Intact Monoclonal Antibody Research Articles

Direct glycosylation analysis of intact monoclonal antibodies combining ESI MS of glycoforms and MALDI-in source decay MS of glycan fragments

Monoclonal antibody (mAb) glycoengineering has the potential to improve the efficacy of biopharmaceuticals by fine-tuning specific biological properties. Glycosylation analysis is key to monitoring the glycoengineering process. Various mass spectrometry (MS)-based methods are available to characterize mAb glycosylation at different structural levels, but comprehensive analysis is typically time-consuming and costly. Here, we present an approach that combines conventional intact mass measurement of glycoforms by direct infusion ESI-MS with an advanced MALDI-in-source decay FT-ICR MS method for direct analysis of glycans in intact mAbs, without the need for enzymatic release and separation. Using a sodium-doped MALDI matrix, glycans were directly released as ISD fragment ions from the intact mAbs during the ionization process. Measurement of 0,2A fragment signals yielded reproducible glycan profiles that were consistent with conventional methods, yet was achieved with unprecedented speed, providing complementary information to that obtained through intact mass measurement. The methods were applied to standard and glycoengineered trastuzumab and rituximab, allowing rapid glycosylation profiling and structural analysis of glycans by tandem MS of selected ISD fragment ions. This fast approach can facilitate the early-phase development of glycoengineering processes by constraining further in-depth analyses. We envision a broader applicability in studies focused on glycosylation changes in mAbs.

Characterization of a Monoclonal Antibody by Native and Denaturing Top-Down Mass Spectrometry.

Established in recent years as an important approach to unraveling the heterogeneity of intact monoclonal antibodies, native mass spectrometry has been rarely utilized for sequencing these complex biomolecules via tandem mass spectrometry. Typically, top-down mass spectrometry has been performed starting from highly charged precursor ions obtained via electrospray ionization under denaturing conditions (i.e., in the presence of organic solvents and acidic pH). Here we systematically benchmark four distinct ion dissociation methods─namely, higher-energy collisional dissociation, electron transfer dissociation, electron transfer dissociation/higher-energy collisional dissociation, and 213 nm ultraviolet photodissociation─in their capability to characterize a therapeutic monoclonal antibody, trastuzumab, starting from denatured and native-like precursor ions. Interestingly, native top-down mass spectrometry results in higher sequence coverage than the experiments carried out under denaturing conditions, with the exception of ultraviolet photodissociation. Globally, electron transfer dissociation followed by collision-based activation of product ions generates the largest number of backbone cleavages in disulfide protected regions, including the complementarity determining regions, regardless of electrospray ionization conditions. Overall, these findings suggest that native mass spectrometry can certainly be used for the gas-phase sequencing of whole monoclonal antibodies, although the dissociation of denatured precursor ions still returns a few backbone cleavages not identified in native experiments. Finally, a comparison of the fragmentation maps obtained under denaturing and native conditions strongly points toward disulfide bonds as the primary reason behind the largely overlapping dissociation patterns.

In Migratio Noncovalent Fluorophore Labeling of Proteins by Propidium Iodide in Sodium Dodecyl Sulfate Capillary Gel Electrophoresis.

Sodium dodecyl sulfate capillary gel electrophoresis is one of the frequently used methods for size-based protein separation in molecular biology laboratories and the biopharmaceutical industry. To increase throughput, quite a few multicapillary electrophoresis systems have been recently developed, but most of them only support fluorescence detection, requiring fluorophore labeling of the sample proteins. To avoid the time-consuming derivatization reaction, we developed an on-column labeling approach utilizing propidium iodide for the first time in SDS-CGE of proteins, a dye only used before for nucleic acid analysis. As a key ingredient of the gel-buffer system, the oppositely migrating positively charged propidium ligand in migratio complexes with the SDS-proteins, therefore, supports in situ labeling during the electrophoretic separation process, not requiring any extra pre- or postcolumn derivatization step. A theoretical treatment is given to shed light on the basic principles of this novel online labeling process, also addressing the influence of propidium iodide on the electroosmotic flow, resulting in reduced retardation. The concept of propidium labeling in SDS-CGE was first demonstrated using a commercially available protein sizing ladder ranging from 6.5 to 200 kDa with different isoelectric points and post-translational modifications. Considering the increasing number of protein therapeutics on the market next, we focused on the labeling optimization of a therapeutic monoclonal antibody and its subunits, including the addition of the nonglycosylated heavy chain. Peak efficiency and resolution were compared between noncovalent and covalent labeling. The effect of ligand concentration on the effective and apparent electrophoretic mobility, the resulting peak area, and the resolution were all evaluated in view of the theoretical considerations. The best detection sensitivity for the intact monoclonal antibody was obtained by using 200 μg/mL propidium iodide in the separation medium (LOD 2 μg/mL, 1.35 × 10-8 M) with excellent detection linearity over 3 orders of magnitude. On the other hand, the resolution between the biopharmaceutical protein test mixture components containing the intact and subunit fragments of the therapeutic monoclonal antibody was very good in the ligand concentration range of 50-200 μg/mL, but using the local maximum at 100 μg/mL for the nonglycosylated/glycosylated heavy chain pair is recommended. The figures of merit, including precision, sensitivity, detection linear range, and resolution for a sample mixture in hand, can be optimized by varying the propidium iodide concentration in the gel-buffer system, as demonstrated in this paper.

Added Value of Internal Fragments for Top-Down Mass Spectrometry of Intact Monoclonal Antibodies and Antibody-Drug Conjugates.

Monoclonal antibodies (mAbs) and antibody-drug conjugates (ADCs) are two of the most important therapeutic drug classes that require extensive characterization, whereas their large size and structural complexity make them challenging to characterize and demand the use of advanced analytical methods. Top-down mass spectrometry (TD-MS) is an emerging technique that minimizes sample preparation and preserves endogenous post-translational modifications (PTMs); however, TD-MS of large proteins suffers from low fragmentation efficiency, limiting the sequence and structure information that can be obtained. Here, we show that including the assignment of internal fragments in native TD-MS of an intact mAb and an ADC can improve their molecular characterization. For the NIST mAb, internal fragments can access the sequence region constrained by disulfide bonds to increase the TD-MS sequence coverage to over 75%. Important PTM information, including intrachain disulfide connectivity and N-glycosylation sites, can be revealed after including internal fragments. For a heterogeneous lysine-linked ADC, we show that assigning internal fragments improves the identification of drug conjugation sites to achieve a coverage of 58% of all putative conjugation sites. This proof-of-principle study demonstrates the potential value of including internal fragments in native TD-MS of intact mAbs and ADCs, and this analytical strategy can be extended to bottom-up and middle-down MS approaches to achieve even more comprehensive characterization of important therapeutic molecules.

High-throughput glycosylation analysis of intact monoclonal antibodies by mass spectrometry coupled with capillary electrophoresis and liquid chromatography.

The analysis of monoclonal antibodies glycosylation is a crucial quality control attribute of biopharmaceutical drugs. High throughput screening approaches for antibody glycoform analysis are required in various stages of process optimization. Here, we present high throughput screening suitable mass spectrometry-based workflows for the analysis of intact antibody glycosylation out of cell supernatants. Capillary electrophoresis and liquid chromatography were coupled with quadrupole time-of-flight mass spectrometry or Orbitrap mass spectrometry. Both separation methods offer fast separation (10-15min) and the capability to prevent the separated cell supernatant matrix to enter the mass spectrometry by post-separation valving. Both mass spectrometry instruments provide comparable results and both are sufficient to determine the glycosylation pattern of the five major glycoforms of the measured antibodies. However, the Orbitrap yields higher sensitivity of 25μg/mL (CE-nanoCEasy-Orbitrap mass spectrometry) and 5μg/mL (liquid chromatography-Orbitrap mass spectrometry). Data processing was optimized for a faster processing and easier detection of low abundant glycoforms based on averaged charge-deconvoluted mass spectra. This approach combines a non-target glycoform analysis while yielding the same glycosylation pattern as the traditional approach based on extracted ion traces. The presented methods enable the high throughput screening of the glycosylation pattern of antibodies down to low μg/mL-range out of cell supernatant without any sample preparation.

Nanobody-based microfluidic human Fc assay for preclinical plasma quantification of IgG1/1.1 and IgG1-Fc-conjugates

BackgroundTherapeutic antibodies and Fc-conjugates are becoming increasingly popular for disease management and accurate and sensitive pharmacokinetic measurements are critical in lead candidate selection in pre-clinical drug discovery. Methods and study designHuman Fc-specific intact monoclonal antibodies, polyclonal antibodies, Fab fragments, aptamers, affibodies and nanobodies were screened for potential as biotinylated capture moieties in a microfluidic assay. Test compounds were Bevacizumab, Rituximab, Infliximab as well as an in-house IgG1.1 and an IgG1-drug conjugate. ResultsCapture molecules were tested for specificity in plasma matrices from beagle dog, rat, mouse, pig, rhesus monkey and cynomolgus monkey. We find that the llama nanobody provides the best selectivity across across species. The assay usability were verified in cynomolgus monkey pharmacokinetic studies of in-house IgG1.1 and IgG1-fusion molecules. ConclusionThe presented generic nanobody-based assay may find relevance in preclinical testing of future human Fc-containing drug conjugates devoid of Fab fragments and intact monoclonal antibodies.

Comparative studies on the substrate specificity and defucosylation activity of three α-l-fucosidases using synthetic fucosylated glycopeptides and glycoproteins as substrates

Core fucosylation is the attachment of an α-1,6-fucose moiety to the innermost N-acetyl glucosamine (GlcNAc) in N-glycans in mammalian systems. It plays a pivotal role in modulating the structural and biological functions of glycoproteins including therapeutic antibodies. Yet, few α-l-fucosidases appear to be capable of removing core fucose from intact glycoproteins. This paper describes a comparative study of the substrate specificity and relative activity of the human α-l-fucosidase (FucA1) and two bacterial α-l-fucosidases, the AlfC from Lactobacillus casei and the BfFuc from Bacteroides fragilis. This study was enabled by the synthesis of an array of structurally well-defined core-fucosylated substrates, including core-fucosylated N-glycopeptides and a few antibody glycoforms. It was found that AlfC and BfFuc could not remove core fucose from intact full-length N-glycopeptides or N-glycoproteins but could hydrolyze only the truncated Fucα1,6GlcNAc-peptide substrates. In contrast, the human α-l-fucosidase (FucA1) showed low activity on truncated Fucα1,6GlcNAc substrates but was able to remove core fucose from intact and full-length core-fucosylated N-glycopeptides and N-glycoproteins. In addition, it was found that FucA1 was the only α-l-fucosidase that showed low but apparent activity to remove core fucose from intact IgG antibodies. The ability of FucA1 to defucosylate intact monoclonal antibodies reveals an opportunity to evolve the human α-l-fucosidase for direct enzymatic defucosylation of therapeutic antibodies to improve their antibody-dependent cellular cytotoxicity.

Exploring frontiers of orbitrap performance for long transients

Experiments on a modified Orbitrap Exploris instrument have shown that even in a compact instrument it is possible to achieve a resolving power in excess of 2,000,000 when appropriate tolerance and tuning requirements are met. Such levels are achieved for a 4-s detection time.A new Orbitrap design with improved pumping allowed, for the first time, isotopic resolution of intact monoclonal antibodies even when using standard nitrogen supply in the Ion Routing Multipole. Longer transients further improve signal-to-noise ratios, approaching levels needed for single-charge resolution in charge detection mass spectrometry. Further steps for improvement of instrumentation are discussed.

Characterization and monitoring of charge variants of a recombinant monoclonal antibody using microfluidic capillary electrophoresis-mass spectrometry

Rigorous characterization of biotherapeutics, and monoclonal antibodies in particular, is a challenging task in terms of ensuring safety, efficacy, and potency of a therapeutic agent because of structural heterogeneity during cell culture, purification and storage. In this work, we used microfluidic capillary electrophoresis-mass spectrometry to analyze intact monoclonal antibody and assess the root cause of increases in acidic and basic variants under stress at high temperature. The antibody was analyzed at multiple levels, including its intact state under native conditions, and subunit and peptide levels. The normal and degraded antibodies at different time points were characterized and compared with each other. We concluded that the basic variants in the unstressed sample were produced C-terminal amidation, while the acidic variants were produced by deamidation. In stressed samples, change in the acidic and main peaks were caused by deamidation, and changes in the basic peaks were caused by both deamidation and oxidation. These results demonstrate that microfluidic capillary electrophoresis-mass spectrometry (CE-MS) is a powerful direct and generic tool for separation and identification of charge heterogeneity of biotherapeutics.

Antibody engineering-driven controllable chemiluminescence resonance energy transfer for immunoassay with tunable dynamic range

The donor-acceptor distance is a critical factor for the occurrence of chemiluminescent resonance energy transfer (CRET). We herein evaluate the donor-acceptor distance and transfer efficiency of CRET immunoassays of a series of donors which contain different sized antibody fragments, intact monoclonal antibody (IgG), antigen binding fragment (Fab), and single chain fragment antibody (scFv). Core/multishell quantum dots were used as the acceptor in three CRET systems. IgG is the maximum antibody fragment, leading to the longest donor-acceptor distance and the lowest transfer efficiency. Donors with Fab and scFv show significantly decreased distance and increased transfer efficiency. These results suggest an inverse correlation between donor size and transfer efficiency and can be used to provide guidance for the construction of controllable CRET. By combining the controllable CRET with immunoassay, we further develop a tunable sulfamethazine analytical system. Three different sized donors based CRET immunoassay show a markedly different sensitivity and dynamic range. Such adjustable detection provides greater flexibility for contaminant detection in different foodstuffs with different residue limits. This work not only illustrates the effect of donor-acceptor distance on regulating the energy transfer efficiency of CRET system, but also provides a guideline for the construction of a tunable immunoassay.

Hydrogel Microsphere Encapsulation Enhances the Flow Properties of Monoclonal Antibody Crystal Formulations

AbstractMonoclonal antibodies are therapeutic molecules known for their high specificity and versatility in the treatment of cancer and autoimmune disorders, but dosage forms are typically limited to low concentrations and large fluid volumes due to formulation challenges. Hydrogel microsphere formulations offer a route to quicker, patient‐friendly dosing regimens for monoclonal antibodies with high loading and favorable flow properties needed for injection through a narrow syringe needle under moderate applied force. Crystals of an intact monoclonal antibody are prepared as a concentrated suspension (>300 mg mL−1) which is then encapsulated within hydrogel microspheres with diameters as small as 30 µm. The hydrogel microspheres contain up to 56 wt% (dry basis) monoclonal antibody and release within 4 days under in vitro dissolution conditions. The hydrogel microspheres are concentrated into densely packed suspensions containing up to 300 mg mL−1 monoclonal antibody to evaluate their flow. These hydrogel formulations shear‐thin and have lower viscosity when compared to both liquid and suspended crystal forms of the monoclonal antibody, demonstrating the potential of hydrogel microsphere encapsulants as a carrier which can mask undesirable flow properties of concentrated antibody therapeutics.

Metabolic understanding of disulfide reduction during monoclonal antibody production.

The disulfide reduction of intact monoclonal antibodies (mAbs) and subsequent formation of low molecular weight (LMW) species pose a direct risk to product stability, potency, and patient safety. Although enzymatic mechanisms of reduction are well established, an understanding of the cellular mechanisms during the bioreactor process leading to increased risk of disulfide reduction after harvest remains elusive. In this study, we examined bench, pilot, and manufacturing-scale batches of two mAbs expressed in Chinese hamster ovary (CHO) cells, where harvested cell culture fluid (HCCF) occasionally demonstrated disulfide reduction. Comparative proteomics highlighted a significant elevation in glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels in a highly reducing batch of HCCF, compared to a non-reducing batch. Analysis during production cell culture showed that increased GAPDH gene and protein expression correlated to disulfide reduction risk in HCCF in every case examined. Additionally, glucose 6-phosphate dehydrogenase (G6PD) activity and an increased (≥ 300%) lactate/pyruvate molar ratio (lac/pyr) during production cell culture correlated to disulfide reduction risk, suggesting a metabolic shift to the pentose phosphate pathway (PPP). In all, these results suggest that metabolic alterations during cell culture lead to changes in protein expression and enzyme activity that in turn increase the risk of disulfide reduction in HCCF. KEY POINTS: • Bioreactor conditions resulted in reduction susceptible harvest material. • GAPDH expression, G6PD activity, and lac/pyr ratio correlated with mAb reduction. • Demonstrated role for cell metabolic changes in post-harvest mAb reduction. Graphical abstract.

Top-Down Characterization of an Intact Monoclonal Antibody Using Activated Ion Electron Transfer Dissociation.

Monoclonal antibodies (mAbs) are important therapeutic glycoproteins, but their large size and structural complexity make them difficult to rapidly characterize. Top-down mass spectrometry (MS) has the potential to overcome challenges of other common approaches by minimizing sample preparation and preserving endogenous modifications. However, comprehensive mAb characterization requires generation of many, well-resolved fragments and remains challenging. While ETD retains modifications and cleaves disulfide bonds-making it attractive for mAb characterization-it can be less effective for precursors having high m/z values. Activated ion electron transfer dissociation (AI-ETD) uses concurrent infrared photoactivation to promote product ion generation and has proven effective in increasing sequence coverage of intact proteins. Here, we present the first application of AI-ETD to mAb sequencing. For the standard NIST mAb, we observe a high degree of complementarity between fragments generated using standard ETD with a short reaction time and AI-ETD with a long reaction time. Most importantly, AI-ETD reveals disulfide-bound regions that have been intractable, thus far, for sequencing with top-down MS. We conclude AI-ETD has the potential to rapidly and comprehensively analyze intact mAbs.

Structural Fingerprints of an Intact Monoclonal Antibody Acquired under Formulated Storage Conditions via 15N Direct Detection Nuclear Magnetic Resonance.

Noninvasive evaluation of tertiary structures is fundamental to the research, development, and use of the biologics. However, few methodologies are currently available for evaluating large molecular weight (MW) biologics, such as therapeutic monoclonal antibodies (mAbs; 150 kDa). Here, we have newly developed a 15N direct detection nuclear magnetic resonance (NMR) technique, the 15N direct detection CRINEPT, which allows the observation of the main chain amide resonances of a nondeuterated protein with MW 150 kDa. The technique not only substantially expands the range of proteins applicable to solution NMR studies but also allows the noninvasive structural analyses of intact mAbs in a wide range of temperature and solvent conditions. As a proof of principle, we successfully acquired the 15N-detected CRINEPT spectra of an intact mAb in its formulated solution at 4 °C. The technique was able to discriminate heterogeneous galactosylation states, demonstrating the benefit of high resolution of the 15N direct detection.

Internal Dosimetry assessment for monoclonal antibodies and antibody fragments labeled by Lutetium-177

In this research, the behaviour of monoclonal antibodies (MAbs) and their fragments labeled by 177Lu after injecting into the body is simulated for diagnostic and therapy. The absorbed doses in organs and tissues with maximum radiation exposure are presented. On the base of reference data in the literature, a biokinetic model is built-in for MAb and their fragments. The cumulative activity of 177Lu in organs and tissues per Bq of administered activity is calculated. Spleen, liver, and red bone marrow have the highest doses when 177Lu associated with intact monoclonal antibodies is injected into human body. The estimated doses on these organs are 1.95, 1.17 and 0.54 mGy/MBq, respectively. For the fragments of monoclonal antibodies is considered with 177Lu, the most exposed organs are the kidneys with the doses of 0.78 mGy/MBq for F(ab’)) and 1.27 mGy/MBq for F(ab′)2.

Coupling Multi-Angle Light Scattering to Reverse-Phase Ultra-High-Pressure Chromatography (RP-UPLC-MALS) for the characterization monoclonal antibodies

Multi-angle light scattering coupled with size-exclusion chromatography (SEC-MALS) is a standard approach for protein characterization. Recently MALS detection has been coupled with ion-exchange chromatography (IEX) which demonstrated the feasibility and high value of MALS in combination with non-sized-based fractionation methods. In this study we coupled reverse-phase ultra-high pressure liquid chromatography (RP-UPLC) with a low-dispersion MALS detector for the characterization of intact monoclonal antibody (mAbs) and their fragments. We confirmed a constant refractive index increment value for mAbs in RP gradients, in good agreement with the values in literature for other classes of proteins. We showed that the impurities eluting from a RP column can often be related to aggregated species and we confirmed that in most cases those oligomers are present also in SEC-MALS. Yet, in few cases small aggregates fractions in RP-UPLC are an artifact. In fact, proteins presenting thermal and physical stability not suitable for the harsh condition applied during the RP separation of mAbs (i.e. organic solvents at high temperature) can aggregate. Further, we applied RP-UPLC-MALS during a long term stability studies. The different principle of separation used in RP-UPLC- MALS provides an additional critical level of protein characterization compared to SEC-MALS and IEX-MALS.

Glycoform-resolved FcɣRIIIa affinity chromatography–mass spectrometry

ABSTRACTDetermination of the impact of individual antibody glycoforms on FcɣRIIIa affinity, and consequently antibody-dependent cell-mediated cytotoxicity (ADCC) previously required high purity glycoengineering. We hyphenated FcɣRIIIa affinity chromatography to mass spectrometry, which allowed direct affinity comparison of glycoforms of intact monoclonal antibodies. The approach enabled reproduction and refinement of known glycosylation effects, and insights on afucosylation pairing as well as on low-abundant, unstudied glycoforms. Our method greatly improves the understanding of individual glycoform structure–function relationships. Thus, it is highly relevant for assessing Fc-glycosylation critical quality attributes related to ADCC.

Assessment of exposure after injection of 99mTc-labeled intact monoclonal antibodies and their fragments into humans.

Human pharmacokinetics and internal radiation dosimetry of normal organs after injection with the 99mTc-labeled monoclonal antibody (intact and fragments) are simulated by the WinAct program and IDAC (Internal Dose Assessment by Computer) software. The WinAct program is used to calculate the cumulative activity in organs and tissues. The calculated cumulative activity is inputted to the IDAC software, an internal dosimetry program for nuclear medicine based on the International Commission on Radiological Protection (ICRP) adult reference voxel phantom, and the absorbed doses by the organs and tissues are estimated. The obtained absorbed doses for the 99mTc-labeled monoclonal antibody (intact and fragments) are compared with the published figures by ICRP-128. The WinAct program method to calculate the cumulative activity is more accurate, as the fraction distribution, Fs, is described and calculated for organs, not only for intake, as in the ICRP model, but also for elimination.

A Middle-Up Approach with Online Capillary Isoelectric Focusing/Mass Spectrometry for In-Depth Characterization of Cetuximab Charge Heterogeneity.

Previously, we reported a new online capillary isoelectric focusing/mass spectrometric (CIEF/MS) method for intact monoclonal antibody (mAb) charge variant analysis that uses an electrokinetically pumped sheath-flow nanospray ion source on a time-of-flight (TOF) MS with a pressure-assisted chemical mobilization. The direct online CIEF/MS method exhibited excellent resolution of charge variants conforming to those of imaged capillary isoelectric focusing with ultraviolet detection (iCIEF/UV). However, for complex mAbs, CIEF/MS spectra of the intact charge variant peaks may be too convoluted to be effectively interpreted. In the present study, we implemented a middle-up approach to enhance the capability of the CIEF/MS method for characterizing complex mAb charge variants by reducing sample complexity. To demonstrate such a strategy, we fragmented cetuximab through IdeS enzymatic cleavage and dithiothreitol (DTT) reduction. For the first time, online CIEF/MS resolved the complex charge variants of cetuximab at subunit level, corroborating the profiles obtained by iCIEF/UV. Furthermore, high-resolution TOF mass spectra with high mass accuracy were obtained for the eight charge variants separated by CIEF/MS after IdeS cleavage and for the 11 charge variants after IdeS digestion with subsequent DTT reduction. In-depth analyses revealed the identities of all charge variants and pinpointed the causes of charge heterogeneity, which are in accord with those reported in the literature. The main sources of charge heterogeneity of cetuximab were identified as terminal lysine on the Fc domain (up to one on each single-chain Fc), glycolylneuraminic acid residues on the Fd' domain (up to two on each Fd'), and likely several deamidation species on the Fd' domain. No charge heterogeneity contribution was found from light chain. The in-depth characterization of complex charge variants for cetuximab demonstrates the remarkable capability of this middle-up CIEF/MS approach. This novel workflow holds great potential for detecting and elucidating charge variants to help understand proteins with complex charge heterogeneity.

Back Cover: Complementary middle-down and intact monoclonal antibody proteoform characterization by capillary zone electrophoresis - mass spectrometry

Back Cover: Complementary middle-down and intact monoclonal antibody proteoform characterization by capillary zone electrophoresis - mass spectrometry