling method. The conventional methods often generate individual peaks that contain multiple components, which may obscure the detection and the quantification of individual critical quality attributes (CQAs). Alternatively, Multi-Attribute Method (MAM) enables detection and quantification of specific CQAs. A high resolution MAM has been developed and qualified to replace several conventional methods in monitoring product quality attributes, such as oxidation, deamidation, clipping, and glycosylation. The qualified MAM was implemented in process characterization, as well as release and stability assays in quality control (QC). In combination with a design-of-experiments (DoE), the MAM method identified multivariate process parameter ranges that yield acceptable CQA level, which provides operational flexibility for manufacturing.