Stability assessment of FDA-approved ramucirumab monoclonal antibody; validated SE-HPLC method for degradation pattern evaluation.

Ramucirumab (RAMU) is a recently US Food and Drug Administration-approved monoclonal antibody that is included in various anticancer protocols. It has a structural complexity and high degradation risk that have a significant effect on its safety and effectiveness. The major aim of this work was to assess the degradation pattern of RAMU based on physicochemical characterization. Mechanical agitation, repeated freeze-thaw cycles, pH and temperature were the selected stress conditions to which RAMU samples were subjected. The SE-HPLC method was applied and validated to monitor the RAMU monomer along with its aggregates and/or fragments. The purity of the separated peaks together with system suitability parameters were determined through the calculation of percentage purity and percentage drop in RAMU concentration. The results were interpreted by correlating them with those of dynamic light scattering and reducing and non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Samples incubated at pH 2.0-10.0 and 37°C for up to 4 weeks were analysed, recording detection of reversed phase (RP) aggregates and low molecular weight peptide fragments. Similarly, samples under short-term storage conditions of 4 weeks at different temperatures (-20, 2-8, 25, 37 and 50°C) showed low molecular weight peptide fragments but to a lesser extent. These results highlight the alarming effect on RAMU multidose vial efficacy and safety.

Fast Afucosylation Profiling of Glycoengineered Antibody Subunits by Middle-Up Mass Spectrometry.

Middle-up LC-MS antibody characterization workflows using reduction or IdeS digestion for a focused assessment of N-glycan profiling of three representative glycoengineered monoclonal antibodies (mAbs), namely, obinutuzumab (GlycomAb technology, Glycart/Roche), benralizumab (Potelligent Technology, BioWa, Kyowa Kirin) and mAb B (kifunensine) and compared to mAb A, produced in a common CHO cell line. In addition, EndoS or EndoS2 enzyme are used for quantitative determination of Fc-glycan core afucosylation and high mannose for these antibodies, as requested by health authorities for Fc-competent therapeutics mAbs critical quality attributes (CQAs).

New wide-pore superficially porous stationary phases with low hydrophobicity applied for the analysis of monoclonal antibodies.

The article describes the development of new stationary phases for the analysis of proteins in reversed phase liquid chromatography (RPLC). The goal was to have columns offering high recovery at low temperature, low hydrophobicity and novel selectivity. For this purpose, three different ligands bound onto the surface of superficially porous silica-based particles were compared, including trimethyl-silane (C1), ethyl-dimethyl-silane (C2) and N-(trifluoroacetomidyl)-propyl-diisopropylsilane (ES-LH). These three phases were compared with two commercial RPLC phases. In terms of protein recovery, the new ES-LH stationary phase clearly outperforms the other phases for any type of biopharmaceutical sample, and can already be successfully used at a temperature of only 60°C. In terms of retention, the new ES-LH and C1 materials were the less retentive ones, requiring lower organic solvent in the mobile phase. However, it is important to mention that the stability of C1 phase was critical under acidic, high temperature conditions. Finally, some differences were observed in terms of selectivity, particularly for the ES-LH column. Besides the chemical nature of the stationary phase, it was found that the nature of organic modifier also plays a key role in selectivity.

Charge variant analysis of protein-based biopharmaceuticals using two-dimensional liquid chromatography hyphenated to mass spectrometry.

The profile of charge variants represents an important critical quality attribute of protein-based biopharmaceuticals, in particular of monoclonal antibodies, and must therefore becontrolled. In this work, 2D-LC methods for charge variant analysis were developed using a strong cation-exchange chromatography (SCX) as first dimension (1D) separation. Non-porous SCX (3 µm) particle columns and different mobile phases were evaluated using a test mixture of some standard proteins of different size and pI (comprising myoglobin, bovine serum albumin, cytochrome c, lysozyme and ß-lactoglobulin) and two monoclonal IgG1 antibodies (NIST mAb and Secukinumab). The most promising 1D eluent for SCX was a salt-mediated pH-gradient system using a ternary mobile phase system with 2-(N-morpholino)ethanesulfonic acid, 1,3-diamino-2-propanol and sodium chloride. For the second dimension (2D), a desalting reversed-phase liquid chromatography (RP-LC) was chosen to enable the hyphenation of the charge variant separation with mass spectrometric (MS) detection. While for intact mAbs the 2D just served for desalting without additional selectivity, the 2D contributed some orthogonal selectivity for the mAb fragment separation. Various core-shell and monolithic columns were tested and variables such as gradient time and flow rate systematically optimized. Unexpectedly, a C4 400 Å column (3.4 µm diameter with 0.2 µm porous shell) provided higher peak capacities compared to the same 1000 Å column (2.7 µm diameter with 0.5 µm porous shell). A thinner shell appeared to be more advantageous than wider pores under high flow regime. An ultra-fast RP-LC method with a run time of one minute was developed using trifluoroacetic acid which was later replaced by formic acid as additive for better MS compatibility. The successful hyphenation of the two orthogonal separation modes, SCX and RP-LC, could be demonstrated in the multiple heart-cutting and the full comprehensive mode. MS analysis using a high-resolution quadrupole time-of-flight instrument enabled to identify different glycoforms and some major charge variants of the antibody at the intact protein level as well as on the subunit level (Fc/2, Lc, Fd') in a middle-up approach by 2D-LC-ESI-MS analysis.

Analytical comparability study of anti-CD20 monoclonal antibodies rituximab and obinutuzumab using a stability-indicating orthogonal testing protocol: Effect of structural optimization and glycoengineering.

Glycoengineering and biosimilarity are the key factors for growing, promising and progressive approaches in monoclonal antibodies development. In this study, the physicochemical stability of anti-CD20 rituximab (RTX); originator and biosimilar was compared to its glycoengineered humanized version; obinutuzumab (OBZ). An orthogonal stability-indicating protocol using a set of validated bioanalytical techniques; size exclusion high performance liquid chromatography (SE-HPLC), reversed phase liquid chromatography (RP-HPLC), quantitative gel electrophoresis by TapeStation, receptor binding assay and dynamic light scattering (DLS) was used to investigate the effect of different stress factors on the pattern and kinetics of degradation. SE-HPLC results supported with spectral purity showed similar degradation extent with a different pattern of degradation between RTX and OBZ. A lower tendency to form degraded fragments and a relatively higher favorability for degradation through aggregate formation has been revealed in case of OBZ. Results were in agreement with those of DLS and receptor binding assay which showed specificity to the intact antibodies in the presence of their degradation products. Furthermore, results were additionally confirmed through denaturing quantitative gel electrophoresis which suggested reducible covalent bonds as the mechanism for aggregates formation. RP-HPLC results showed two oxidized forms via excessive oxidation of RTX and OBZ with nearly the same degradation percent. Comparability data of RTX and OBZ using the applied methodologies showed that although glycoengineering; carried out to enhance the therapeutic and biological activity of OBZ altered the pattern of degradation but did not significantly affect the overall stability. Results showed also consistent stability profile between the biosimilar and its originator RTX products.

Simple Addition of Glycine in Trifluoroacetic Acid-Containing Mobile Phases Enhances the Sensitivity of Electrospray Ionization Mass Spectrometry for Biopharmaceutical Characterization.

Trifluoroacetic acid (TFA) is a commonly used mobile phase additive in liquid chromatography-mass spectrometry (LC-MS)-based biopharmaceutical characterization to enhance reversed-phase chromatographic performance of peptide separation; however, it leads to significant mass spectrometry signal suppression during electrospray ionization. Previous studies have shown that introducing large amounts of carboxylic acids or ammonium hydroxide to LC eluents postcolumn can improve MS sensitivity. In this study, we discovered glycine as a simple additive for TFA mobile phases, which mitigates ion suppression through a similar mechanism but in a more effective way than weak acid or weak base and boosts mass spectrometry responses (signal-to-noise ratio) of peptides by more than 1 order of magnitude on average after directly adding a small amount (e.g., 2 mM) into TFA mobile phases without compromising the chromatographic performance of peptide separation. Other small molecule additives in TFA mobile phases, such as amino acids containing extended side chains with different chemical properties and glycine-based variants, were also evaluated. The results demonstrated that glycine offered the best response boosting on peptides. The discovery of this glycine additive in TFA mobile phases provides a simple and conventional approach to achieve greater mass spectrometry detection sensitivity than TFA mobile phases for LC-MS-based characterization of biopharmaceuticals.

Incidence and Management of Therapeutic Monoclonal Antibody Interference in Monoclonal Gammopathy Monitoring.

BACKGROUND: The treatment of multiple myeloma (MM) has been revolutionized by the introduction of therapeutic monoclonal antibodies (tmAbs). Daratumumab, a human IgG1/κ tmAb against CD38 on plasma cells, has improved overall survival in refractory MM and was recently approved as a frontline therapy for MM. Work on tmAb interference with serum protein electrophoresis (SPE) during MM monitoring has failed to provide information for laboratories on incidence of interference and effective methods of managing the interference at a practicable level. We aimed to evaluate daratumumab and elotuzumab interference in a large academic hospital setting and implement immediate solutions. METHODS: We identified and chart reviewed all cases of possible daratumumab interference by electrophoretic pattern (120 of 1317 total cases over 3 months). We retrospectively reviewed SPE cases in our laboratory to assess clinical implications of tmAb interference before the laboratory was aware of tmAb treatment. We supplemented samples with daratumumab and elotuzumab to determine the limits of detection and run free light chain analysis. RESULTS: Approximately 9% (120 of 1317) of tested cases have an SPE and/or immunofixation electrophoresis (IFE) pattern consistent with daratumumab, but only approximately 47% (56) of these cases were associated with daratumumab therapy. Presence of daratumumab led to physician misinterpretation of SPE/IFE results. Limits of daratumumab detection varied with total serum gammaglobulin concentrations, but serum free light chain analysis was unaffected. CONCLUSIONS: Clinical laboratories currently rely on interference identification by electrophoretic pattern, which may be insufficient and is inefficient. Critical tools in preventing misinterpretation efficiently include physician education, pharmacy notifications, separate order codes, and interpretive comments.

Middle Level IM-MS and CIU Experiments for Improved Therapeutic Immunoglobulin Subclass Fingerprinting.

Most of the current FDA and EMA approved therapeutic monoclonal antibodies (mAbs) are based on humanized or human IgG1, 2, or 4 subclasses and engineered variants. On the structural side, these subclasses are characterized by specific interchain disulfide bridge connections. Different analytical techniques have been reported to assess intact IgGs subclasses, with recently special interest in native ion mobility (IM) and collision induced unfolding (CIU) mass spectrometry (MS). However, these two techniques exhibit significant limitations to differentiate mAb subclasses at the intact level. In the present work, we aimed at developing a unique IM-MS-based approach for the characterization of mAb subclasses at the middle level. Upon IdeS-digestion, the unfolding patterns of the F(ab')2 and Fc domains were simultaneously analyzed in a single run to provide deeper structural insights of the mAb scaffold. The unfolding patterns associated with the F(ab')2 domains are completely different in terms of unfolding energies and number of transitions. Thereby, F(ab')2 regions are the diagnostic domain to provide specific unfolding signatures to differentiate IgG subclasses and provide more confident subclass categorization than CIU on intact mAbs. In addition, the potential of middle-level CIU was evaluated through the characterization of eculizumab, a hybrid therapeutic IgG2/4 mAb. The unfolding signatures of both domains were allowed to corroborate, within a single run, the hybrid nature of eculizumab as well as specific subclass domain assignments to the F(ab')2 and Fc regions. Altogether, our results confirm the suitability of middle-level CIU of F(ab')2 domains for subclass categorization of canonical and more complex new generation engineered antibodies and related products.

Evaluating an easy sampling method using dried blood spots to determine vedolizumab concentrations.

An association between vedolizumab (VDZ) trough concentrations and therapeutic outcome has been observed in patients with inflammatory bowel diseases. VDZ samples are typically collected via venous sampling for therapeutic drug monitoring (TDM), but can alternatively be collected via dried blood spot (DBS) samples, which can be used for intensive sampling to investigate pharmacokinetic profiles. Therefore, we have developed a DBS method for determining VDZ concentrations and validated this method by comparing VDZ measurements in paired DBS and venous patient samples. First, VDZ was spiked in citrated whole blood and spotted on filter paper. After drying, DBS were extracted and VDZ concentrations were determined in the extracts using ELISA. For clinical validation, 41 paired DBS and serum samples were collected from 19 VDZ-treated patients. VDZ concentrations measured in DBS extracts strongly correlated with serum concentrations (Pearson r = 0.978, p < 0.0001). No significant impact of the hematocrit value was observed on the VDZ DBS/serum concentration ratios. Additionally, the VDZ DBS/serum ratio was calculated in nine individual patients, which was, in general, shown to be stable over time. VDZ DBS sampling is a robust method that can be used as an alternative to venous blood collection for TDM of VDZ. VDZ concentrations in DBS highly correlated with VDZ serum concentrations over a broad concentration range, allowing DBS to be used for intensive sampling to gain more insight in VDZ pharmacokinetics.

Demonstrating Analytical Similarity of Trastuzumab Biosimilar HLX02 to Herceptin® with a Panel of Sensitive and Orthogonal Methods Including a Novel FcγRIIIa Affinity Chromatography Technology.

BACKGROUND: A biosimilar needs to demonstrate its similarity to the originator reference product (RP) in terms of structural and functional properties as well as nonclinical and clinical outcomes. OBJECTIVES: The aim was to assess the analytical similarity between the trastuzumab biosimilar HLX02 and Europe-sourced Herceptin® (EU-Herceptin®) and China-sourced Herceptin® (CN-Herceptin®) following a quality-by-design (QbD) quality study and tier-based quality attribute evaluation. METHODS: A panel of highly sensitive and orthogonal methods, including a novel Fc gamma receptor IIIa (FcγRIIIa) affinity chromatography technique that enables quantitative comparison of glycan effects on effector function, was developed for the assessment. To ensure the full product variability was captured, ten batches of HLX02 were compared with 39 RP batches with expiry dates from August 2017 to March 2021. RESULTS: The extensive three-way similarity assessment demonstrated that HLX02 is highly similar to the RPs. Furthermore, the %afucose, %galactose, and FcγRIIIa affinity of the RPs were observed to first decrease and then return to the original level in relation to their expiry dates, and the RP batches can be subgrouped by their FcγRIIIa affinity chromatograms. HLX02 is demonstrated to be more similar to the RPs of the high FcγRIIIa affinity group. CONCLUSION: Besides having an overall high analytical similarity to both EU-Herceptin® and CN-Herceptin®, HLX02 is more similar to Herceptin® with high FcγRIIIa affinity, a result that demonstrates the power of the novel FcγRIIIa affinity chromatography technology in biosimilarity evaluation.

An in-line enzymatic microreactor for the middle-up analysis of monoclonal antibodies by capillary electrophoresis.

Monoclonal antibodies (mAbs) are undergoing rapid growth in the pharmaceutical industry due to their clinical efficiency. Concomitantly, robust, cost-effective, and high throughput analytical methods are needed for their quality control. Among all analytical techniques, capillary electrophoresis (CE) presents alternative and attractive features because the capillary can be used both as a microreactor and as a support for separation. Transverse diffusion of laminar flow profiles was applied for the middle-up analysis of mAbs for the first time. Infliximab was selected as the model mAb. All middle-up analysis steps (enzymatic digestion, electrophoretic separation and UV detection) were integrated into the same capillary. The conditions for the separation of infliximab subunits (pH, ionic strength, and type of background electrolyte) and in-line digestion parameters (reactant injection conditions, time, temperature and enzyme/mAb ratio) were optimized. The in-line methodology was compared to the off-line methodology and evaluated in terms of proteolysis efficiency, repeatability, and applicability to different mAbs. Finally, the methodology was transferred to capillary electrophoresis coupled to mass spectrometry (sheathless interface) to identify infliximab subunits. The in-line methodology was successfully implemented with a simplified injection scheme, temperature control, fast enzymatic reaction and high resolution of separation of infliximab subunits under pseudo-native MS compatible conditions. In comparison with the off-line methodology, reactant consumption was reduced by a factor of 1000, and the numbers of theoretical plates were increased by a factor of 2.

PNL2: A Useful Adjunct Biomarker to HMB45 in the Diagnosis of Uterine Perivascular Epithelioid Cell Tumor (PEComa).

Perivascular epithelioid cell tumors (PEComa) are rare neoplasms characterized by co-expression of melanocytic and muscle markers. HMB45 and Melan-A are used to confirm a PEComa diagnosis; however, both are often focally expressed and sensitivity for Melan-A is low. PNL2 is a reliable biomarker for epithelioid melanoma and renal angiomyolipoma/PEComa. The objective of this study was to determine PNL2 utility in diagnosing uterine PEComas as well as distinguishing PEComas from uterine smooth muscle tumors (SMTs). Twenty-one uterine PEComas and 45 SMTs were analyzed for PNL2; a subset was also stained for HMB45, Melan-A, Cathepsin-K, Desmin, and h-Caldesmon. Cases were scored as negative (0), focal (<10% of tumor cells), or patchy to diffusely positive (>10% of tumor cells). PEComas were positive for PNL2, HMB45, and Melan-A in 86%, 100%, and 57% of cases, respectively. In PEComas, PNL2 was patchy to diffusely positive more frequently (10/18, 56%) than Melan-A (4/12, 33%). In contrast, 2 of 45 (4%) SMTs were focally PNL2 positive; HMB45 was focally positive in 4 SMTs (11%) and all were negative for Melan-A. Desmin and h-Caldesmon were positive in 90% and 57% of PEComas, and 91% and 82% of SMTs. Cathepsin-K was positive in 100% of PEComas and 93% of SMTs. PNL2 is a useful biomarker for the diagnosis of uterine PEComa, with comparable sensitivity and specificity to HMB45. In contrast, PNL2 stains more PEComas when compared with Melan-A. Cathepsin-K, Desmin, and h-Caldesmon are of little utility for distinguishing PEComas and SMTs; however, lack of Cathepsin-K argues against PEComa. These results suggest that PNL2 should be used in conjunction with HMB45 in the diagnosis of PEComa of the uterine corpus.

Vedolizumab quantitation using high-resolution accurate mass-mass spectrometry middle-up protein subunit: method validation.

Background While quantitation methods for small-molecule and tryptic peptide bottom-up mass spectrometry (MS) have been well defined, quantitation methods for top-down or middle-up MS approaches have not been as well defined. Therapeutic monoclonal antibodies (t-mAbs) are a group of proteins that can be used to both demonstrate the advantages of top-down or middle-up detection methods over classic tryptic peptide bottom-up along with the growing need for robust quantitation strategies/software for these top-down or middle-up methods. Bottom-up proteolytic digest methods for the t-mAbs tend to suffer from challenges such as limited peptide selection due to potential interference from the polyclonal immunoglobulin background, complicated workflows, and inadequate sensitivity and specificity without laborious purification steps, and therefore have prompted the search for new detection and quantitation methods. Time-of-flight along with Orbitrap MS have recently evolved from the research and/or pharmaceutical setting into the clinical laboratory. With their superior mass measurement accuracy, resolution and scanning speeds, these are ideal platforms for top-down or middle-up characterization and quantitation. Methods We demonstrate a validated, robust, middle-up protein subunit detection and quantitation method for the IgG1 t-mAb, vedolizumab (VEDO), which takes advantage of the high resolution of the Orbitrap MS detection and quantitation software to increase specificity. Results Validated performance characteristics met pre-defined acceptance criteria with simple workflows and rapid turnaround times: characteristics necessary for implementation into a high-volume clinical MS laboratory. Conclusions While the extraction method can easily be used with other IgG1 t-mAbs, the detection and quantitation method may become an option for measurement of other proteins.

A cell-based immunogenicity assay to detect antibodies against chimeric antigen receptor expressed by tisagenlecleucel.

The use of T-cells expressing Chimeric Antigen Receptors (CARs) offers new opportunities for cancer treatment, as well as new challenges for the bioanalysis of this new class of drugs. The analysis of humoral immunogenicity (anti-drug antibodies) against CARs could be performed with a bridging ELISA, using labeled CAR fragments. However, outside of its native cell membrane environment and without potential interaction partners on the cell surface, a labeled or coated recombinant CAR fragment may structurally differ from the membrane-bound CAR expressed on CAR-T cells. Consequently, immunogenicity to CARs may be missed due to the artificial nature of a ligand binding assay setup. T-cell lines expressing the CAR offer the opportunity to measure anti-drug antibodies to the CAR in its natural cell environment, as an alternative to ligand-binding assays. Here we describe a novel, flow cytometry-based humoral immunogenicity assay for tisagenlecleucel (CTL019, Kymriah®) using a human T-cell line that expresses murine CAR19. The assay described here was fully validated according to health authority guidelines for the development and validation of immunogenicity assays and has a sensitivity of 100 ng/mL. A good correlation of screening assay signal strengths to titer assay results was observed while exploring options to increase titration assay throughput. Pre-existing antibodies against the cell line used in the assay as well as against the CAR itself complicate the assay and data interpretation.

Laboratory coagulation tests and emicizumab treatment A United Kingdom Haemophilia Centre Doctors' Organisation guideline.

INTRODUCTION: The factor VIII mimetic emicizumab (Hemlibra, Hoffman-la Roche, Basel, Switzerland) has a novel mode of action that affects the laboratory monitoring of patients receiving this treatment. AIM: This guideline from the United Kingdom Haemophilia Centre Doctors Organisation (UKHCDO) aims to provide advice for clinical and laboratory staff on appropriate use of laboratory assays in patients with Haemophilia A treated with emicizumab. METHODOLOGY: The guideline was prepared by a review of the available literature and discussion and revision by the authors. RESULTS: The guideline describes the effect of emicizumab on commonly used coagulations tests and provides recommendations on the use of assays for measurement of factor VIII and factor VIII inhibitor in the presence of emicizumab. The guideline also provides recommendations on measurement of emicizumab. CONCLUSION: Knowledge of the effect of emicizumab on coagulation tests and factor assays is required to ensure appropriate testing and monitoring of therapy in patients receiving this drug.

Analytical validation of an alternative method to quantify specific antibodies in 3 applications.

The detection and the quantification of specific antibodies represent essential tools for the diagnosis and for the biological monitoring of immune humoral response in many clinical situations in particular in autoimmune diseases or in the context of immunotherapy using monoclonal antibodies. This article focuses on the development of a specific antibody measuring method (Patent n°PCT/IB2014/064437). The principle of this method is based on the combined use of a monoclonal antibody as standard and the protein G as immunoglobulins detecting agent. We performed a complete analytical validation of this method for the quantification of antibodies in three different applications: autoantibodies, alloantibodies and therapeutic monoclonal antibody. The results showed good performances compatible with the use of these assays as diagnostic tools. This method allows avoiding the use of products from human origin as reagent that causes ethical and infectious concerns but also storage and long term stock management problems. Moreover, this approach is particularly useful when no commercial reagent is available, especially in the case of rare diseases.

Enzyme linked immunosorbent assay for the quantification of nivolumab and pembrolizumab in human serum and cerebrospinal fluid.

Immunotherapy with monoclonal antibodies targeting the programmed-death-1 (PD-1) receptor has become standard of care for an increasing number of tumor types. Pharmacokinetic studies may help to optimize anti-PD-1 therapy. Therefore, accurate and sensitive determination of antibody concentrations is essential. Here we report an enzyme linked immunosorbent assay (ELISA) capable of measuring nivolumab and pembrolizumab concentrations in serum and cerebrospinal fluid (CSF) with high sensitivity and specificity. The assay was developed and validated based on the specific capture of nivolumab and pembrolizumab by immobilized PD-1, with subsequent enzymatic chemiluminescent detection by anti-IgG4 coupled with horse radish peroxidase (HRP). The lower limit of quantification for serum and CSF was 2 ng/mL for both anti-PD-1 agents. The ELISA method was validated and showed long term sample stability of >1 year. This method is reliable, relatively inexpensive and can be used in serum and CSF from pembrolizumab and nivolumab treated patients.

A Novel Online Four-Dimensional SEC×SEC-IM×MS Methodology for Characterization of Monoclonal Antibody Size Variants.

The determination of size variants is a major critical quality attribute of a therapeutic monoclonal antibody (mAb that may affect the drug product safety, potency, and efficacy. Size variant characterization often relies on size-exclusion chromatography (SEC), which could be hampered by difficult identification of peaks. On the other hand, mass spectrometry (MS)-based techniques performed in nondenaturing conditions have proven to be valuable for mAb-related compound characterization. On the basis of the observation that limited SEC performance was observed in nondenaturing MS compatible ammonium acetate buffer compared with classical phosphate salts, a multidimensional analytical approach was proposed. It combines comprehensive online two-dimensional chromatography (SEC×SEC), with ion mobility and mass spectrometry (IM-MS) in nondenaturing conditions for the characterization of a variety of mAbs. We first exemplify the versatility of our approach for simultaneous detection, identification, and quantitation of adalimumab size variants. Benefits of the SEC×SEC-native IM×MS were further highlighted on forced degraded pembrolizumab and bevacizumab samples, for which the 4D setup was mandatory to obtain an extensive and unambiguous identification, and accurate quantitation of unexpected high/low molecular weight species (HMWS and LMWS). In this specific context, monomeric conformers were detected by IM-MS as HMWS or LMWS. Altogether, our results emphasize the power of comprehensive 2D LC×LC setups hyphenated to IM×MS in nondenaturing conditions with unprecedented performance including: (i) maintaining optimal SEC performance (under classical nonvolatile salt conditions), (ii) performing online native MS identification, and (iii) providing IM-MS conformational characterization of all separated size variants.