Conformational stability and aggregation of therapeutic monoclonal antibodies studied with ANS and thioflavin T binding

Characterization of aggregation profiles of monoclonal antibodies (mAb) is gaining importance because an increasing number of mAb-based therapeutics are entering clinical studies and gaining marketing approval. To develop a successful formulation, it is imperative to identify the critical biochemical properties of each potential mAb drug candidate. We investigated the conformational change and aggregation of a human IgG1 using external dye-binding experiments with fluorescence spectroscopy and compared the aggregation profiles obtained to the results of size-exclusion chromatography. We show that using an appropriate dye at selected mAb concentration, unfolding or aggregation can be studied. In addition, dye-binding experiments may be used as conventional assays to study therapeutic mAb stability.Key words: therapeutic monoclonal antibody, protein aggregation, conformational change, stability and shelf-life prediction, accelerated studiesMonoclonal antibodies (mAbs) have emerged as a novel class of protein drugs and are utilized for a variety of mostly incurable and debilitating diseases such as cancer and rheumatoid arthritis.^1–4 For treatment of chronic diseases, it is desirable for these drugs to be administered subcutaneously, in which case high protein concentrations (>100 mg/mL) are generally needed.^5,6 Protein-based drugs containing mAbs must contain minimum amounts of aggregation and fragmentation and conserve their structural integrity during storage because degraded or aggregated protein may induce immunogenicity or reduce efficacy. Currently, size-exclusion chromatography-high performance liquid chromatography (SEC-HPLC) is the most commonly used method to characterize mAb aggregation profiles;⁷ however it is time consuming, expensive and requires expertise. SEC-HPLC cannot be used to obtain accurate biophysical profiles of mAbs at high concentrations because dilution during the experiment might lead to reversible aggregation. Furthermore, the potential interaction of aggregates with surfaces, e.g., needle, tubing, column, will lead to the loss of sample and thus an inaccurate analysis.^8,9 Additional drawbacks of the technique are that different conformations such as partially unfolded monomers also cannot be distinguished by SEC-HPLC and large aggregates may be totally excluded during the injection into the column.External dye binding assays have been used to characterize protein stability and aggregation,^10–12 and studies involving biopharmaceuticals have been reported recently, e.g., for thermostability screening¹⁰ and detection of aggregation.^11–14 These methods are not limited by protein quantity and are more sensitive because they are fluorescence-based. We studied the accelerated unfolding of an IgG1 mAb with the hydrophobic dye 1-anilino-8-naphthale-nesulfonate (ANS), and its accelerated aggregation with aggregate specific Thioflavin T (ThT). We have also conducted accelerated aggregation studies with SEC-HPLC7 and compared the findings to the ThT binding results. We hypothesize that key structures formed during mAb aggregation can be probed selectively by the appropriate dyes (Fig. 1) with specific mAb concentrations.Open in a separate windowFigure 1Key structures of the mAb probed by fluorescent dyes. N and U are native and unfolded monomers, respectively. “n” reactive monomers form aggregates.     

Glycosylation influences on the aggregation propensity of therapeutic monoclonal antibodies

Monoclonal antibodies are the fastest growing class of biologics in the pharmaceutical industry. The correlation between mAb glycosylation and aggregation has not been elucidated in detail, yet understanding the structure-stability relationship involving glycosylation is critical for developing successful drug formulations. We conducted studies of temperature-induced aggregation and compared the stability of both glycosylated and aglycosylated forms of a human IgG1. In parallel, we also performed molecular dynamics simulations of the glycosylated full antibody to gain an understanding of the polysaccharide surroundings at the molecular level. Aglycosylated mAbs are somewhat less stable and therefore aggregate more easily than the glycosylated form at the temperatures studied. Glycosylation seems to enhance solubility and stability of these therapeutics and thus might be important for long-term storage.     

Conformational changes in oxidatively stressed monoclonal antibodies studied by hydrogen exchange mass spectrometry

Oxidation of methionine residues in biopharmaceuticals is a common and often unwanted modification that frequently occurs during their manufacture and storage. It often results in a lack of stability and biological function of the product, necessitating continuous testing for the modification throughout the product shelf life. A major class of biopharmaceutical products are monoclonal antibodies (mAbs), however, techniques for their detailed structural analysis have until recently been limited. Hydrogen/deuterium exchange mass spectrometry (HXMS) has recently been successfully applied to the analysis of mAbs. Here we used HXMS to identify and localise the structural changes that occurred in a mAb (IgG1) after accelerated oxidative stress. Structural alterations in a number of segments of the Fc region were observed and these related to oxidation of methionine residues. These included a large change in the hydrogen exchange profile of residues 247–253 of the heavy chain, while smaller changes in hydrogen exchange profile were identified for peptides that contained residues in the interface of the C_H2 and C_H3 domains.     

Study on the binding of Thioflavin T to beta-sheet-rich and non-beta-sheet cavities

Amyloid fibril formation plays a role in more than 20 diseases including Alzheimer's disease. In vitro detection of these fibrils is often performed using Thioflavin T (ThT), though the ThT binding mode is largely unknown. In the present study, spectral properties of ThT in binding environments representing beta-sheet-rich and non-beta-sheet cavities were examined. Acetylcholinesterase and gamma-cyclodextrin induced a characteristic ThT fluorescence similar to that with amyloid fibrils, whereas beta-cyclodextrin and the beta-sheet-rich transthyretin did not. The cavities of acetylcholinesterase and gamma-cyclodextrin were of similar diameter and only these cavities could accommodate two ThT ions according to molecular modelling. Binding stoichiometry studies also showed a possible binding of two ThT ions. Thus, the characteristic ThT fluorescence is induced in cavities with a diameter of 8-9A and a length able to accommodate the entire length of the ThT ion. The importance of a cavity diameter capable of binding two ThT ions, among others, indicates that an excimer formation is a plausible mechanism for the characteristic fluorescence. We propose a similar ThT binding mode in amyloid fibrils, where cavities of an appropriate size running parallel to the fibril axis have previously been proposed in several amyloid fibril models.     

Effects of acid exposure on the conformation, stability, and aggregation of monoclonal antibodies

Exposure of antibodies to low pH is often unavoidable for purification and viral clearance. The conformation and stability of two humanized monoclonal antibodies (hIgG4-A and -B) directed against different antigens and a mouse monoclonal antibody (mIgG1) in 0.1M citrate at acidic pH were studied using circular dichroism (CD), differential scanning calorimetry (DSC), and sedimentation velocity. Near- and far-UV CD spectra showed that exposure of these antibodies to pH 2.7-3.9 induced only limited conformational changes, although the changes were greater at the lower pH. However, the acid conformation is far from unfolded or so-called molten globule structure. Incubation of hIgG4-A at pH 2.7 and 3.5 at 4 degrees C over the course of 24 h caused little change in the near-UV CD spectra, indicating that the acid conformation is stable. Sedimentation velocity showed that the hIgG4-A is largely monomeric at pH 2.7 and 3.5 as well as at pH 6.0. No time-dependent changes in sedimentation profile occurred upon incubation at these low pHs, consistent with the conformational stability observed by CD. The sedimentation coefficient of the monomer at pH 2.7 or 3.5 again suggested that no gross conformational changes occur at these pHs. DSC analysis of the antibodies showed thermal unfolding at pH 2.7-3.9 as well as at pH 6.0, but with decreased melting temperatures at the lower pH. These results are consistent with the view that the antibodies undergo limited conformational change, and that incubation at 4 degrees C at low pH results in no time-dependent conformational changes. Titration of hIgG4-A from pH 3.5 to 6.0 resulted in recovery of native monomeric proteins whose CD and DSC profiles resembled those of the original sample. However, titration from pH 2.7 resulted in lower recovery of monomeric antibody, indicating that the greater conformational changes observed at this pH cannot be fully reversed to the native structure by a simple pH titration.     

Conformational changes in diphtheria toxoids. Analysis with monoclonal antibodies

Monoclonal antibodies (Mab) were raised against CRM197, a non-toxic mutant of diphtheria toxin (DT). The ability of four Mabs to bind DT and the six functional mutants CRM197, CRM176, CRM228, CRM1001, CRM45 and CRM30 was assessed by immunoblotting and by a radioimmunoassay in which the protein antigen in solution competes with labeled CRM197 for the Mab binding site. The results show that the peptides recognized by Mab11.3, Mab53 and Mab23 are accessible in the mutant molecules in solution but not when they are part of the native DT structure, which could therefore be described for this purpose as 'closed' in contrast with an 'open' conformation of CRM197, CRM176 and CRM228. In particular, the behaviour of Mab53 indicates that the single amino acid substitutions in the A fragments of CRM197 and CRM176 also affect the conformation of their B fragments.     

Cross-reactions among carbonic anhydrases I,II, and III studied by binding tests and with monoclonal antibodies

Cross-reactions among carbonic anhydrases (CAs) I, II, and III were studied using a variety of antisera: (1) a rabbit antiserum to bovine CA III, (2) mouse antisera to human CA I, CA II, and CA III; and (3) five monoclonal antibodies prepared by the hybridoma technique using splenocytes from a mouse immunized with human CAs I and II and bovine CA III. Cross-reactions between CAs were readily found by binding assays using these antisera. Human CA I, but not human CA II, inhibited the reaction of the rabbit anti-CA III with its homologous antigen. Mouse antisera to CA I or CA II bound the homologous I or II with nearly as great efficiency as the autologous isozyme and sometimes weakly bound CA III. Mouse antisera to CA III frequently bound CA I or II. These cross-reactions were confirmed by the first use of hybridoma-prepared, monoclonal antibodies to CAs. The mouse monoclonal antibodies to CA isozymes varied in the amount of cross-reactivity among I, II, and III: at one extreme, one monoclonal was highly specific for the autologous CA III; at the other extreme, one monoclonal weakly reacted with some examples of CAs I, II, and III.This work was supported by NIH Grant GM-24681 and a grant from the National Foundation-March of Dimes.     

Conformational changes in C1q after binding to immune complexes: detection of neoantigens with monoclonal antibodies

The formation of neoantigens within the C1q molecule after the binding of C1r and C1s to C1q and the binding of C1q to immune complexes is described. The neoantigens were detected by different monoclonal anti-C1q antibodies. This immunochemical study supports the hypothesis drawn from functional studies that the activation of the classical C pathway results from conformational changes within the C1q molecule leading to the activation of C1r and subsequently C1s.     

Thioflavin T fluorescence anisotropy: an alternative technique for the study of amyloid aggregation

The process of amyloid polymerisation raises keen interest in particular because of the biomedical impact of this process. A variety of analytical methods have been developed to monitor amyloid formation. Thioflavin T (ThT) is the most commonly used dye for detection of amyloid aggregation. Nevertheless, ThT fluorescence enhancement is strongly dependent of fibril morphology. In this study using the HET-s prion fibril model, we show that amyloid formation can be monitored by measuring ThT fluorescence anisotropy. Kinetic parameters obtained by this method are identical to those determined by CD spectrometry. We propose that ThT anisotropy represent an interesting, simple and alternative technique to analyze the amyloid formation process.     

Conformational effects of ligand binding on the beta 2 subunit of Escherichia coli tryptophan synthase analyzed with monoclonal antibodies

Five monoclonal antibodies recognizing five different epitopes of the native beta 2 subunit of Escherichia coli tryptophan synthase (EC 4.1.2.20) were used to analyze the conformational changes occurring upon ligand binding or chemical modifications of the enzyme. For this purpose, the affinities of each antibody for the different forms of the enzyme were determined by using an enzyme-linked immunosorbent assay which allows measurement of the dissociation constant of antigen-antibody equilibrium in solution. The fixation of the coenzyme pyridoxal 5'-phosphate and the substrate L-serine modifies the affinity constants of most of the antibodies for the enzyme, thus showing the existence of extended conformational rearrangements of the protein. The association of the alpha subunit with the beta 2 subunit, which brings about an increase of the tryptophan synthase activity and abolishes the serine deaminase activity of beta 2, is accompanied by an important conformational change of the N-terminal domain of beta 2 (F1) since none of the anti-F1 monoclonal antibodies can bind to alpha 2 beta 2. Similarly, chemical modifications of beta 2 which are known to produce significant effects on the enzymatic activities of beta 2 result in changes of the affinities of the monoclonal antibodies which can be interpreted as the acquisition of different conformational states of the enzyme.     

Conformational stability and binding properties of porcine odorant binding protein.

Apparently homogeneous odorant binding protein purified from pig nasal mucosa (pOBP) exhibited subunit molecular masses of 17 223, 17 447, and 17 689 (major component) Da as estimated by ESI/MS. According to gel filtration, this protein, its truncated forms, and/or its variants are homodimeric under physiologic conditions (pH 6-7, 0.1 M NaCl). The dimer if monomer equilibrium shifts toward a prevalent monomeric form at pH <4.5. Velocity sedimentation reveals a monomeric state of OBP at both pH 7.2 and 3.5, indicating a pressure-induced dissociation of the homodimer. High-sensitivity differential scanning calorimetry (HS-DSC) shows that the unfolding transition of pOBP is reversible at neutral pH. It is characterized by the transition temperature of 69.23 degrees C and an enthalpy of 391.1 kJ/mol per monomer. The transition heat capacity curve of pOBP is well-approximated by the two-state model on the level of subunit, indicating that the two monomers behave independently. Isothermal titration calorimetry (ITC) shows that at physiological pH pOBP binds 2-isobutyl-3-methoxypyrazine (IBMP) and 3,7-dimethyloctan-1-ol (DMO) with association constants of 3.19 x 10(6) and 4.94 x 10(6) M(-)(1) and enthalpies of -97.2 and -87.8 kJ/mol, respectively. The binding stoichiometry of both ligands is nearly one molecule of ligand per homodimer of pOBP. The interaction of pOBP with both ligands is enthalpically driven with an unfavorable change of entropy. The binding affinity of pOBP with IBMP does not change significantly at acidic pH, while the binding stoichiometry is nearly halved. According to HS-DSC data, the interaction with IBMP and DMO leads to a substantial stabilization of the pOBP folded structure, which is manifested by the increase in the unfolding temperature and enthalpy. The calorimetric data allow us to conclude that the mechanism of binding of the studied odorants to pOBP is not dominated by a hydrophobic effect related to any change in the hydration state of protein and ligand groups but, most likely, is driven by polar and van der Waals interactions.     

Diagnostic and therapeutic applications of antiplatelet monoclonal antibodies

The interaction of platelets with natural and artificial surfaces is briefly reviewed, emphasizing the role of the platelet glycoprotein Ib and IIb/IIIa receptors. Studies utilizing monoclonal antibodies to these receptors for the diagnosis and therapy of hemorrhagic and thrombotic disorders are described, indicating the potential of such agents as platelet inhibitors.     

The surface antigens of Rickettsia prowazekii studied with monoclonal antibodies]

R. prowazekii antigens have been tested with the use of monoclonal antibodies (McAb) to different epitopes of the microorganism. As revealed in these tests, McAb B4/4 and A-3/D, active against species-specific thermolabile antigen, interact with protein having a molecular weight of 90-120 KD. McAb C5/2, active against thermostable group antigen common with that of Rickettsia typhi, interact with LPS-like antigen having a molecular weight of 30 KD. Ultrastructural immunochemical studies have revealed that both R. prowazekii antigens are located on surface structures of rickettsiae, such as the microcapsule and cell wall.     

Conformational transitions of antibodies induced by hapten binding

The conformational changes of antibody structure induced by hapten molecule binding were investigated by means of thermal perturbation difference spectroscopy. The studies of the free rabit anti-dinitrophenyl antibodies show the conformational transition at temperatures between 25 and 35 degrees C. The changes occurring at the higher temperature are accompanied by the screening of the significant part of exposed tyrosine residues. Binding of the hapten molecules induces a similar transition to that which occurs between the two temperature dependent states of the free antibody. In contrast to our previous results with anti-dansyl rabbit antibodies the dinitrophenyl lysine stabilizes the "low temperature" native state of the protein. The investigation of the MOPC-315 mouse immunoglobulin A myeloma protein possessing anti-dinitrophenyl activity indicates no conformational transition at temperatures between 25 and 35 degrees C and only a small decrease of tyrosine exposure induced by the hapten binding. Our present and previous results indicate that most of the free immunoglobulins exist in two native conformational states which have a small difference in free energy. Hapten binding causes the transition in equilibrium between the two states towards the one of better binding. It is possible that this transition is necessary but not sufficient step for inducing the effector function of antibodies.     

Conformational dynamics and thermodynamics of protein-ligand binding studied by NMR relaxation

Protein conformational dynamics can be critical for ligand binding in two ways that relate to kinetics and thermodynamics respectively. First, conformational transitions between different substates can control access to the binding site (kinetics). Secondly, differences between free and ligand-bound states in their conformational fluctuations contribute to the entropy of ligand binding (thermodynamics). In the present paper, I focus on the second topic, summarizing our recent results on the role of conformational entropy in ligand binding to Gal3C (the carbohydrate-recognition domain of galectin-3). NMR relaxation experiments provide a unique probe of conformational entropy by characterizing bond-vector fluctuations at atomic resolution. By monitoring differences between the free and ligand-bound states in their backbone and side chain order parameters, we have estimated the contributions from conformational entropy to the free energy of binding. Overall, the conformational entropy of Gal3C increases upon ligand binding, thereby contributing favourably to the binding affinity. Comparisons with the results from isothermal titration calorimetry indicate that the conformational entropy is comparable in magnitude to the enthalpy of binding. Furthermore, there are significant differences in the dynamic response to binding of different ligands, despite the fact that the protein structure is virtually identical in the different protein-ligand complexes. Thus both affinity and specificity of ligand binding to Gal3C appear to depend in part on subtle differences in the conformational fluctuations that reflect the complex interplay between structure, dynamics and ligand interactions.     

Epitopes in human growth hormone and chorionic somatomammotropin studied with monoclonal antibodies

The immune complexes formed by human growth hormone or human chorionic somatomammotropin and various monoclonal antibodies have been studied by gel filtration and polyacrylamide gel electrophoresis. Two of the monoclonal antibodies gave rise to complexes with molecular weights suggesting an antigen:antibody 1:1 ratio. When both antibodies were simultaneously incubated with human growth hormone the ratio estimated for the new complex was 1:2, indicating the existence of two nonoverlapping epitopes in the antigen. The other monoclonal antibodies exhibited a more intricate behavior: incubated separately with human growth hormone they gave rise to both types of the aforementioned complexes. A similar phenomenon could be demonstrated with human chorionic somatomammotropin. The study of the immunoreactivity of a synthetic peptide indicates that the involved epitopes are localized within the region limited by amino acid residues 44 and 128 of human growth hormone.     

Dextrose-mediated aggregation of therapeutic monoclonal antibodies in human plasma: Implication of isoelectric precipitation of complement proteins

Many therapeutic monoclonal antibodies (mAbs) are clinically administered through intravenous infusion after mixing with a diluent, e.g., saline, 5% dextrose. Such a clinical setting increases the likelihood of interactions among mAb molecules, diluent, and plasma components, which may adversely affect product safety and efficacy. Avastin® (bevacizumab) and Herceptin® (trastuzumab), but not Remicade® (infliximab), were shown to undergo rapid aggregation upon dilution into 5% dextrose when mixed with human plasma in vitro; however, the biochemical pathways leading to the aggregation were not clearly defined. Here, we show that dextrose-mediated aggregation of Avastin or Herceptin in plasma involves isoelectric precipitation of complement proteins. Using mass spectrometry, we found that dextrose-induced insoluble aggregates were composed of mAb itself and multiple abundant plasma proteins, namely complement proteins C3, C4, factor H, fibronectin, and apolipoprotein. These plasma proteins, which are characterized by an isoelectronic point of 5.5–6.7, lost solubility at the resulting pH in the mixture with formulated Avastin (pH 6.2) and Herceptin (pH 6.0). Notably, switching formulation buffers for Avastin (pH 6.2) and Remicade (pH 7.2) reversed their aggregation profiles. Avastin formed little, if any, insoluble aggregates in dextrose-plasma upon raising the buffer pH to 7.2 or above. Furthermore, dextrose induced pH-dependent precipitation of plasma proteins, with massive insoluble aggregates being detected at pH 6.5–6.8. These data show that isoelectric precipitation of complement proteins is a prerequisite of dextrose-induced aggregation of mAb in human plasma. This finding highlights the importance of assessing the compatibility of a therapeutic mAb with diluent and human plasma during product development.     

Dextrose-mediated aggregation of therapeutic monoclonal antibodies in human plasma: Implication of isoelectric precipitation of complement proteins

Many therapeutic monoclonal antibodies (mAbs) are clinically administered through intravenous infusion after mixing with a diluent, e.g., saline, 5% dextrose. Such a clinical setting increases the likelihood of interactions among mAb molecules, diluent, and plasma components, which may adversely affect product safety and efficacy. Avastin® (bevacizumab) and Herceptin® (trastuzumab), but not Remicade® (infliximab), were shown to undergo rapid aggregation upon dilution into 5% dextrose when mixed with human plasma in vitro; however, the biochemical pathways leading to the aggregation were not clearly defined. Here, we show that dextrose-mediated aggregation of Avastin or Herceptin in plasma involves isoelectric precipitation of complement proteins. Using mass spectrometry, we found that dextrose-induced insoluble aggregates were composed of mAb itself and multiple abundant plasma proteins, namely complement proteins C3, C4, factor H, fibronectin, and apolipoprotein. These plasma proteins, which are characterized by an isoelectronic point of 5.5–6.7, lost solubility at the resulting pH in the mixture with formulated Avastin (pH 6.2) and Herceptin (pH 6.0). Notably, switching formulation buffers for Avastin (pH 6.2) and Remicade (pH 7.2) reversed their aggregation profiles. Avastin formed little, if any, insoluble aggregates in dextrose-plasma upon raising the buffer pH to 7.2 or above. Furthermore, dextrose induced pH-dependent precipitation of plasma proteins, with massive insoluble aggregates being detected at pH 6.5–6.8. These data show that isoelectric precipitation of complement proteins is a prerequisite of dextrose-induced aggregation of mAb in human plasma. This finding highlights the importance of assessing the compatibility of a therapeutic mAb with diluent and human plasma during product development.     

A screening tool for therapeutic monoclonal antibodies: Identifying the most stable protein and its best formulation based on thioflavin T binding

The lack of a fast selection method to identify the most stable protein is one of the major challenges for developing successful therapeutic protein formulations more rapidly. The swift and accurate detection of small amounts of aggregates is another problem since aggregates may trigger an immunological response and the aggregation decreases the biological activity of the antibody. Here we present an alternative method for initial screening of the aggregation propensity of proteins, using monoclonal antibodies (mAb) as an example and thioflavin T (ThT) binding. The major advantage of ThT binding is the short duration of testing compared with size-exclusion chromatography (SEC) measurements that can take 6 months or more even under accelerated conditions. The tendency to aggregate of each therapeutic human mAb probed with the ThT assay, together with SEC, is employed to formulate the ranking of mAb aggregation. ThT binding can determine the propensity of proteins to aggregate in a few days, illustrating that ThT binding would be a valuable screening tool.     

Covert cross reactants in a two-site immunoassay studied with monoclonal antibodies

A one-step two-site immunoradiometric assay for the measurement of free β subunit of human chorionic gonadotrophin (β-hCG) was developed using monoclonal antibodies. The immobilized antibody was specific for free β subunit and the radiolabeled antibody recognized both intact human chorionic gonadotrophin (hCG) and free β subunit. Although the level of hCG “cross-reaction” was low when studied using conventional techniques, the apparent β-hCG content of samples was found to be inversely proportional to the hCG level. From both experimental evidence and computer simulation studies this was found to be due to the binding of hCG to the limited amount of ¹²⁵I-labeled antibody present. The term covert cross reactants has been introduced to describe substances which bind to only one of the antibodies in a two-site immunoassay. When establishing such an assay the effect of covert cross reactants on the response of an analyte should be investigated.