Intact Antibody Research Articles

Dynamics of Intact Immunoglobulin G Explored by Drift‐Tube Ion‐Mobility Mass Spectrometry and Molecular Modeling

AbstractCollision cross‐sections (CCS) of immunoglobulins G1 and G4 have been determined using linear drift‐tube ion‐mobility mass spectrometry. Intact antibodies and Fc‐hinge fragments present with a larger range of CCS than proteins of comparable size. This is rationalized with MD simulations, which indicate significant in vacuo dynamics between linked folded domains. The IgG4 subclass presents over a wider CCS range than the IgG1 subclass.

Dynamics of intact immunoglobulin G explored by drift-tube ion-mobility mass spectrometry and molecular modeling.

Collision cross-sections (CCS) of immunoglobulins G1 and G4 have been determined using linear drift-tube ion-mobility mass spectrometry. Intact antibodies and Fc-hinge fragments present with a larger range of CCS than proteins of comparable size. This is rationalized with MD simulations, which indicate significant in vacuo dynamics between linked folded domains. The IgG4 subclass presents over a wider CCS range than the IgG1 subclass.

Functional expression of a single-chain antibody fragment against human epidermal growth factor receptor 2 (HER2) inEscherichia coli

The human epidermal growth factor receptor (HER) family plays an important role in cell growth and signaling and alteration of its function has been demonstrated in many different kinds of cancer. Receptor dimerization is necessary for the HER signal transduction pathway and tyrosine kinase activity. Recently, several monoclonal antibodies have been developed to directly interfere with ligand-HER receptor binding and receptor dimerization. A single chain variable fragment (ScFv) is a valuable alternative to an intact antibody. This report describes the production and purification of an ScFv specific for domain II of the HER2 receptor in Escherichia coli BL21 (DE3) cytoplasm. The majority of expressed of anti-her2his-ScFv protein was produced as inclusion bodies. A Ni-NTA affinity column was used to purify the anti-her2his-ScFv protein. The molecular weight of anti-her2his-ScFv protein was estimated to be approximately 27 kDa, as confirmed by SDS-PAGE and Western blotting assay. The anti-her2his-ScFv showed near 95 % purity and reached a yield of approximately 29 mg/l in flask fermentation. The purified anti-her2his-ScFv showed its biological activity by binding to HER2 receptor on the surface of BT-474 cells. This ScFv may be a potential pharmaceutical candidate for targeting tumour cells overexpressing HER2 receptor.

Peripheral blood monocyte-derived chemokine blockade prevents murine transfusion-related acute lung injury (TRALI)

AbstractTransfusion-related acute lung injury (TRALI) is the leading cause of transfusion-related mortality and can occur with any type of transfusion. TRALI is thought to be primarily mediated by donor antibodies activating recipient neutrophils resulting in pulmonary endothelial damage. Nonetheless, details regarding the interactions between donor antibodies and recipient factors are unknown. A murine antibody-mediated TRALI model was used to elucidate the roles of the F(ab′)2 and Fc regions of a TRALI-inducing immunoglobulin G anti–major histocompatibility complex (MHC) class I antibody (34.1.2s). Compared with intact antibody, F(ab′)2 fragments significantly increased serum levels of the neutrophil chemoattractant macrophage inflammatory protein 2 (MIP-2); however, pulmonary neutrophil levels were only moderately increased, and no pulmonary edema or mortality occurred. Fc fragments did not modulate any of these parameters. TRALI induction by intact antibody was completely abrogated by in vivo peripheral blood monocyte depletion by gadolinium chloride (GdCl3) or chemokine blockade with a MIP-2 receptor antagonist but was restored upon repletion with purified monocytes. The results suggest a two-step process for antibody-mediated TRALI induction: the first step involves antibody binding its cognate antigen on blood monocytes, which generates MIP-2 chemokine production that is correlated with pulmonary neutrophil recruitment; the second step occurs when antibody-coated monocytes increase Fc-dependent lung damage.

One siRNA pool targeting the λ constant region stops λ light-chain production and causes terminal endoplasmic reticulum stress

AbstractIn systemic light-chain amyloidosis, λ light chains produced by clonal plasma cells cause organ damage and early death. In pursuit of novel therapy, we developed 1 pool of short interfering RNA (siRNA) targeting the constant region of λ light chains that substantially and promptly reduces λ-light-chain production and secretion by human plasma cells regardless of sequence diversity. In clones producing intact immunoglobulin G (IgG) λ antibodies (containing paired heavy and light chains), the secretion of intact antibodies is reduced, and all 3 branches of the unfolded protein response are activated by accumulation of unpaired IgG heavy chains in the endoplasmic reticulum (ER). Moreover, an ER stress response can then become terminal with effector caspase activity mediated in part by the transcription of the Bcl-2 homology 3 domain only family member NOXA. This pool of siRNA can be used to reduce pathological λ-light-chain production and cause apoptosis in human plasma cells making intact IgGλ antibodies.

SU‐E‐T‐45: Antibody Mean Residence Time in Blood and Its Correlation with Protein Molecular Weight

Purpose:Animal biodistribution data are required prior to introducing a new radiopharmaceutical into clinical trials. Protein engineering, using recombinant DNA techniques can produce a large number of related (cognate) antibodies to a given molecular target. Thus, it is important that these constructs be numerically related to one another via a single criterion. In the following, we use the mean residence time (MRT) in murine blood as this criterion.Methods:Five cognate anti‐CEA (Carcinoembryonic Antigen) antibodies were compared with regard to their MRT in whole blood of CEA‐positive tumor‐bearing (LS174T) mice. MRT was defined by blood AUC (area under the curve) divided by the initial blood uptake value; all in units of percent injected dose per gram (%ID/g). Cognates included single chain scFv (25 kDa), diabody (50 kDa), minibody (80 kDa), F(ab')2 (120 kDa), and intact (155 kDa) forms of the murine cT84.66 antibody against CEA. All were labeled with radioactive iodine.Results:The agents, in the sequence listed, exhibited MRT values of 1.16 +/‐ 0.01 h, 0.99 h, 5.06 +/‐ 0.70 h, 6.61 +/‐ 0.36 h, and 59.3 +/‐ 2.4 h respectively. Because of the monotonic nature of the sequence, a linear correlation analysis was performed between molecular weight (MW) and MRT or ln(MRT) of the 5 proteins. Probability of random correlation was 0.10 for MRT and 0.01 for ln(MRT).Conclusion:MRT values of cognate anti‐CEA antibodies were found to be a monotonically increasing sequence with respect to MW. Cognate MW values correlated best to ln(MRT) of the protein species. Thus MRT was proportional to an exponential function of molecular weight. The extended intact antibody circulation time presumably reflected its relatively maximal MW. Presence of an intact FC segment on this native antibody may also have influenced these results.

Structural analysis of an intact monoclonal antibody by online electrochemical reduction of disulfide bonds and Fourier transform ion cyclotron resonance mass spectrometry.

Structural confirmation and quality control of recombinant monoclonal antibodies (mAbs) by top-down mass spectrometry is still challenging due to the size of the proteins, disulfide content, and post-translational modifications such as glycosylation. In this study we have applied electrochemistry (EC) to overcome disulfide bridge complexity in top-down analysis of mAbs. To this end, an electrochemical cell was coupled directly to an electrospray ionization (ESI) source and a Fourier transform ion cyclotron resonance (FTICR) mass spectrometer (MS) equipped with a 15 T magnet. By performing online EC-assisted reduction of interchain disulfide bonds in an intact mAb, the released light chains could be selected for tandem mass spectrometry (MS/MS) analysis without interference from heavy-chain fragments. Moreover, the acquisition of full MS scans under denaturing conditions allowed profiling of all abundant mAb glycoforms. Ultrahigh-resolution FTICR-MS measurements provided fully resolved isotopic distributions of intact mAb and enabled the identification of the most abundant adducts and other interfering species. Furthermore, it was found that reduction of interchain disulfide bonds occurs in the ESI source dependent on capillary voltage and solvent composition. This phenomenon was systematically evaluated and compared with the results obtained from reduction in the electrochemical cell.

Noninvasive Imaging of PSMA in prostate tumors with (89)Zr-Labeled huJ591 engineered antibody fragments: the faster alternatives.

Engineered antibody fragments offer faster delivery with retained tumor specificity and rapid clearance from nontumor tissues. Here, we demonstrate that positron emission tomography (PET) based detection of prostate specific membrane antigen (PSMA) in prostatic tumor models using engineered bivalent antibodies built on single chain fragments (scFv) derived from the intact antibody, huJ591, offers similar tumor delineating properties but with the advantage of rapid targeting and imaging. 89Zr-radiolabeled huJ591 scFv (dimeric scFv-CH3; 89Zr-Mb) and cysteine diabodies (dimeric scFv; 89Zr-Cys-Db) demonstrated internalization and similar Kds (∼2 nM) compared to 89Zr-huJ591 in PSMA(+) cells. Tissue distribution assays established the specificities of both 89Zr-Mb and 89Zr-Cys-Db for PSMA(+) xenografts (6.2 ± 2.5% ID/g and 10.2 ± 3.4% ID/g at 12 h p.i. respectively), while minimal accumulation in PSMA(−) tumors was observed. From the PET images, 89Zr-Mb and 89Zr-Cys-Db exhibited faster blood clearance than the parent huJ591 while tumor-to-muscle ratios for all probes show comparable values across all time points. Ex vivo autoradiography and histology assessed the distribution of the probes within the tumor. Imaging PSMA-expressing prostate tumors with smaller antibody fragments offers rapid tumor accumulation and accelerated clearance; hence, shortened wait periods between tracer administration and high-contrast tumor imaging and lower dose-related toxicity are potentially realized.

Highly Sensitive Nanomechanical Immunosensor Using Half Antibody Fragments

The improvement of sensitivity is of great significance to the application of biochemical sensor. In this study, we propose a micocantilever-based immunosensor in surface stress mode using half antibody fragments as receptor molecules. The thiol-containing half antibody fragment was obtained with a low loss of antibody biological activity and then was covalently and orientedly immobilized on the gold surface of microcantilevers via two native thiol groups. Such a one-step reaction and immobilization of receptor molecule simplify the preparation process of micocantilever immunosensor. Using shortened and highly oriented half antibody fragments as receptor molecules, the generation of surface stress and the transmission of stress from the interaction region of molecules to the surface of the microcantilever have been elevated significantly. The limit of detection (LOD) of the presented sensor has been significantly lowered to 1 pg/mL, or 1.1 pM in equivalence, which is a 500-fold improvement when compared with intact full antibody coated conventional micocantilever sensors. The results indicate that the half antibody fragment is well suited for the functionalization of the microcantilever surface and is generally applicable to all microcantilever immunosensor development, and this principle will help to design a functional film of devices with significantly lower LOD.

A Mutated Anti-CA19-9 scFv-Fc for Positron Emission Tomography of Human Pancreatic Cancer Xenografts

Intact antibodies have a long serum persistence resulting in high background signal that inhibits their direct translation as imaging agents. Engineering of antibody fragments through the introduction of mutations in the fragment crystallizable (Fc) region can dramatically reduce serum persistence. We sought to develop a Fc-mutated, anti-CA19-9 antibody fragment (anti-CA 19-9 scFv-Fc H310A) to provide micro-positron emission tomography (microPET) imaging of pancreatic cancer xenografts. The anti-CA19-9 scFv-Fc H310A was successfully expressed and purified. Biochemical characterization included size exclusion chromatography, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western blot, and flow cytometry. The antibody fragment was labeled with iodine-124 ((124)I) and injected into mice containing human pancreatic cancer xenografts. MicroPET/CT images were then obtained. Blood, organ, and tumor radioactivity was measured and expressed as the percent of injected dose per gram of tissue (%ID/g). Biochemical characterization was consistent with the creation of a 105kD dimer containing a human Fc region. Flow cytometry demonstrated antigen-specific binding, and cell-based ELISA further established a dissociation constant (K D) of 10.7nM. (124)I-labeled scFv-Fc H310A localized to the antigen-positive tumor xenografts as detected by microPET. Objective confirmation of targeting was demonstrated by higher %ID/g in the antigen-positive tumor compared to the blood, antigen-negative tumor, and liver. We successfully engineered and produced an anti-CA19-9 scFv-Fc H310A antibody fragment that retains similar affinity when compared to the parental intact murine antibody. Additionally, our engineered and mutated fragment exhibited antigen-specific microPET imaging of both subcutaneous and orthotopic pancreatic cancer xenografts at early time points secondary to decreased serum half-life.

Endo F3 glycosynthase mutant enables chemoenzymatic synthesis of core fucosylated triantennary complex type glycopeptides and glycoproteins (1007.7)

Chemoenzymatic synthesis of glycoproteins, which enables the production of glycoproteins with homogenous glycans, is an essential tool for structural and functional studies of glycoproteins. The endo‐β‐N‐acetylglucosaminidase from Elizabethkingia meningosepticum, Endo F3, is an endoglycosidase that can hydrolyze complex type N‐glycans and also possess transglycosylation activity. We have generated glycosynthase mutants by site‐directed mutagenesis and have found that the glycosynthases were capable of efficiently transferring complex type N‐glycans to GlcNAc‐peptide/protein without product hydrolysis. Our experimental data have further shown that the Endo‐F3 glycosynthases are able to use tri‐antennary glycan oxazolines as substrates for transglycosylation, which were unable to achieve by previously reported endoglycosidase mutants derived from Endo‐A, Endo‐M, Endo‐D and Endo‐S. The usefulness of the Endo‐F3 mutants was demonstrated by the synthesis of CD52 antigen and by the efficient Fc glycosylation remodeling of monoclonal antibody rituximab. The binding of these novel glycoforms of rituximab to FcγRIIIa and galectin‐3 is being analyzed. This chemoenzymatic approach permits an efficient synthesis of core fucosylated bi‐ and tri‐antennary glycopeptides and the glycosylation remodeling of intact monoclonal antibodies, making it possible to probe the structural and functional roles of the attached glycans in more details.Grant Funding Source: Supported by NIH R01 GM080374

Measurement of in Vivo Drug Load Distribution of Cysteine-Linked Antibody–Drug Conjugates Using Microscale Liquid Chromatography Mass Spectrometry

Analysis of samples containing intact antibody-drug conjugates (ADC) using mass spectrometry provides a direct measurement of the drug-load distribution. Once dosed, the drug load distribution changes due to a combination of biological and chemical factors. Liquid chromatography-mass spectrometry (LC-MS) methods to measure the in vivo drug load distribution have been established for ADCs containing native disulfide bonds (lysine-linked or cysteine-linked). However, because of an IgG reduction step in conjugation processes, using LC-MS to analyze intact cysteine-linked ADCs requires native conditions, thus limiting sensitivity. While this limitation has been overcome at the analytical scale, to date, these methods have not been translated to a smaller scale that is required for animal or clinical doses/sampling. In this manuscript, we describe the development of ADC specific affinity capture reagents for processing in vivo samples and optimization of native LC-MS methods at a microscale. These methods are then used to detect the changing drug load distribution over time from a set of in vivo samples, representing to our knowledge the first native mass spectra of cysteine-linked ADCs from an in vivo source.

Intratracheal exposure to Fab fragments of an allergen‐specific monoclonal antibody regulates asthmatic responses in mice

Fab fragments (Fabs) maintain the ability to bind to specific antigens but lack effector functions due to the absence of the Fc portion. In the present study, we tested whether Fabs of an allergen-specific monoclonal antibody (mAb) were able to regulate asthmatic responses in mice. Asthmatic responses were induced in BALB/c mice by passive sensitization with anti-ovalbumin (OVA) polyclonal antibodies (pAbs) (day 0) and by active sensitization with OVA (days 0 and 14), followed by intratracheal (i.t.) challenge with OVA on day 1 and days 28, 29, 30 and 35. Fabs prepared by the digestion of an anti-OVA IgG1 (O1-10) mAb with papain were i.t. administered only once 30 min before antigenic challenge on day 1 or day 35. The results showed that i.t. administration of O1-10 Fabs with OVA markedly suppressed the early and/or late phases of asthmatic responses caused by passive and active sensitization. Similar results were obtained when Fabs of anti-OVA IgG2b mAb (O2B-3) were i.t. administered. In contrast, neither i.t. injection of intact 01-10/O2B-3 nor systemic injection of O1-10 Fabs suppressed the asthmatic responses. In vitro studies revealed that the capture of OVA by O1-10 Fabs prevented the subsequent binding of intact anti-OVA pAbs to the captured OVA. These results suggest that asthmatic responses may be down-regulated by the i.t. exposure to Fabs of an allergen-specific mAb via a mechanism involving the capture of allergen by Fabs in the respiratory tract before the interaction of intact antibody and allergen essential for the induction of asthmatic responses.

Ion-pair reversed-phase high performance liquid chromatography method for the quantification of isoaspartic acid in a monoclonal antibody

Isomerization of aspartic acid residues is one of the major causes of chemical degradation during the shelf life of biological pharmaceuticals. Monoclonal antibody biopharmaceuticals are typically stored at mildly acidic pH conditions, which can lead to the isomerization reaction. The mechanism of this non-enzymatic chemical reaction has been studied in great detail. However, the identification and quantification of the isomerization sites in a given protein still remains a challenge. We developed an ion-pair reversed-phase HPLC method for the separation of an intact monoclonal antibody variant containing a single isoaspartic acid residue from its native counterpart. We identified and characterized the isomerization site using ion-pair reversed-phase HPLC mass spectrometry methods of the reduced and alkylated antibody and the enzymatically cleaved antibody. Lys-C followed by Asp-N digestion of the antibody was used for the identification of the isomerization site. Electron transfer dissociation (ETD) mass spectrometry was used to confirm the isomerization site at a DY motif at an aspartic acid residue in the CDR-H3 region of the antibody. Tyrosine at the C-terminus of an aspartic acid residue is typically not regarded as a hot spot for isomerization. Our findings suggest that it is not possible to predict isomerization sites in proteins with confidence and all aspartic acid residues located in the CDR regions of antibodies must be considered as potential isomerization site due to the solvent exposure or the flexibility of these regions of the molecule. Additionally, the effect of the pH on the isomerization rate was evaluated using the ion-pair reversed-phase HPLC method, showing that at a lower pH the isomerization rate is faster. Storage at 25°C for 6 months resulted in an increase of the amount of isoaspartic acid to 6.6% at pH 5.4, 6.0% at pH 5.8, and 5.6% at pH 6.2.

Numerical Comparison of Iodine-Based and Indium-Based Antibody Biodistributions

Single-step iodination is often performed in preference to use of a radiometal label for initial animal biodistributions. Yet loss of iodine occurs in vivo so that it is important to measure uptake (%ID/g) differences between radiolabels. Murine biodistributions of four radioiodinated and (111)In-labeled cognate anti-carcinoembryonic antigen antibodies were compared. Uptakes were obtained in athymic mice out to 96 hours for diabody, minibody, scFv-Fc, and intact humanized (M5A) versions of the T84.66 antibody. Tissues included liver, spleen, kidneys, lungs, and human LS174T colorectal xenografts. Ratios (R) of iodine uptake to indium uptake were calculated. For all cognates, no significant differences were found in the blood uptakes between the two labels. In normal solid organs, iodine was generally cleared more rapidly with decreasing molecular weight (MW). In liver and spleen by 24 hours, the R value was 5% for diabody and minibody, whereas a value of 50% was seen for the intact mAb. Renal differences were even more marked for the two lower MW species. Tumor losses, however, were found to be essentially independent of MW and were modest; 50% by 48 hours. To test the generality of the results, comparisons were then made for normal organs and tumors of the R values found above for M5A and MN-14 intact antibody literature results. Good agreement, within estimated errors in R, was seen between these two cases, although (111)In and (88)Y were the respective radiometals. Loss of iodine from labeled antibodies can be marked in normal organs and that correction (by 1/R) of the iodine biodistribution to estimate the associated radiometal result may be possible. Explicit differences between the two types of biodistribution also imply that separate uptake results need to be considered when evaluating the impact on imaging and therapy using various possible radiolabels.

Suppression of Collagen-Induced Arthritis in Mice by Anti-Collagen Antibody Fab Fragments

Fabs fragments (Fabs) maintain binding ability to specific antigens, but lack the binding site for complements and for receptors on leukocytes that play a crucial role in Rheumatoid Arthritis (RA). In the present study, we investigated whether type II Collagen (CII)-Induced Arthritis (CIA) in mice was suppressed by anti-CII Fabs prepared by papain digestion of anti-CII antibodies. CIA was induced in DBA/1J mice by immunization with chicken CII and completes Freund’s adjuvant. To investigate the effect of anti-CII Fabs on the CIA model, mice were injected intraperitoneally with anti-CII Fabs 1 day before the first immunization. As a result, CIA was markedly inhibited by anti-CII Fabs; furthermore, the histological features of anti-CII Fabs-untreated mice included severe hyperplatic synnovium, cartilage and joint destruction, and leukocytic infiltration, whereas animals given anti-CII Fabs showed significant reduction of these histological changes. Additionally, antigen-specific suppression of CIA by anti-CII Fabs was related to the reduced levels of complement C3a in serum. Meanwhile, in vitro studies revealed that anti-CII Fabs significantly blocked the binding of intact anti-CII antibodies and also decreased complement activation. Collectively, the development of CIA was suppressed by anti-CII Fabs, which prevented the binding of anti-CII antibodies to antigen followed by the inhibition of complement activation, suggesting that autoimmune arthritic diseases such as RA may be specifically treated with pathogenic antibody Fabs.

Relationships between nitrate uptake and nitrate reductase activity in Cucumis sativus L.

Anti-NR IgG fragments obtained after papain digestion of polyclonal antibodies gave the positive immunological reaction with both, a soluble and plasma membrane-bound nitrate reductase. Anti-NR antibody as well as IgG fragments almost totally inhibited the nitrate reductase activity in cytosol proving a crossreaction of antibody with the catalytic site of a soluble NR. Anti-NR IgG fragments, but not undigested polyclonal antibodies affected the activity of the nitrate reductase associated with plasma membranes. Discrepancy in the action of intact antibodies and fragments obtained after they digestion were interpreted as a consequence of same differences in the ability of those molecules to the penetration through the membrane. Undigested anti-NR antibody have no effect on the nitrate uptake by intact plants, as well as by the right-side plasma membrane vesicles. On the other hand, IgG fragments of polyclonal antibodies abolished almost totally the nitrate uptake in the case of intact seedlings, but have only slight effect on the N03 uptake in plasma membranes. On the basis of above findings, some relations between nitrate uptake and its assimilation inside the cell are suggested. Since IgG fragments only slightly changed the N03 absorption in vesicles whereas the activity of plasmalemma associated nitrate reductase was strongly repressed, we concluded that the PM-NR is not structurally involved in the nitrate transport through the membrane.

Accumulation of secreted antibodies in plant cell cultures varies according to the isotype, host species and culture conditions

Nicotiana tabacum suspension cells have been widely used to produce monoclonal antibodies, but the yield of secreted antibodies is usually low probably because of proteolytic degradation. Most IgGs that have been expressed in suspension cells have been of the human IgG1 isotype. In this study, we examined whether other isotypes displayed the same sensitivity to proteolytic degradation and whether the choice of plant host species mattered. Human serum IgG displayed different degradation profiles when incubated in spent culture medium from N. tabacum, Nicotiana benthamiana or Arabidopsis thaliana suspension cells. Zymography showed that the protease profile was host species dependent. Three human isotypes, IgG1, IgG2 and IgG4, and a mouse IgG2a were provided with the same heavy- and light-chain variable regions from an anti-human IgM antibody and expressed in N. tabacum cv. BY-2 and A. thaliana cv. Col-0 cells. Although all tested isotypes were detected in the extracellular medium using SDS-PAGE and a functional ELISA, up to 10-fold differences in the level of intact antibody were found according to the isotype expressed, to the host species and to the culture conditions. In the best combination (BY-2 cells secreting human IgG1), we reported accumulation of more than 30 mg/L of intact antibody in culture medium. The possibility of using IgG constant regions as a scaffold to allow stable accumulation of antibodies with different variable regions was demonstrated for human IgG2 and mouse IgG2a.

Complex mixtures of antibodies generated from a single production qualitatively and quantitatively evaluated by native Orbitrap mass spectrometry

Composite antibody mixtures designed to combat diseases present a new, rapidly emerging technology in the field of biopharmaceuticals. The combination of multiple antibodies can lead to increased effector response and limit the effect of escape variants that can propagate the disease. However, parallel development of analytical technologies is required to provide fast, thorough, accurate, and robust characterization of these mixtures. Here, we evaluate the utility of native mass spectrometry on an Orbitrap platform with high mass resolving power to characterize composite mixtures of up to 15 separate antibodies. With this technique, unambiguous identification of each antibody in the mixtures was achieved. Mass measurements of the intact antibodies varied 7 ppm on average, allowing highly reproducible identification and quantitation of each compound in these complex mixtures. We show that with the high mass-resolving power and robustness of this technology, high-resolution native mass spectrometry can be used efficiently even for batch-to-batch characterization.

In-depth qualitative and quantitative analysis of composite glycosylation profiles and other micro-heterogeneity on intact monoclonal antibodies by high-resolution native mass spectrometry using a modified Orbitrap

Here, we describe a fast, easy-to-use, and sensitive method to profile in-depth structural micro-heterogeneity, including intricate N-glycosylation profiles, of monoclonal antibodies at the native intact protein level by means of mass spectrometry using a recently introduced modified Orbitrap Exactive Plus mass spectrometer. We demonstrate the versatility of our method to probe structural micro-heterogeneity by describing the analysis of three types of molecules: (1) a non-covalently bound IgG4 hinge deleted full-antibody in equilibrium with its half-antibody, (2) IgG4 mutants exhibiting highly complex glycosylation profiles, and (3) antibody-drug conjugates. Using the modified instrument, we obtain baseline separation and accurate mass determination of all different proteoforms that may be induced, for example, by glycosylation, drug loading and partial peptide backbone-truncation. We show that our method can handle highly complex glycosylation profiles, identifying more than 20 different glycoforms per monoclonal antibody preparation and more than 30 proteoforms on a single highly purified antibody. In analyzing antibody-drug conjugates, our method also easily identifies and quantifies more than 15 structurally different proteoforms that may result from the collective differences in drug loading and glycosylation. The method presented here will aid in the comprehensive analytical and functional characterization of protein micro-heterogeneity, which is crucial for successful development and manufacturing of therapeutic antibodies