Insights into the Conformation and Self-Association of a Concentrated Monoclonal Antibody using Isothermal Chemical Denaturation and Nuclear Magnetic Resonance.

The purpose of this investigation was to highlight the utility of nuclear magnetic resonance (NMR) as a multi-attribute method for the characterization of therapeutic antibodies. In this case study, we compared results from isothermal chemical denaturation (ICD) and NMR with standard methods to relate conformational states of a model monoclonal antibody (mAb1) with protein-protein interactions (PPI) that lead to self - association in concentrated solutions. The increase in aggregation rate and relative viscosity for mAb1 was found to be both concentration and pH dependent. The free energy of unfolding (∆G°) from ICD and thermal analysis in dilute solutions indicated that although the native state predominated between pH 4 - pH 7, it was disrupted at the CH2 and unfolded noncooperatively under acidic conditions. One-dimensional (1D) 1H NMR and two-dimensional (2D) 13C-1H NMR performed, in concentrated solutions, confirmed that PPI between pH 4-7 occurred while mAb1 was in the native state. NMR corroborated that mAb1 maintained a dominant native state at formulation-relevant conditions at the tested pH range, had increased global molecular tumbling dynamics at lower pH and confirmed increased PPI at higher pH conditions. This report aligns and compares typical characterization of an IgG1 with assessment of structure by NMR and provided a more precise assessment and deeper insight into the conformation of an IgG1 in concentrated solutions.

Higher order structures of Adalimumab, Infliximab and their complexes with TNFα revealed by electron microscopy.

Adalimumab and Infliximab are recombinant IgG1 monoclonal antibodies (mAbs) that bind and neutralize human tumor necrosis factor alpha (TNFα). TNFα forms a stable homotrimer with unique surface-exposed sites for Adalimumab, Infliximab, and TNF receptor binding. Here, we report the structures of Adalimumab-TNFα and Infliximab-TNFα complexes modeled from negative stain EM and cryo-EM images. EM images reveal complex structures consisting of 1:1, 1:2, 2:2, and 3:2 complexes of Adalimumab-TNFα and Infliximab-TNFα. The 2:2 complex structures of Adalimumab-TNFα and Infliximab-TNFα show diamond-shaped profiles and the 2D class averages reveal distinct orientations of the Fab domains, indicating different binding modes by Adalimumab and Infliximab to TNFα. After separation by size exclusion chromatography and analysis by negative stain EM, the 3:2 complexes of Adalimumab-TNFα or Infliximab-TNFα complexes are more complicated but retain features recognized in the 2:2 complexes. Preliminary cryo-EM analysis of 3:2 Adalimumab-TNFα complex generated a low-resolution density consistent with a TNFα trimer bound with three Fab domains from three individual antibody molecules, while each antibody molecule binds to two molecules of TNFα trimer. The Fc domains are not visible in the reconstruction. These results show the two mAbs form structurally distinct complexes with TNFα.

When Good Intentions Go Awry: Modification of a Recombinant Monoclonal Antibody in Chemically Defined Cell Culture by Xylosone, an Oxidative Product of Ascorbic Acid.

With the advent of new initiatives to develop chemically defined media, cell culture scientists screen many additives to improve cell growth and productivity. However, the introduction or increase of supplements, typically considered beneficial or protective on their own, to the basal media or feed stream may cause unexpected detrimental consequences to product quality. For instance, because cultured cells are constantly under oxidative stress, ascorbic acid (vitamin C, a potent natural reducing agent) is a common additive to cell culture media. However, as reported herein, a recombinant monoclonal antibody (adalimumab) in cell culture was covalently modified by xylosone (molecular weight 148), an oxidative product of ascorbate. Containing reactive carbonyl groups, xylosone modifies various amines (e.g., the N-termini of the heavy and light chains and susceptible lysines), forming either hemiaminal (+148 Da) or Schiff base (imine, +130 Da) products. Our findings show, for the first time, that ascorbate-derived xylosone can contribute to an increase in molecular heterogeneity, such as acidic species. Our work serves as a reminder that additives to cell culture and their metabolites may become reactive and negatively impact the overall product quality and should be carefully monitored with any changes in cell culture conditions.

Discovery of a chemical modification by citric acid in a recombinant monoclonal antibody.

Recombinant therapeutic monoclonal antibodies exhibit a high degree of heterogeneity that can arise from various post-translational modifications. The formulation for a protein product is to maintain a specific pH and to minimize further modifications. Generally Recognized as Safe (GRAS), citric acid is commonly used for formulation to maintain a pH at a range between 3 and 6 and is generally considered chemically inert. However, as we reported herein, citric acid covalently modified a recombinant monoclonal antibody (IgG1) in a phosphate/citrate-buffered formulation at pH 5.2 and led to the formation of so-called "acidic species" that showed mass increases of 174 and 156 Da, respectively. Peptide mapping revealed that the modification occurred at the N-terminus of the light chain. Three additional antibodies also showed the same modification but displayed different susceptibilities of the N-termini of the light chain, heavy chain, or both. Thus, ostensibly unreactive excipients under certain conditions may increase heterogeneity and acidic species in formulated recombinant monoclonal antibodies. By analogy, other molecules (e.g., succinic acid) with two or more carboxylic acid groups and capable of forming an anhydride may exhibit similar reactivities. Altogether, our findings again reminded us that it is prudent to consider formulations as a potential source for chemical modifications and product heterogeneity.

Arginine modifications by methylglyoxal: discovery in a recombinant monoclonal antibody and contribution to acidic species.

Heterogeneity is common among protein therapeutics. For example, the so-called acidic species (charge variants) are typically observed when recombinant monoclonal antibodies (mAbs) are analyzed by weak-cation exchange chromatography (WCX). Several protein post-translational modifications have been established as contributors but still cannot completely account for all heterogeneity. As reported herein, an unexpected modification by methylglyoxal (MGO) was identified, for the first time, in a recombinant monoclonal antibody expressed in Chinese hamster ovary (CHO) cells. Modifications of arginine residues by methylglyoxal lead to two adducts (dihydroxyimidazolidine and hydroimidazolone) with increases of molecular weights of 72 and 54 Da, respectively. In addition, the modification by methylglyoxal causes the antibody to elute earlier in the weak cation exchange chromatogram. Consequently, the extent to which an antibody was modified at multiple sites corresponds to the degree of shift in elution time. Furthermore, cell culture parameters also affected the extent of modifications by methylglyoxal, a highly reactive metabolite that can be generated from glucose or lipids or other metabolic pathways. Our findings again highlight the impact that cell culture conditions can have on the product quality of recombinant protein pharmaceuticals.

Comparison of the in vitro and in vivo stability of a succinimide intermediate observed on a therapeutic IgG1 molecule.

Deamidation of asparagine residues, a post-translational modification observed in proteins, is a common degradation pathway in monoclonal antibodies (mAbs). The kinetics of deamidation is influenced by primary sequence as well as secondary and tertiary folding. Analytical hydrophobic interaction chromatography (HIC) is used to evaluate hydrophobicity of candidate mAbs and uncover post-translational modifications. Using HIC, we discovered atypical heterogeneity in a highly hydrophobic molecule (mAb-1). Characterization of the different HIC fractions using LC/MS/MS revealed a stable succinimide intermediate species localized to an asparagine-glycine motif in the heavy chain binding region. The succinimide intermediate was stable in vitro at pH 7 and below and increased on storage at 25°C and 40°C. Biacore evaluation showed a decrease in binding affinity of the succinimide intermediate compared with the native asparagine molecule. In vivo studies of mAb-1 recovered from a pharmacokinetic study in cynomolgus monkeys revealed an unstable succinimide species and rapid conversion to aspartic/iso-aspartic acid. Mutation from asparagine to aspartic acid led to little loss in affinity. This study illustrates the importance of evaluating modifications of therapeutic mAbs both in vitro and in serum, the intended environment of the molecule. Potential mechanisms that stabilize the succinimide intermediate in vitro are discussed.

The structure of dual-variable-domain immunoglobulin molecules alone and bound to antigen.

A dual-specific, tetravalent immunoglobulin G-like molecule, termed dual variable domain immunoglobulin (DVD-Ig™), is engineered to block two targets. Flexibility modulates Fc receptor and complement binding, but could result in undesirable cross-linking of surface antigens and downstream signaling. Understanding the flexibility of parental mAbs is important for designing and retaining functionality of DVD-Ig™ molecules. The architecture and dynamics of a DVD-Ig™ molecule and its parental mAbs was examined using single particle electron microscopy. Hinge angles measured for the DVD-Ig™ molecule were similar to the inner antigen parental mAb. The outer binding domain of the DVD-Ig™ molecule was highly mobile and three-dimensional (3D) analysis showed binding of inner antigen caused the outer domain to fold out of the plane with a major morphological change. Docking high-resolution X-ray structures into the 3D electron microscopy map supports the extraordinary domain flexibility observed in the DVD-Ig™ molecule allowing antigen binding with minimal steric hindrance.

A high throughput capillary electrophoresis method to obtain pharmacokinetics and quality attributes of a therapeutic molecule in circulation.

Therapeutic proteins circulating in blood are in a highly crowded, redox environment at high temperatures of ~37°C. These molecules circulate in the presence of enzymes and other serum proteins making it difficult to predict from in vitro studies the stability, aggregation or pharmacokinetics of a therapeutic protein in vivo. Here, we describe use of a high throughput capillary electrophoresis based microfluidic device (LabChip GXII) to obtain pharmacokinetics (PK) of a fluorescently labeled human mAb directly from serum. The non-labeled and labeled mAbs were evaluated in single dose rat PK studies using a traditional ELISA method or LabChip GXII, respectively. The fluorescent dye did not significantly alter clearance of this particular mAb, and PK parameters were comparable for labeled and unlabeled molecules. Further, from the CE profile we concluded that the mAb was resistant to fragmentation or aggregation during circulation. In a follow-up experiment, dimers were generated from the mAb using photo-induced cross-linking of unmodified proteins (PICUP) and labeled with the same fluorophore. The extent of dimerization was incomplete and some monomer and higher molecular weight species were found in the preparation. In rat PK studies, the serum concentration-time profile of the three entities present in the dimer preparation could be followed simultaneously with the GXII technology. While further studies are warranted, we believe this method could be adapted to obtain PK of different forms of antibodies (oxidized, deamidated or various glycosylated species) and other proteins.

Increased serum clearance of oligomannose species present on a human IgG1 molecule.

The role of Fc glycans on clearance of IgG molecule has been examined by various groups in experiments where specific glycans have been enriched or the entire spectrum of glycans was studied after administration in pre-clinical or clinical pharmacokinetic (PK) studies. The overall conclusions from these studies are inconsistent, which may result from differences in antibody structure or experimental design. In the present study a well-characterized recombinant monoclonal IgG1 molecule (mAb-1) was analyzed from serum samples obtained from a human PK study. mAb-1 was recovered from serum using its ligand cross-linked to Sepharose beads. The overall purity and recovery of all isoforms were carefully evaluated using a variety of methods. Glycans were then enzymatically cleaved, labeled using 2-aminobenzamide and analyzed by normal phase high performance liquid chromatography. The assays for recovering mAb-1 from serum and subsequent glycan analysis were rigorously qualified at a lower limit of quantitation of 15 µg/mL, thus permitting analysis to day 14 of the clinical PK study. Eight glycans were monitored and classified into two groups: (1) the oligomannose type structures (M5, M6 and M7) and (2) fucosylated biantennary oligosaccharides (FBO) structures (NGA2F, NA1F, NA2F, NA1F-GlcNAc and NGA2F-GlcNAc). We observed that the oligomannose species were cleared at a much faster rate (40%) than FBOs and conclude that high mannose species should be carefully monitored and controlled as they may affect PK of the therapeutic; they should thus be considered an important quality attribute. These observations were only possible through the application of rigorous analytical methods that we believe will need to be employed when comparing innovator and biosimilar molecules.

Comparability analysis of protein therapeutics by bottom-up LC-MS with stable isotope-tagged reference standards.

Comparability studies lie at the heart of assessments that evaluate differences amongst manufacturing processes and stability studies of protein therapeutics. Low resolution chromatographic and electrophoretic methods facilitate quantitation, but do not always yield detailed insight into the effect of the manufacturing change or environmental stress. Conversely, mass spectrometry (MS) can provide high resolution information on the molecule, but conventional methods are not very quantitative. This gap can be reconciled by use of a stable isotope-tagged reference standard (SITRS), a version of the analyte protein that is uniformly labeled (13)C6-arginine and (13)C6-lysine. The SITRS serves as an internal control that is trypsin-digested and analyzed by liquid chromatography (LC)-MS with the analyte sample. The ratio of the ion intensities of each unlabeled and labeled peptide pair is then compared to that of other sample(s). A comparison of these ratios provides a readily accessible way to spot even minute differences among samples. In a study of a monoclonal antibody (mAb) spiked with varying amounts of the same antibody bearing point mutations, peptides containing the mutations were readily identified and quantified at concentrations as low as 2% relative to unmodified peptides. The method is robust, reproducible and produced a linear response for every peptide that was monitored. The method was also successfully used to distinguish between two batches of a mAb that were produced in two different cell lines while two batches produced from the same cell line were found to be highly comparable. Finally, the use of the SITRS method in the comparison of two stressed mAb samples enabled the identification of sites susceptible to deamidation and oxidation, as well as their quantitation. The experimental results indicate that use of a SITRS in a peptide mapping experiment with MS detection enables sensitive and quantitative comparability studies of proteins at high resolution.

Effect of the light chain C-terminal serine residue on disulfide bond susceptibility of human immunoglobulin G1λ.

The light chain cysteine residue that forms an interchain disulfide bond with the cysteine residue in the heavy chain in IgG1κ is the last amino acid. The cysteine residue is followed by a serine residue in IgG1λ. Effect of the serine residue on the susceptibility of disulfide bonds to reduction was investigated in the current study using a method including reduction, differential alkylation using iodoacetic acid with either natural isotopes or enriched with carbon-13, and mass spectrometry analysis. This newly developed method allowed an accurate determination of the susceptibility of disulfide bonds in IgG antibodies. The effect of the serine residue on disulfide bond susceptibility was compared using three antibodies with differences only in the light chain last amino acid, which was either a serine residue, an alanine residue or deleted. The results demonstrated that the presence of the amino acid (serine or alanine) increased the susceptibility of the inter light and heavy chain disulfide bonds to reduction. On the other hand, susceptibility of the two inter heavy chain disulfide bonds and intrachain disulfide bonds was not changed significantly.

Ranking the susceptibility of disulfide bonds in human IgG1 antibodies by reduction, differential alkylation, and LC-MS analysis.

One of the basic structural features of human IgG1 is the arrangement of the disulfide bond structure, 4 inter chain disulfide bonds in the hinge region and 12 intra chain disulfide bonds associated with twelve individual domains. Disulfide bond structure is critical for the structure, stability, and biological functions of IgG molecules. It has been known that inter chain disulfide bonds are more susceptible to reduction than intra chain disulfide bonds. However, a complete ranking of the susceptibility of disulfide bonds in IgG1 molecules is lacking. A method including reduction, differential alkylation, and liquid chromatography-mass spectrometry (LC-MS) analysis was developed and employed to investigate the complete ranking order of the susceptibility of disulfide bonds in two recombinant monoclonal antibodies. The results confirmed that inter chain disulfide bonds were more susceptible than intra chain disulfide bonds. In addition, it was observed that the disulfide bonds between the light chain and heavy chain were more susceptible than disulfide bonds between the two heavy chains. The upper disulfide bond of the two inter heavy chain disulfide bonds was more susceptible than the lower one. Furthermore, disulfide bonds in the CH2 domain were the most susceptible to reduction. Disulfide bonds in VL, CL, VH, and CH1 domains had similar and moderate susceptibility, while disulfide bonds in the CH3 domain were the least susceptible to reduction. Interestingly, a difference between IgG1kappa and IgG1lambda was also observed. The difference in the susceptibility of inter light heavy chain disulfide bonds and inter heavy chain disulfide bonds was smaller in IgG1kappa than in IgG1lambda. The intra chain disulfide bonds in the Fab region of IgG1kappa were also less susceptible than disulfide bonds in the Fab region of IgG1lambda.

Elevated cleavage of human immunoglobulin gamma molecules containing a lambda light chain mediated by iron and histidine.

Monoclonal antibodies in liquid formulation undergo nonenzymatic hydrolysis when stored at 5 degrees C for extended periods. This hydrolysis is enhanced at extreme pH and high temperature. In this study we discover that iron in the presence of histidine also enhanced cleavage of human immunoglobulin gamma (IgG) molecules containing a lambda light chain when incubated at 40 degrees C. The level of cleavage was concentration dependent on both iron and histidine levels. Enhanced cleavage with iron and histidine was not observed on IgG molecules containing a kappa light chain. Using CE-SDS to quantify levels of Fab+Fc, the Fab arm, and free light chain (LC) and heavy chain (HC) fragments, we show that cleavage resulted in elevated levels of free light and heavy chain fragments. Using MS we find elevated scission between residues E/C on the LC resulting in LC fragment 1-215. We also observed enhanced cleavage between S/C residues of the HC resulting in HC fragment 1-217. The corresponding Fab+Fc fragment beginning with cys-218 was not found. Instead, we find elevation of a Fab+Fc fragment that began with aspartic acid (cleavage between C/D). Further studies to understand how iron and histidine enhance cleavage of lambda light chain IgG molecules are ongoing.

Mass spectrometry analysis of in vitro nitration of a recombinant human IgG1 monoclonal antibody.

Nitration of a recombinant human monoclonal antibody was carried out in vitro by incubating the antibody with the nitrating reagent tetranitromethane (TNM). The susceptible sites of nitration were identified using high-performance liquid chromatography/mass spectrometry (HPLC/MS). In general, tyrosine residues in the variable domains of the antibody are more susceptible to nitration, while tyrosine residues in the constant domains are relatively resistant to nitration. However, one tyrosine residue in the CH1 domain and one tyrosine residue in the CH2 domain are highly susceptible to nitration. Interestingly, the susceptible tyrosine residue in the CH2 domain is followed by the conserved asparagine residue that is glycosylated.

Monoclonal antibody classification based on epitope-binding using differential antigen disruption.

Currently, classifying a population of specific antigen-reactive monoclonal antibodies (mAbs) according to their epitope-binding properties has been limited to competition assays. Such assays are time consuming, labor intensive and restricted to the number of mAbs in the experiment. To overcome this problem, a differential antigen disruption-based antibody profiling procedure was developed. This procedure rapidly classifies specific antigen-reactive mAbs into epitope-related groups by measuring the binding signal of the antibodies to a set of structurally disrupted antigens and then clustering the antibodies according to the similarity of their binding profiles. The clustering results generated by differential antigen disruption showed a significant concordance with those generated by competition experiments. Therefore, differential antigen disruption method opens an opportunity to assess the entire population of antigen-reactive mAbs according to their epitope-binding properties. In doing so, a set of representative antibodies can be drawn to describe the epitope complexity for systematically exploring their functions.

VEGF-A stimulates lymphangiogenesis and hemangiogenesis in inflammatory neovascularization via macrophage recruitment.

Lymphangiogenesis, an important initial step in tumor metastasis and transplant sensitization, is mediated by the action of VEGF-C and -D on VEGFR3. In contrast, VEGF-A binds VEGFR1 and VEGFR2 and is an essential hemangiogenic factor. We re-evaluated the potential role of VEGF-A in lymphangiogenesis using a novel model in which both lymphangiogenesis and hemangiogenesis are induced in the normally avascular cornea. Administration of VEGF Trap, a receptor-based fusion protein that binds and neutralizes VEGF-A but not VEGF-C or -D, completely inhibited both hemangiogenesis and the outgrowth of LYVE-1(+) lymphatic vessels following injury. Furthermore, both lymphangiogenesis and hemangiogenesis were significantly reduced in mice transgenic for VEGF-A(164/164) or VEGF-A(188/188) (each of which expresses only one of the three principle VEGF-A isoforms). Because VEGF-A is chemotactic for macrophages and we demonstrate here that macrophages in inflamed corneas release lymphangiogenic VEGF-C/VEGF-D, we evaluated the possibility that macrophage recruitment plays a role in VEGF-A-mediated lymphangiogenesis. Either systemic depletion of all bone marrow-derived cells (by irradiation) or local depletion of macrophages in the cornea (using clodronate liposomes) prior to injury significantly inhibited both hemangiogenesis and lymphangiogenesis. We conclude that VEGF-A recruitment of monocytes/macrophages plays a crucial role in inducing inflammatory neovascularization by supplying/amplifying signals essential for pathological hemangiogenesis and lymphangiogenesis.

Angiopoietins have distinct modular domains essential for receptor binding, dimerization and superclustering.

Angiopoietins are a recently discovered family of angiogenic factors that interact with the endothelial receptor tyrosine kinase Tie2, either as agonists (angiopoietin-1) or as context-dependent agonists/antagonists (angiopoietin-2). Here we show that angiopoietin-1 has a modular structure unlike any previously characterized growth factor. This modular structure consists of a receptor-binding domain, a dimerization motif and a superclustering motif that forms variable-sized multimers. Genetic engineering of precise multimers of the receptor-binding domain of angiopoietin-1, using surrogate multimerization motifs, reveals that tetramers are the minimal size required for activating endothelial Tie2 receptors. In contrast, engineered dimers can antagonize endothelial Tie2 receptors. Surprisingly, angiopoietin-2 has a modular structure and multimerization state similar to that of angiopoietin-1, and its antagonist activity seems to be a subtle property encoded in its receptor-binding domain.

Cytokine traps: multi-component, high-affinity blockers of cytokine action.

Cytokines can initiate and perpetuate human diseases, and are among the best-validated of therapeutic targets. Cytokines can be blocked by the use of soluble receptors; however, the use of this approach for cytokines such as interleukin (IL)-1, IL-4, IL-6 and IL-13 that use multi-component receptor systems is limited because monomeric soluble receptors generally exhibit low affinity or function as agonists. We describe here a generally applicable method to create very high-affinity blockers called 'cytokine traps' consisting of fusions between the constant region of IgG and the extracellular domains of two distinct cytokine receptor components involved in binding the cytokine. Traps potently block cytokines in vitro and in vivo and represent a substantial advance in creating novel therapeutic candidates for cytokine-driven diseases.

Binding of discoidin domain receptor 2 to collagen I: an atomic force microscopy investigation.

Collagens have recently been identified as ligands for discoidin domain receptors (DDR1 and DDR2), generating an interest in studying the properties of binding of DDR to its ligand. We are interested in the interaction of DDR2 with collagen I because of its potential role in liver fibrosis. Our in vitro binding assay utilizes DDR2-Fc fusion proteins, which can be clustered (multimerized) by use of antibodies to form DDR2 complexes. Binding of DDR2 complexes to collagen I coated on plastic plates was established by a microplate-based assay using Eu(3+)-labeled proteins and time-resolved fluorometry. Clustering of the DDR2-Fc with antibody was found to be requisite for binding to collagen in vitro. Using atomic force microscopy (AFM) in an aqueous environment, we characterized the surface topographies of DDR2 complexes and collagen I, and investigated binding of this receptor-ligand pair. We were able to image and identify binding of DDR2 complexes onto individual molecules of triple-helical collagen and provide insight into the number and locations of binding sites on collagen I. In most cases, a single receptor complex bound to a single collagen molecule and there were preferred DDR2 binding sites on the collagen I triple helix. These data were validated by rotary-replication transmission electron microscopy (TEM) of glycerol-sprayed samples.

VEGF-Trap: a VEGF blocker with potent antitumor effects.

Vascular endothelial growth factor (VEGF) plays a critical role during normal embryonic angiogenesis and also in the pathological angiogenesis that occurs in a number of diseases, including cancer. Initial attempts to block VEGF by using a humanized monoclonal antibody are beginning to show promise in human cancer patients, underscoring the importance of optimizing VEGF blockade. Previous studies have found that one of the most effective ways to block the VEGF-signaling pathway is to prevent VEGF from binding to its normal receptors by administering decoy-soluble receptors. The highest-affinity VEGF blocker described to date is a soluble decoy receptor created by fusing the first three Ig domains of VEGF receptor 1 to an Ig constant region; however, this fusion protein has very poor in vivo pharmacokinetic properties. By determining the requirements to maintain high affinity while extending in vivo half life, we were able to engineer a very potent high-affinity VEGF blocker that has markedly enhanced pharmacokinetic properties. This VEGF-Trap effectively suppresses tumor growth and vascularization in vivo, resulting in stunted and almost completely avascular tumors. VEGF-Trap-mediated blockade may be superior to that achieved by other agents, such as monoclonal antibodies targeted against the VEGF receptor.