Evaluation of effects of pH and ionic strength on colloidal stability of IgG solutions by PEG-induced liquid-liquid phase separation.

Colloidal stability of IgG antibody solutions is important for pharmaceutical and medicinal applications. Solution pH and ionic strength are two key factors that affect the colloidal stability of protein solutions. In this work, we use a method based on the PEG-induced liquid-liquid phase separation to examine the effects of pH and ionic strength on the colloidal stability of IgG solutions. We found that at high ionic strength (≥0.25M), the colloidal stability of most of our IgGs is insensitive to pH, and at low ionic strength (≤0.15M), all IgG solutions are much more stable at pH 5 than at pH 7. In addition, the PEG-induced depletion force is less efficient in causing phase separation at pH 5 than at pH 7. In contrast to the native inter-protein interaction of IgGs, the effect of depletion force on phase separation of the antibody solutions is insensitive to ionic strength. Our results suggest that the long-range electrostatic inter-protein repulsion at low ionic strength stabilizes the IgG solutions at low pH. At high ionic strength, the short-range electrostatic interactions do not make a significant contribution to the colloidal stability for most IgGs with a few exceptions. The weaker effect of depletion force at lower pH indicates a reduction of protein concentration in the condensed phase. This work advances our basic understanding of the colloidal stability of IgG solutions and also introduces a practical approach to measuring protein colloidal stability under various solution conditions.

Quantitative evaluation of colloidal stability of antibody solutions using PEG-induced liquid-liquid phase separation.

Colloidal stability of antibody solutions, i.e., the propensity of the folded protein to precipitate, is an important consideration in formulation development of therapeutic monoclonal antibodies. In a protein solution, different pathways including crystallization, colloidal aggregation, and liquid-liquid phase separation (LLPS) can lead to the formation of precipitates. The kinetics of crystallization and aggregation are often slow and vary from protein to protein. Due to the diverse mechanisms of these protein condensation processes, it is a challenge to develop a standardized test for an early evaluation of the colloidal stability of antibody solutions. LLPS would normally occur in antibody solutions at sufficiently low temperature, provided that it is not preempted by freezing of the solution. Poly(ethylene glycol) (PEG) can be used to induce LLPS at temperatures above the freezing point. Here, we propose a colloidal stability test based on inducing LLPS in antibody solutions and measuring the antibody concentration of the dilute phase. We demonstrate experimentally that such a PEG-induced LLPS test can be used to compare colloidal stability of different antibodies in different solution conditions and can be readily applied to high-throughput screening. We have derived an equation for the effects of PEG concentration and molecular weight on the results of the LLPS test. Finally, this equation defines a binding energy in the condensed phase, which can be determined in the PEG-induced LLPS test. This binding energy is a measure of attractive interactions between antibody molecules and can be used for quantitative characterization of the colloidal stability of antibody solutions.

Phase separation in solutions of monoclonal antibodies and the effect of human serum albumin.

We report the observation of liquid-liquid phase separation in a solution of human monoclonal antibody, IgG2, and the effects of human serum albumin, a major blood protein, on this phase separation. We find a significant reduction of phase separation temperature in the presence of albumin, and a preferential partitioning of the albumin into the antibody-rich phase. We provide a general thermodynamic analysis of the antibody-albumin mixture phase diagram and relate its features to the magnitude of the effective interprotein interactions. Our analysis suggests that additives (HSA in this report), which have moderate attraction with antibody molecules, may be used to forestall undesirable protein condensation in antibody solutions. Our findings are relevant to understanding the stability of pharmaceutical solutions of antibodies and the mechanisms of cryoglobulinemia.

Elucidation of acid-induced unfolding and aggregation of human immunoglobulin IgG1 and IgG2 Fc.

Understanding the underlying mechanisms of Fc aggregation is an important prerequisite for developing stable and efficacious antibody-based therapeutics. In our study, high resolution two-dimensional nuclear magnetic resonance (NMR) was employed to probe structural changes in the IgG1 Fc. A series of (1)H-(15)N heteronuclear single-quantum correlation NMR spectra were collected between pH 2.5 and 4.7 to assess whether unfolding of C(H)2 domains precedes that of C(H)3 domains. The same pH range was subsequently screened in Fc aggregation experiments that utilized molecules of IgG1 and IgG2 subclasses with varying levels of C(H)2 glycosylation. In addition, differential scanning calorimetry data were collected over a pH range of 3-7 to assess changes in C(H)2 and C(H)3 thermostability. As a result, compelling evidence was gathered that emphasizes the importance of C(H)2 stability in determining the rate and extent of Fc aggregation. In particular, we found that Fc domains of the IgG1 subclass have a lower propensity to aggregate compared with those of the IgG2 subclass. Our data for glycosylated, partially deglycosylated, and fully deglycosylated molecules further revealed the criticality of C(H)2 glycans in modulating Fc aggregation. These findings provide important insights into the stability of Fc-based therapeutics and promote better understanding of their acid-induced aggregation process.

Nondenaturing size-exclusion chromatography-mass spectrometry to measure stress-induced aggregation in a complex mixture of monoclonal antibodies.

During therapeutic candidate selection, diverse panels of monoclonal antibodies (mAbs) are routinely subjected to various stress conditions, and assayed for biophysical and biochemical stability. A novel high throughput method has been developed to differentiate candidate molecules in a mixture based on their propensity for forming aggregates when subjected to agitation (vortexing) stress. Protein monomers are separated from soluble and insoluble aggregates using size exclusion chromatography, under nondenaturing conditions, and the individual components in the mixture are identified by mass spectrometry and quantitated relative to an unstressed control. An internal standard was added to the mixture after stress, and used to correct for differences in ionization between samples. Treatment of the samples with the enzyme IdeS (FabRICATOR) significantly reduces sample complexity, and allows for a large number of candidate molecules to be assessed in a single analysis. Simple and robust, the method is well suited for measuring relative aggregation propensity (RAP) in conjunction with molecule selection and coformulation development.

Phase transitions in human IgG solutions.

Protein condensations, such as crystallization, liquid-liquid phase separation, aggregation, and gelation, have been observed in concentrated antibody solutions under various solution conditions. While most IgG antibodies are quite soluble, a few outliers can undergo condensation under physiological conditions. Condensation of IgGs can cause serious consequences in some human diseases and in biopharmaceutical formulations. The phase transitions underlying protein condensations in concentrated IgG solutions is also of fundamental interest for the understanding of the phase behavior of non-spherical protein molecules. Due to the high solubility of generic IgGs, the phase behavior of IgG solutions has not yet been well studied. In this work, we present an experimental approach to study IgG solutions in which the phase transitions are hidden below the freezing point of the solution. Using this method, we have investigated liquid-liquid phase separation of six human myeloma IgGs and two recombinant pharmaceutical human IgGs. We have also studied the relation between crystallization and liquid-liquid phase separation of two human cryoglobulin IgGs. Our experimental results reveal several important features of the generic phase behavior of IgG solutions: (1) the shape of the coexistence curve is similar for all IgGs but quite different from that of quasi-spherical proteins; (2) all IgGs have critical points located at roughly the same protein concentration at ~100 mg/ml while their critical temperatures vary significantly; and (3) the liquid-liquid phase separation in IgG solutions is metastable with respect to crystallization. These features of phase behavior of IgG solutions reflect the fact that all IgGs have nearly identical molecular geometry but quite diverse net inter-protein interaction energies. This work provides a foundation for further experimental and theoretical studies of the phase behavior of generic IgGs as well as outliers with large propensity to condense. The investigation of the phase diagram of IgG solutions is of great importance for the understanding of immunoglobulin deposition diseases as well as for the understanding of the colloidal stability of IgG pharmaceutical formulations.

Acid-induced aggregation of human monoclonal IgG1 and IgG2: molecular mechanism and the effect of solution composition.

The prevention of aggregation in therapeutic antibodies is of great importance to the biopharmaceutical industry. In our investigation, acid-induced aggregation of monoclonal IgG1 and IgG2 antibodies was studied at pH 3.5 as a function of salt concentration and buffer type. The extent of aggregation was estimated using a native cation-exchange chromatography (CEX) method based on the loss of soluble monomer. This approach allowed quantitative analysis of antibody aggregation kinetics for individual and mixed protein solutions. Information regarding the aggregation mechanism was gained by assessing stabilities of intact antibodies relative to their Fc and Fab fragments. The role of protein thermodynamic stability in aggregation was deduced from differential scanning calorimetry (DSC). The rate of aggregation under conditions mimicking the viral inactivation step during monoclonal antibody (mAb) processing was found to be strongly dependent on the antibody subclass (IgG1 vs IgG2). At 25 °C, IgG1s were resistant to low pH aggregation, but IgG2s aggregated readily in the presence of salt. The observed distinction between IgG1 and IgG2 aggregation resulted from differential stability of the corresponding C(H)2 domains. This was further confirmed by experimenting with an IgG1 molecule containing an aglycosylated C(H)2 domain. Interestingly, comparative analysis of two buffer systems (based on acetic acid vs citric acid) revealed differences in mAb aggregation under identical pH conditions. Evidence is provided for the importance of the total acid concentration for antibody aggregation at low pH. The effects of C(H)2 instability and solution composition on aggregation are significant and deserve careful consideration during the development of mAb- or Fc-based therapeutics.

Denaturant-dependent conformational changes in a beta-trefoil protein: global and residue-specific aspects of an equilibrium denaturation process.

Conformational properties of the folded and unfolded ensembles of human interleukin-1 receptor antagonist (IL-1ra) are strongly denaturant-dependent as evidenced by high-resolution two-dimensional nuclear magnetic resonance (NMR), limited proteolysis, and small-angle X-ray scattering (SAXS). The folded ensemble was characterized in detail in the presence of different urea concentrations by (1)H-(15)N HSQC NMR. The beta-trefoil fold characteristic of native IL-1ra was preserved until the unfolding transition region beginning at 4 M urea. At the same time, a subset of native resonances disappeared gradually starting at low denaturant concentrations, indicating noncooperative changes in the folded state. Additional evidence of structural perturbations came from the chemical shift analysis, nonuniform and bell-shaped peak intensity profiles, and limited proteolysis. In particular, the following nearby regions of the tertiary structure became progressively destabilized with increasing urea concentrations: the beta-hairpin interface of trefoils 1 and 2 and the H2a-H2 helical region. These regions underwent small-scale perturbations within the native baseline region in the absence of populated molten globule-like states. Similar regions were affected by elevated temperatures known to induce irreversible aggregation of IL-1ra. Further evidence of structural transitions invoking near-native conformations came from an optical spectroscopy analysis of its single-tryptophan variant W17A. The increase in the radius of gyration was associated with a single equilibrium unfolding transition in the case of two different denaturants, urea and guanidine hydrochloride (GuHCl). However, the compactness of urea- and GuHCl-unfolded molecules was comparable only at high denaturant concentrations and deviated under less denaturing conditions. Our results identified the role of conformational flexibility in IL-1ra aggregation and shed light on the nature of structural transitions within the folded ensembles of other beta-trefoil proteins, such as IL-1beta and hFGF-1.

Quantitative Evaluation of Protein Solubility in Aqueous Solutions by PEG-Induced Liquid-Liquid Phase Separation.

This chapter describes an experimental method to quantitatively evaluate the solubility of proteins in aqueous solutions. Measurement of protein solubility can be challenging because low solubility can be manifested through various pathways (e.g., crystallization, aggregation, gelation, and liquid-liquid phase separation), some of which may occur over long periods of time. In the method described here, a nonionic polymer, polyethylene glycol (PEG), is added to a protein solution of interest to induce instantaneous formation of protein-rich liquid droplets. After incubation at a given temperature, the samples are centrifuged. The protein concentration in the supernatant is measured at various PEG concentrations to calculate an equilibrium binding free energy, which provides a measure of protein solubility. Based on the first principles of thermodynamics, this method is highly reproducible and applicable to various proteins and buffer conditions.

Biophysical characterization of structural properties and folding of interleukin-1 receptor antagonist.

Structural properties and folding of interleukin-1 receptor antagonist (IL-1ra), a therapeutically important cytokine with a symmetric beta-trefoil topology, are characterized using optical spectroscopy, high-resolution NMR, and size-exclusion chromatography. Spectral contributions of two tryptophan residues, Trp17 and Trp120, present in the wild-type protein, have been determined from mutational analysis. Trp17 dominates the emission spectrum of IL-1ra, while Trp120 is quenched presumably by the nearby cysteine residues in both folded and unfolded states. The same Trp17 gives rise to two characteristic negative peaks in the aromatic CD. Urea denaturation of the wild-type protein is probed by measuring intrinsic and extrinsic (binding of 1-anilinonaphthalene-8-sulfonic acid) fluorescence, near- and far-UV CD, and 1D and 2D ((1)H-(15)N heteronuclear single quantum coherence (HSQC)) NMR. Overall, the data suggest an essentially two-state equilibrium denaturation mechanism with small, but detectable structural changes within the pretransition region. The majority of the (1)H-(15)N HSQC cross-peaks of the folded state show only a limited chemical shift change as a function of the denaturant concentration. However, the amide cross-peak of Leu31 demonstrates a significant urea dependence that can be fitted to a two-state binding model with a dissociation constant of 0.95+/-0.04 M. This interaction has at least a five times higher affinity than reported values for nonspecific urea binding to denatured proteins and peptides, suggesting that the structural context around Leu31 stabilizes the protein-urea interaction. A possible role of denaturant binding in inducing the pretransition changes in IL-1ra is discussed. Urea unfolding of wild-type IL-1ra is sufficiently slow to enable HPLC separation of folded and unfolded states. Quantitative size-exclusion chromatography has provided a hydrodynamic view of the kinetic denaturation process. Thermodynamic stability and unfolding kinetics of IL-1ra resemble those of structurally and evolutionary close IL-1beta, suggesting similarity of their free energy landscapes.

Structural characterization of an equilibrium unfolding intermediate in cytochrome c.

Although the denaturant-induced unfolding transition of cytochrome c was initially thought to be a cooperative process, recent spectroscopic studies have shown deviations from two-state behavior consistent with accumulation of an equilibrium intermediate. However, little is known about the structural and thermodynamic properties of this state, and whether it is stabilized by the presence of non-native heme ligands. We monitored the reversible denaturant-induced unfolding equilibrium of oxidized horse cytochrome c using various spectroscopic probes, including fluorescence, near and far-UV CD, heme absorbance bands in the Soret, visible and near-IR regions of the spectrum, as well as 2D NMR. Global fitting techniques were used for a quantitative interpretation of the results in terms of a three-state model, which enabled us to determine the intrinsic spectroscopic properties of the intermediate. A well-populated intermediate was observed in equilibrium experiments at pH 5 using either guanidine-HCl or urea as a denaturant, both for wild-type cytochrome c as well as an H33N mutant chosen to prevent formation of non-native His-heme ligation. For a more detailed structural characterization of the intermediate, we used 2D 1H-15N correlation spectroscopy to follow the changes in peak intensity for individual backbone amide groups. The equilibrium state observed in our optical and NMR studies contains many native-like structural features, including a well-structured alpha-helical sub-domain, a short Trp59-heme distance and solvent-shielded heme environment, but lacks the native Met80 sulfur-iron linkage and shows major perturbations in side-chain packing and other tertiary interactions. These structural properties are reminiscent of the A-state of cytochrome c, a compact denatured form found under acidic high-salt conditions, as well as a kinetic intermediate populated at a late stage of folding. The denaturant-induced intermediate also resembles alkaline forms of cytochrome c with altered heme ligation, suggesting that disruption of the native methionine ligand favors accumulation of structurally analogous states both in the presence and absence of non-native ligands.

Free fatty acid particles in protein formulations, part 2: contribution of polysorbate raw material.

Polysorbate 20 (PS20) is a nonionic surfactant frequently used to stabilize protein biopharmaceuticals. During the development of mAb formulations containing PS20, small clouds of particles were observed in solutions stored in vials. The degree of particle formation was dependent on PS20 concentration. The particles were characterized by reversed-phase HPLC after dissolution and labeling with the fluorescent dye 1-pyrenyldiazomethane. The analysis showed that the particles consisted of free fatty acids (FFAs), with the distribution of types consistent with those found in the PS20 raw material. Protein solutions formulated with polysorbate 80, a chemically similar nonionic surfactant, showed a substantial delay in particle formation over time compared with PS20. Multiple lots of polysorbates were evaluated for FFA levels, each exhibiting differences based on polysorbate type and lot. Polysorbates purchased in more recent years show a greater distribution and quantity of FFA and also a greater propensity to form particles. This work shows that the quality control of polysorbate raw materials could play an important role in biopharmaceutical product quality.

Remediating agitation-induced antibody aggregation by eradicating exposed hydrophobic motifs.

Therapeutic antibodies must encompass drug product suitable attributes to be commercially marketed. An undesirable antibody characteristic is the propensity to aggregate. Although there are computational algorithms that predict the propensity of a protein to aggregate from sequence information alone, few consider the relevance of the native structure. The Spatial Aggregation Propensity (SAP) algorithm developed by Chennamsetty et. al. incorporates structural and sequence information to identify motifs that contribute to protein aggregation. We have utilized the algorithm to design variants of a highly aggregation prone IgG2. All variants were tested in a variety of high-throughput, small-scale assays to assess the utility of the method described herein. Many variants exhibited improved aggregation stability whether induced by agitation or thermal stress while still retaining bioactivity.

Native-state solubility and transfer free energy as predictive tools for selecting excipients to include in protein formulation development studies.

In the present report, two formulation strategies, based on different aggregation models, were compared for their ability to quickly predict which excipients (cosolutes) would minimize the aggregation rate of an immunoglobulin G1 monoclonal antibody (mAb-1) stored for long term at refrigerated and room temperatures. The first formulation strategy assumed that a conformational change to an aggregation-prone intermediate state was necessary to initiate the association process and the second formulation strategy assumed that protein self-association was instead controlled by the solubility of the native state. The results of these studies indicate that the stabilizing effect of excipients formulated at isotonic concentrations is derived from their ability to solubilize the native state, not by the increase of protein conformational stability induced by their presence. The degree the excipients solvate the native state was determined from the apparent transfer free energy of the native state from water into each of the excipients. These values for mAb-1 and two additional therapeutic antibodies correlated well to their long-term 4°C and room temperature aggregation data and were calculated using only the literature values for the apparent transfer free energies of the amino acids into the various excipients and the three-dimensional models of the antibodies.

Effect of benzyl alcohol on recombinant human interleukin-1 receptor antagonist structure and hydrogen-deuterium exchange.

Benzyl alcohol, a preservative commonly added to multidose therapeutic protein formulations, can accelerate aggregation of recombinant human interleukin-1 receptor antagonist (rhIL-1ra). To investigate the interactions between benzyl alcohol and rhIL-1ra, we used nuclear magnetic resonance to observe the effect of benzyl alcohol on the chemical shifts of amide resonances of rhIL-1ra and to measure hydrogen-deuterium exchange rates of individual rhIL-1ra residues. Addition of 0.9% benzyl alcohol caused significant chemical shifts of amide resonances for residues 90-97, suggesting that these solvent-exposed residues participate in the binding of benzyl alcohol. In contrast, little perturbation of exchange rates was observed in the presence of either sucrose or benzyl alcohol.

The use of native cation-exchange chromatography to study aggregation and phase separation of monoclonal antibodies.

This study introduces a novel analytical approach for studying aggregation and phase separation of monoclonal antibodies (mAbs). The approach is based on using analytical scale cation-exchange chromatography (CEX) for measuring the loss of soluble monomer in the case of individual and mixed protein solutions. Native CEX outperforms traditional size-exclusion chromatography in separating complex protein mixtures, offering an easy way to assess mAb aggregation propensity. Different IgG1 and IgG2 molecules were tested individually and in mixtures consisting of up to four protein molecules. Antibody aggregation was induced by four different stress factors: high temperature, low pH, addition of fatty acids, and rigorous agitation. The extent of aggregation was determined from the amount of monomeric protein remaining in solution after stress. Consequently, it was possible to address the role of specific mAb regions in antibody aggregation by co-incubating Fab and Fc fragments with their respective full-length molecules. Our results revealed that the relative contribution of Fab and Fc regions in mAb aggregation is strongly dependent on pH and the stress factor applied. In addition, the CEX-based approach was used to study reversible protein precipitation due to phase separation, which demonstrated its use for a broader range of protein-protein association phenomena. In all cases, the role of Fab and Fc was clearly dissected, providing important information for engineering more stable mAb-based therapeutics.

Investigation of degradation processes in IgG1 monoclonal antibodies by limited proteolysis coupled with weak cation-exchange HPLC.

A new cation-exchange high-performance liquid chromatography (HPLC) method that separates fragment antigen-binding (Fab) and fragment crystallizable (Fc) domains generated by the limited proteolysis of monoclonal antibodies (mAbs) was developed. This assay has proven to be suitable for studying complex degradation processes involving various immunoglobulin G1 (IgG1) molecules. Assignment of covalent degradations to specific regions of mAbs was facilitated by using Lys-C and papain to generate Fab and Fc fragments with unique, protease-dependent elution times. In particular, this method was useful for characterizing protein variants formed in the presence of salt under accelerated storage conditions. Two isoforms that accumulated during storage were readily identified as Fab-related species prior to mass-spectrometric analysis. Both showed reduced biological activity likely resulting from modifications within or in proximity of the complementarity-determining regions (CDRs). Utility of this assay was further illustrated in the work to characterize light-induced degradations in mAb formulations. In this case, a previously unknown Fab-related species which populated upon light exposure was observed. This species was well resolved from unmodified Fab, allowing for direct and high-purity fractionation. Mass-spectrometric analysis subsequently identified a histidine-related degradation product associated with the CDR2 of the heavy chain. In addition, the method was applied to assess the structural organization of a noncovalent IgG1 dimer. A new species corresponding to a Fab-Fab complex was found, implying that interactions between Fab domains were responsible for dimerization. Overall, the data presented demonstrate the suitability of this cation-exchange HPLC method for studying a wide range of covalent and noncovalent degradations in IgG1 mAbs.

High throughput thermostability screening of monoclonal antibody formulations.

Differential scanning fluorimetry (DSF) was employed to increase the throughput of the thermostability screening of monoclonal antibody (mAb) formulations. The method consists of measuring the fluorescence intensity of a polarity sensitive probe at gradually increasing temperatures, and obtaining the transition temperature of exposure of the hydrophobic regions of proteins (T(h)). The change in fluorescence intensity was directly related to protein unfolding levels and temperatures. The results from thermostability measurements were compared with the data acquired using differential scanning calorimetry (DSC), and a good correlation between T(h) and the temperature of protein unfolding or melting (T(m)) was observed. The method was applied to screen four mAb molecules in 84 different buffers. The studies revealed a good correlation of T(h) values with the known effects of pH and excipients on protein stability in solution. Specifically, the elevated aggregation levels induced by salt, low pH, and high protein concentrations could be successfully predicted by this thermal stability screening. This method is efficient, with high throughput capability, and could be widely applied in the biopharmaceutical industry for formulation and process development, and characterization.

Characterization of a unique IgG1 mAb CEX profile by limited Lys-C proteolysis/CEX separation coupled with mass spectrometry and structural analysis.

The unique cation exchange chromatography (CEX) charge variant profile of mAb1 is characterized by a combination of mass spectrometry, limited Lys-C digestion followed by CEX separation and structural analysis. During CEX method development, mAb1 showed several unexpected phenomena, including a unique profile containing two main species (acidic 2 and main) and significant instability during stability studies of the main species. Reduced Lys-C peptide mapping identified a small difference in one of the heavy chain peptides (H4) in acidic 2 and further mass analysis identified this difference as Asn55 deamidation. However, the amount of Asn55 deamidation in acidic 2 could account for only half of the species present in this peak. Lys-C limited digest followed by CEX separated several unique peaks in the acidic peak 2 including two pre Fab peaks (LCC1 and LCC2). Whole protein mass analysis suggested that both LCC1 and LCC2 were potentially deamidated species. Subsequent peptide mapping with MS/MS determined that LCC1 contained isoAsp55 and LCC2 contained Asp55. Combining LCC1 and LCC2 CEX peak areas could account for nearly all of the species present in acidic peak 2. Subsequent detailed sequence analysis combined with molecular modeling identified Asn55 and its surrounding residues are responsible for the different CEX behavior and instability of mAb1 following forced degradation at high pH. Overall, the combinatorial approach used in this study proved to be a powerful tool to understand the unique charge variant and stability profile of a monoclonal antibody.

Succinimide formation at Asn 55 in the complementarity determining region of a recombinant monoclonal antibody IgG1 heavy chain.

We investigated the formation and stability of succinimide, an intermediate of deamidation events, in recombinant monoclonal antibodies (mAbs). During the course of an analytical development study of an IgG1 mAbs, we observed that a specific antibody population could be separated from the main product by cation-exchange (CEX) chromatography. The cell-based bioassay measured a approximately 70% drop in potency for this fraction. Liquid chromatography time-of-flight mass spectrometry (LC-TOF/MS) and tandem mass spectrometry (LC-MS/MS) analyses showed that the modified CEX fraction resulted from the formation of a succinimide intermediate at Asn 55 in the complementarity determining region (CDR) of the heavy chain. Biacore assay revealed a approximately 50% decrease in ligand binding activity for the succinimide-containing Fab with respect to the native Fab. It was found that the succinimide form existed as a stable intermediate with a half-life of approximately 3 h at 37 degrees C and pH 7.6. Stress studies indicated that mildly acidic pH conditions (pH 5) favored succinimide accumulation, causing a gradual loss in potency. Hydrolysis of the succinimide resulted in a further drop in potency. The implications of the succinimide formation at Asn 55, a highly conserved residue among IgG1 (mAbs), are discussed.