Capillary electrophoresis-sodium dodecyl sulfate (CE-SDS), a high-resolution and high-sensitivity analytical technique, is an essential tool for analyzing the critical quality attributes (CQAs) of monoclonal antibodies (mAbs) and their derivatives, including antibody-drug conjugates (ADCs) and bispecific antibodies (bsAbs). This study systematically reviews the applications of CE-SDS in analyzing the purity and fragments of mAbs, characterizing positional isomers of ADCs, and identifying mismatch impurities in bsAbs. Focusing on the core technical challenge that CE-SDS cannot be directly coupled with mass spectrometry (MS) for fragment structure identification, the study summarizes technical solutions based on indirect identification approaches and offline/online coupling strategies with Capillary Zone Electrophoresis-Mass Spectrometry (CZE-MS). In addition, from a regulatory science perspective, this study details the key considerations for method validation, establishment of quality standards, and preparation of regulatory submissions for CE-SDS. This study aims to provide a systematic reference for the development and quality control of related biopharmaceuticals, highlighting future development directions, including high-throughput analysis, coupling techniques, and degradation prediction.

The present review aims to provide comprehensive bench-to-bedside insights into ADC-related ocular toxicity for drug designers, pharmaceutical manufacturers, toxicologists, and medical staff, thereby enhancing the safety of ADC therapeutic applications. The review comprehensively analyzes the recent progress in the pathological mechanisms of ADC-related ocular toxicity, evaluates existing non-clinical risk assessment strategies based on animal toxicological studies, and highlights future optimization directions. It also summarizes clinical adverse events to demonstrate the typical profile of ocular surface toxicity and provides clinical management strategies. ADC ocular toxicity primarily affects the ocular surface via on-target (antibody-mediated) and off-target (non-specific uptake) mechanisms. Key determinants include payload type (e.g., MMAF and DM4, which exhibit higher toxicity due to intracellular retention), linker properties (cleavable linkers mitigate off-target effects), and ADCs' physicochemical characteristics. Non-clinical models effectively predict corneal injury but poorly recapitulate conjunctival responses. Clinical management relies on early ophthalmic monitoring and dose adjustment, with 42.9

In antibody–drug conjugate research, the small molecule impurities requiring evaluation include not only free payloads, but also potential payload-related small molecule impurities. In this study, high-resolution mass spectrometry and triple quadrupole mass spectrometry were employed for systematically and comprehensively investigate small molecule related impurities in antibody–drug conjugate product. First, the quantitation of free payload was analyzed. High-resolution mass spectrometry enabled quantification at both precursor ion and product ion levels, the free payload exhibited good linearity over the concentration range of 0.05–100 ng/mL (R² ≥ 0.991), with a lower limit of quantification of 0.05 ng/mL. The accuracy ranged from 80% to 120%, and the precision was below 5%. The high-resolution mass spectrometry results were confirmed using triple quadrupole mass spectrometry. Second, other potential payload related small molecule impurities in the antibody–drug conjugate was investigated in depth. Multiple possible hydrolysis pathways of the payload were predicted based on molecular structure analysis, and the corresponding payload related impurities were subsequently identified using triple quadrupole mass spectrometry in accordance with the predicted precursor–product ion information. Third, stability studies of the antibody–drug conjugate were conducted under forced degradation conditions. Qualitative analyses were performed to characterize unknown payload related degradation products generated under stress conditions, including light exposure, heat, acidic, and alkaline environments. The major degradation products and their relative abundances were identified, with alkaline conditions exerting the greatest impact on sample stability. Clinical trial number: not applicable.

Loncastuximab tesirine is a cysteine-linked antibody–drug conjugate composed of an anti-CD19 monoclonal antibody linked to the PBD dimer payload SG3249 via a protease-cleavable linker, enabling targeted release of a highly cytotoxic DNA cross-linking agent. NAC-SG3249, a thiol adduct formed with N-acetylcysteine, serves as an important indicator of payload release and ADC stability. In this study, mass spectrometry techniques were employed to systematically investigate the stability and related small-molecule impurities of this ADC. Quantitative analysis of the free payload showed good linearity over 0.05–100 ng/mL (R² ≥ 0.991), with an LLOQ of 0.05 ng/mL, accuracy of 80

Antibody–drug conjugates (ADCs) are a pivotal technology for precision cancer therapy, harnessing the synergistic effects of antibody targeting and toxin delivery. However, traditional ADCs encounter limitations in efficacy that stem from tumor resistance, heterogeneity, and intense target competition. Dual-payload ADCs (DP-ADCs) represent a promising solution to these challenges, as they leverage dual mechanisms of action that mitigate acquired drug resistance and enhance adaptability to tumor heterogeneity. The complex structure of DP-ADCs presents substantial quality control hurdles. In this manuscript, we review the current payload selection and conjugation strategies of DP-ADCs and examine recent advances in quality control research. Specifically, we analyze the analytical challenges related to the quantification of free toxins, the determination of the total antibody content, and the characterization of the drug-to-antibody ratio and its distribution. Ultimately, the aim of this work is to provide valuable guidance for future DP-ADC quality control analyses to facilitate their clinical translation and application.

Background/Objectives: This study comprehensively characterized the O- and N-glycosylation profiles of bispecific antibodies (BsAbs) via advanced analytical techniques to evaluate their structural and functional implications. Methods: High-resolution MS revealed O-xylosylation at Ser468 within the (G4S)4 linker peptide, which was identified as xylose with a molecular weight of 132.042 Da. HILIC-HPLC analysis of N-glycosylation revealed glycan species engineered to eliminate Fc effector functions. O-glycosylation analysis via β-elimination followed by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) identified xylose as the predominant glycan. Results: O-xylosylation does not affect the binding of BsAbs to either antigen Programmed Death-1 (PD-1) or Vascular Endothelial Growth Factor (VEGF). Notably, O-xylosylation interactions with mannose receptor represent the first discovery highlighting potential immunomodulatory roles. Conclusions: This study highlights the critical importance of monitoring comprehensive glycosylation characterization during the development of BsAb with (G4S)n linkers to ensure optimal therapeutic efficacy, safety, and reduced immunogenic potential.

Identification of sequence variants that may affect the safety and efficacy is critical in cell line development and process optimization for therapeutic antibodies. Protein-level sequence variant analysis typically employs LC-MS/MS peptide mapping. However, incorrect variant identification often arises from mismatched mass spectral assignments. Furthermore, the sample preparation workflow, particularly the proteolytic digestion steps, may inadvertently generate nonphysiological sequence variants that are not readily distinguishable from authentic variants due to their correct peptide matches. Here we report a type of artifactual sequence variant (e.g., C425S → C425R variant in the antibody Fc region) and elucidate its formation mechanism. Our findings demonstrate that such byproducts originate from digested amino acid transfer to the C-terminus of nonspecifically cleaved peptides, such as the transfer of Arg to W417QQGNVFSC425 in Trypsin/Lys-C mix-digested antibodies, resulting in W417QQGNVFSC425R. The significantly higher cleavage efficiency at carbamidomethyl-C425S compared to carbamidomethyl-C425R facilitates byproduct accumulation. This study establishes that such artifacts arise through nonspecific cleavage-associated transpeptidation, enabling their identification as analytical artifacts during data processing.

CD39 is a novel therapeutic target involved in antitumor immunity that can be manipulated through the ATP-adenosine signaling pathway. As a promising cancer treatment option, several drugs targeting CD39 have recently progressed into clinical trials. In this study, we describe the development and validation of a novel method to evaluate the relative bioactivity of anti-CD39 antibodies based on CD39-expressing CHO-K1-huCD39-10B6 cells. The method uses a commercially available CellTiter-Glo luminescent cell viability assay kit to generate luciferase signals, thereby providing convenient and rapid sample analysis. The method was comprehensively validated following the ICH Q2 guidelines, and the results showed that this method demonstrated high specificity, accuracy, precision, and linearity, with a range of 60-140 %. Additionally, the CHO-K1-huCD39-10B6 cell line exhibited relative stability and robustness within 15 generations. Therefore this method has potential use in anti-CD39 antibody bioactivity assays for lot release and stability studies.

Pyrogen, often as a contaminant, is a key indicator affecting the safety of almost all parenteral drugs (including biologicals, chemicals, traditional Chinese medicines and medical devices). It has become a goal to completely replace the in vivo rabbit pyrogen test by using the in vitro pyrogen test based on the promoted ‘reduction, replacement and refinement’ principle, which has been highly considered by regulatory agencies from different countries. We used NF-κB, a central signalling molecule mediating inflammatory responses, as a pyrogenic marker and the monocyte line THP-1 transfected with a luciferase reporter gene regulated by NF-κB as an in vitro model to detect pyrogens by measuring the intensity of a fluorescence signal. Here, we show that this test can quantitatively and sensitively detect endotoxin (lipopolysaccharide from different strains) and nonendotoxin (lipoteichoic acid, zymosan, peptidoglycan, lectin and glucan), has good stability in terms of NF-κB activity and cell phenotypes at 39 cell passages and can be applied to detect pyrogens in biologicals (group A & C meningococcal polysaccharide vaccine; basiliximab; rabies vaccine (Vero cells) for human use, freeze-dried; Japanese encephalitis vaccine (Vero cells), inactivated; insulin aspart injection; human albumin; recombinant human erythropoietin injection (CHO Cell)). The within-laboratory reproducibility of the test in three independent laboratories was 85%, 80% and 80% and the interlaboratory reproducibility among laboratories was 83.3%, 95.6% and 86.7%. The sensitivity (true positive rate) and specificity (true negative rate) of the test were 89.9% and 90.9%, respectively. In summary, the test provides a novel alternative for pyrogen detection.

Objective To perform methodological validation of non-reduced capillary electrophoresis-sodium dodecyl sulfate(nrCE-SDS)for monoclonal antibodies,providing a reference for the methodological validation of corresponding quality control methods in the industry.Methods Samples were analyzed according to the general chapter<3127>determination method for monoclonal antibody molecular size variants in the Pharmacopoeia of the People's Republic of China(2020 edition).Two laboratory technicians performed experiments over three days,using the same detection method daily to analyze the test samples,alongside comprehensive methodological validation.Results The method demonstrated good specificity,with the accuracy of the main peaks of all analytical samples reaching 98% to 102%,and the accuracy of total fragments and specific impurities reaching 70% to 130% .The relative standard deviation(RSD)for repeatability and intermediate precision was≤15% .The ranges for the main peak,total fragment,and specific impurity were≥89.9%,0.66% to 10.10%,and 0.36% to 4.80%,respectively.The limit of quantification of the method was 0.33% .Conclusion Full validation demonstrates good accuracy,precision,linearity,range,limit of quantification,and specificity of the method,providing a reference for the scientific and rational design of corresponding methodological validations.

Fever is a systemic inflammatory response of the body to pyrogens. Nuclear factor κB (NF-κB) is a central signalling molecule that causes the excessive secretion of various proinflammatory factors induced by pyrogens. This study explored the feasibility of a novel reporter gene assay (RGA) for pyrogen detection using RAW 264.7 cells stably transfected with the NF-κB reporter gene as a pyrogenic marker. Pyrogen was incubated with the transgenic cells, and the intensity of the fluorescence signal generated by luciferase secreted by the reporter gene was used to reflect the degree of activation of NF-κB, so as to quantitatively detect the pyrogens. The RGA could detect different types of pyrogens, including the lipopolysaccharide (LPS) of gram-negative bacteria, the lipoteichoic acid (LTA) of gram-positive bacteria, and the zymosan of fungi, and a good dose-effect relationship was observed in terms of NF-κB activity. The limits of detection of the RGA to those pyrogens were 0.03 EU/ml, 0.001 μg/ml, and 1 μg/ml, respectively. The method had good precision and accuracy and could be applied to many biological products (e.g., nivolumab, rituximab, bevacizumab, etanercept, basiliximab, haemophilus influenzae type b conjugate vaccine, 23-valent pneumococcal polysaccharide vaccine, and group A and group C meningococcal conjugate vaccine). The results of this study suggest that the novel RGA has a wide pyrogen detection spectrum and is sufficiently sensitive, stable, and accurate for various applications. Importance Pyrogen testing is mandatory and a critical method to ensure the safety of parenteral products including vaccines. Currently, only two pharmacological tests, including the rabbit pyrogen test and the bacterial endotoxins test (BET), are applied to evaluate pyrogenic contamination in parenteral pharmaceuticals by most of state pharmacopoeias. Although generally reliable, both of these assays have shortcomings. The rabbit test is not quantitative but is expensive and involves the use of animals. It can also produce varying responses depending on the strain, age and housing conditions of the rabbits. The BET, however, does not detect pyrogens other than gram-negative bacterial endotoxins and is often problematic when used to test solutions with a high protein content. To overcome these shortcomings and satisfy the growing need for new methods prompted by the constantly increasing production of biological compounds, it is necessary to develop the novel assay for pyrogen detection. Highlights This novel reporter gene assay can detect different types of pyrogens, including the lipopolysaccharide of gram-negative bacteria, the lipoteichoic acid of gram-positive bacteria, and the zymosan of fungi. The novel reporter gene assay is sufficiently sensitive, stable, and accurate for various applications.

针对抗PD-1单抗的报告基因活性测定法开展实验室间的联合验证,以研究该方法多实验室间的可应用和可转移性.本文采用两种协作方案,分别对该方法的检测内、检测间和实验室间精密度以及线性和准确性进行了研究.结果 显示:该方法的检测内精密度的95％可置信区间为(1.72～16.89)％,检测间精密度为(2.63～17.67)％,实验室间精密度为(9.00～l4.26)％;线性的相关系数均大于0.99,对于不同效价水平准确性的95％可置信区间,50％为(91.83～104.40)％,75％为(90.40～101.40)％,100％为(94.71～105.60)％,125％为(94.00～102.00)％,150％为(96.73～104.30)％.联合验证结果证明,该针对抗PD-1单抗的报告基因测活法,其精密度、线性和准确性均良好,可应用于不同实验室抗PD-1单抗的放行检测及稳定性分析.

RANKL (receptor activator of nuclear factor κB ligand) plays a key role in the differentiation, activation and survival of osteoclasts. Denosumab, which targets RANKL, is approved for osteoporosis or bone loss that has a high risk for fracture and bone metastases from solid tumors. Bioactivity determination is essential for the safety and efficacy of therapeutic antibodies. At present, the mechanism of action (MOA) based bioassay for anti-RANKL monoclonal antibodies (mAbs) is the measurement of tartrate resistant acid phosphatase (TRAP) activity, which takes about five days and has complex operation and relatively high variation. In this study, we developed a reporter gene assay (RGA) based on a RAW264.7 cell line stably expressing luciferase reporter under the control of nuclear factor-κB (NF-κB) response elements. After optimizing the key parameters, the validation results based on ICH-Q2 not only show superior specificity, precision, linearity, accuracy and passage stability, but also a short duration and simple operation. These results demonstrate the RGA based on the RANKL-RANK-NF-κB pathway can be an excellent alternative for measuring the bioactivity of anti-RANKL mAbs.

IL-6 has an important role in the pathogenesis of autoimmunity and chronic inflammation. Several mAbs that target IL-6 or the IL-6 receptor (IL-6R) have been established and approved for the treatment of various diseases such as multicentric Castleman's disease and rheumatoid arthritis. Quality control of therapeutic antibodies requires accurate determination of bioactivity. However, current cell-based anti-proliferation assays are tedious, time consuming, and result in high variation. We therefore developed a reporter gene assay (RGA) based on an IL-6-dependent DS-1 cell line that stably expressed the reporter luciferase controlled by the serum-induced element (SIE) response element, which was a key element located downstream of the IL-6 signaling pathway. The RGA method demonstrated good performance characteristics after careful optimization, including high specificity, stability, accuracy, precision, and robustness. It also had superior precision and sensitivity. The assay is simple compared with the traditional anti-proliferation assay. This novel RGA based on the IL-6-IL-6R-STAT3 pathway can be useful, in conjunction with the anti-proliferation bioassay, to determine the bioactivity of anti-IL-6/anti-IL-6R therapeutic mAbs.

Objective:To explore the effect of different sampling methods on sub-visible particle testing of smallvolume therapeutic antibodies by light obscuration method.Methods:Sub-visible particles in 65 lots of therapeutic antibodies against 19 targets were detected using light obscuration method.The volumes of adopted antibodies were all below 25 mL.The detection channels of ≥ 2 μm,≥ 10 μm and ≥ 25 μm were included.The antibodies were divided into 3 groups,and the corresponding sampling methods were single dose with different sampling volumes (0.5,1,3 mL) and combined doses (sample volume 5 mL);combined doses with different sampling volumes (0.5,1,3,5 mL);combined doses with different dilutions (20 times,10 times,5 times) and combined doses without dilution.Results:For sub-visible particles of ≥ 2 μm,≥ 10 μm and ≥ 25 μm,the testing results of different sampling volumes with single dose or combined doses were not significantly different with combined doses with sampling volume of 5 mL;and the result variation of ≥ 2 μm particles was smaller than that of ≥ 10 μm and ≥ 25 μm particles.However,there was significant difference between results of combined doses with different dilutions and combined doses without dilution.And the results of diluted samples were higher than those of without dilution;the larger the dilution multiple,the higher the result is.Conclusion:When light obscuration method is applied in testing sub-visible particles of therapeutic antibodies with volumes of less than 25 mL,small sampling volume of less than 5 mL was the prior consideration in order to save samples and reduce exogenous particles,for both single dose and combined doses;while method of combined doses with different dilutions should be used with caution.