Understanding the Increased Aggregation Propensity of a Light-Exposed IgG1 Monoclonal Antibody Using Hydrogen Exchange Mass Spectrometry,Biophysical Characterization,and Structural Analysis

This work compares the conformational stability, backbone flexibility, and aggregation propensity of monomer and dimer fractions of an IgG1 monoclonal antibody (mAb) generated on UVA light exposure for up to 72 h collected by preparative size-exclusion chromatography, compared with unstressed control. UVA light exposure induced covalent aggregation, and fragmentation as measured by size-exclusion chromatography, sodium dodecyl sulfate polyacrylamide gel electrophoresis, and extensive oxidation of specific methionine residues (Met 257, Met 433, and Met 109) in both size fractions identified by reverse phase chromatography coupled to mass spectrometry. Compared with unstressed mAb, both the monomer and dimer fractionated from 72 h UVA light–exposed mAb had decreased thermal melting temperatures (T_m1) by 1.4°C as measured by differential scanning calorimetry, minor changes in tertiary structure as measured by near-UV CD, increased monomer loss, and aggregation on accelerated storage at 35°C. Hydrogen/deuterium exchange mass spectrometry identified local segments with increased flexibility in C_H2 and C_H3 domains of both size fractions, and decreased flexibility in few segments of F_ab and C_H1 domains in the dimer fraction. Segment 247-256 in heavy chain, an established aggregation hotspot in IgG1 mAbs had large increase in flexibility in both size fractions compared with unstressed mAb.     

Investigating Monoclonal Antibody Aggregation Using a Combination of H/DX-MS and Other Biophysical Measurements

To determine how structural changes in antibodies are connected with aggregation, the structural areas of an antibody prone to and/or impacted by aggregation must be identified. In this work, the higher-order structure and biophysical properties of two different monoclonal antibody (mAb) monomers were compared with their simplest aggregated form, that is, dimers that naturally occurred during normal production and storage conditions. A combination of hydrogen/deuterium exchange mass spectrometry and other biophysical measurements was used to make the comparison. The results show that the dimerization process for one of the mAb monomers (mAb1) displayed no differences in its deuterium uptake between monomer and dimer forms. However, the other mAb monomer (mAb2) showed subtle changes in hydrogen/deuterium exchange as compared with its dimer form. In this case, differences observed were located in specific functional regions of the C_H2 domain and the hinge region between C_H1 and C_H2 domains. The importance and the implications of these changes on the antibody structure and mechanism of aggregation are discussed.     

The Use of a GroEL-BLI Biosensor to Rapidly Assess Preaggregate Populations for Antibody Solutions Exhibiting Different Stability Profiles

An automated method using biotinylated GroEL-streptavidin biosensors with biolayer interferometry (GroEL-BLI) was evaluated to detect the formation of transiently formed, preaggregate species in various pharmaceutically relevant monoclonal antibody (mAb) samples. The relative aggregation propensity of various IgG1 and IgG4 mAbs was rank ordered using the GroEL-BLI biosensor method, and the least stable IgG4 mAb was subjected to different stresses including elevated temperatures, acidic pH, and addition of guanidine HCl. The GroEL-BLI biosensor detects mAb preaggregate formation mostly before, or sometimes concomitantly with, observing soluble aggregates and subvisible particles using size-exclusion chromatography and microflow imaging, respectively. A relatively unstable bispecific antibody (Bis-3) was shown to bind the GroEL biosensor even at low temperatures (25^°C). During thermal stress (50°C, 1 h), increased Bis-3 binding to GroEL-biosensors was observed prior to aggregation by size-exclusion chromatography or microflow imaging. Transmission electron microscopy analysis of Bis-3 preaggregate GroEL complexes revealed, in some cases, potential hydrophobic interaction sites between the Fc domain of the Bis-3 and GroEL protein. The automated BLI method not only enables detection of transiently formed preaggregate species that initiate protein aggregation pathways but also permits rapid mAb formulation stability assessments at low volumes and low protein concentrations.     

Deciphering the High Viscosity of a Therapeutic Monoclonal Antibody in High Concentration Formulations by Microdialysis-Hydrogen/Deuterium Exchange Mass Spectrometry

High concentration formulations of therapeutic monoclonal antibodies (mAbs) are highly desired for subcutaneous injection. However, high concentration formulations can exhibit unusual molecular behaviors, such as high viscosity or aggregation, that present challenges for manufacturing and administration. To understand the molecular mechanism of the high viscosity exhibited by high concentration protein formulations, we analyzed a human IgG4 (mAb1) at high protein concentrations using sedimentation velocity analytical ultracentrifugation (SV-AUC), X-ray crystallography, hydrogen/deuterium exchange mass spectrometry (HDX-MS), and protein surface patches analysis. Particularly, we developed a microdialysis HDX-MS method to determine intermolecular interactions at different protein concentrations. SV-AUC revealed that mAb1 displayed a propensity for self-association of Fab-Fab, Fab-Fc, and Fc-Fc. mAb1 crystal structure and HDX-MS results demonstrated self-association between complementarity-determining regions (CDRs) and Fc through electrostatic interactions. HDX-MS also indicated Fab-Fab interactions through hydrophobic surface patches constructed by mAb1 CDRs. Our multi-method approach, including fast screening of SV-AUC as well as interface analysis by X-ray crystallography and HDX-MS, helped to elucidate the high viscosity of mAb1 at high concentrations as induced by self-associations of Fab-Fc and Fab-Fab.     

Correlating Excipient Effects on Conformational and Storage Stability of an IgG1 Monoclonal Antibody with Local Dynamics as Measured by Hydrogen/Deuterium-Exchange Mass Spectrometry

The effects of sucrose and arginine on the conformational and storage stability of an IgG1 monoclonal antibody (mAb) were monitored by differential scanning calorimetry (DSC) and size-exclusion chromatography (SEC), respectively. Excipient effects on protein physical stability were then compared with their effects on the local flexibility of the mAb in solution at pH 6, 25°C using hydrogen/deuterium-exchange mass spectrometry (H/D-MS). Compared with a 0.1 M NaCl control, sucrose (0.5 M) increased conformational stability (T_m values), slowed the rate of monomer loss, reduced the formation of insoluble aggregates, and resulted in a global trend of small decreases in local flexibility across most regions of the mAb. In contrast, the addition of arginine (0.5 M) decreased the mAb's conformational stability, increased the rate of loss of monomer with elevated levels of soluble and insoluble aggregates, and led to significant increases in the local flexibility in specific regions of the mAb, most notably within the constant domain 2 of the heavy chain (C_H2). These results provide new insights into the effect of sucrose and arginine on the local dynamics of IgG1 domains as well as preliminary correlations between local flexibility within specific segments of the C_H2 domain (notably heavy chain 241–251) and the mAb's overall physical stability. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:2136–2151, 2013     

Reformulation and Thermal Stability of a Therapeutic Anti-Cocaine mAb

We concentrated and reformulated the anti-cocaine mAb, h2E2, to reduce the amount of sucrose and histidine buffer infused with the mAb, to satisfy FDA maximum exposure levels for those components for use in clinical trials. After concentration of the original 20 mg/ml mAb, 4 reformulation buffers were evaluated for suitability. The concentration of histidine was reduced from 10 mM to 3 or 0 mM, and the concentration of sucrose reduced from 10% to 2, 4, or 6%. The approximately 100 mg/ml reformulated mAb samples were analyzed for oligomer formation, aggregation, concentration of the emulsifier polysorbate 80, and thermal stability. These reformulated mAb samples were also assessed for their stability at 40°C from 1 day to 12 weeks. As expected, long term thermal resistance to oligomer formation increased as a function of increasing sucrose concentration. Interestingly, unbuffered reformulated mAb displayed a less than or equal to tendency to form oligomers and aggregates, compared to the histidine buffered samples. Importantly, even after 12 weeks at 40°C, all the reformulated samples displayed little aggregation, and bound their antigen (cocaine) with identical affinities and thermodynamics, as measured by isothermal titration calorimetry (ITC). These ITC thermodynamic binding parameters are consistent with recently published values for the original formulation of this mAb. In all reformulated samples there was a slight decrease in the number of cocaine binding sites after 12 weeks at 40°C, likely due to the parallel small increase in soluble oligomeric antibody, suggesting that soluble oligomeric mAb may no longer bind cocaine with high affinity.     

Clip Formation in the Complementarity Determining Region of Bevacizumab Lowers Monomer Stability and Affinity for Both FcRn and FcγR: A Comprehensive Characterization of the Clipped Variant Including its Higher Order Structure

The presence of monoclonal antibody (mAb) fragments in pharmaceutical mAb products is a critical quality attribute and should be controlled for safety. Several mAb fragments derived from clip formation in the complementarity determining regions (CDRs), as well as from cleavage in the hinge region, have been reported. However, the properties of CDR-clipped variants are not fully understood because of difficulties in separating them from intact mAbs under non-denaturing conditions due to similarities in size. We have established a method for separating CDR-clipped variants under non-denaturing conditions using an appropriate size exclusion chromatography column.¹ In this report, we provide a comprehensive characterization of a CDR-clipped variant from bevacizumab. The variant exhibited a lower pI, a higher tendency to form dimers, and a lower affinity for both neonatal Fc receptor (FcRn) and Fcγ receptor (FcγR). The effects of clip formation in CDR H3 on the higher order structure were analyzed by hydrogen/deuterium exchange mass spectrometry, and the observed changes in the structures of the VH, CH2, and VL domains were in agreement with the lowered affinity for antigen, FcRn, and FcγR. These findings suggest that clip formation in the CDR may affect the efficacy, safety, and pharmacokinetics of pharmaceutical mAbs.     

Kinetics and Characterization of Non-enzymatic Fragmentation of Monoclonal Antibody Therapeutics

Purpose

To understand non-enzymatic hydrolytic fragmentation of a monoclonal antibody therapeutic under temperature stressed conditions and investigating possible mechanism for the same.

Methods

The mAb therapeutic was incubated at 50°C in phosphate buffer at pH 6.5 and fragmentation was monitored at different ionic strengths under stressed conditions. The incubated mAb was sampled at regular time intervals by analytical Size Exclusion Chromatography (SEC).

Results

It was observed that 57% of the mAb product fragmented over 4 days into two fragment species – Fc-Fab and Fab with molecular weights of 97 KDa and 47 KDa, respectively, as measured by mass spectrometry (MS) and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The fragmentation rate was slow initially and then accelerated with time. No change in % aggregate level was observed in this duration, implying that degradation was primarily via fragmentation at high temperature. Kinetics of hydrolytic fragmentation was hypothesized and SEC data was fitted to estimate the kinetic rate constants. While degradation of the monomer into fragment species was non-Arrhenius with a negative activation energy, further degradation of Fab-Fc fragments into Fab or Fc fragments followed Arrhenius Law with an activation energy of 2.1 and 15.38 kcal/mol, respectively.

Conclusion

High temperature (50°C) causes mAb to cleave at the hinge region to form Fab-Fc and Fab/Fc, as confirmed by dynamic light scattering, SDS-PAGE, SEC, and MS. A kinetic model for hydrolytic fragmentation has been proposed. The results are expected to assist end users in formulation development as well as in monitoring stability of biotherapeutic products.

     

Effects of Temperature and Osmolytes on Competing Degradation Routes for an IgG1 Antibody

Addition of excipients is a common strategy to slow protein aggregation during storage. Excipient effects on the mechanism(s) and temperature (T) dependence of aggregation for a monoclonal antibody solution were tested using size-exclusion chromatography, differential scanning calorimetry (DSC), temperature scanning monomer loss (TSML), and laser light scattering; previous work in buffer-only conditions had shown non-Arrhenius behavior and implicated Fab and/or C_H3 unfolding as a key step in aggregation. Excipients included citrate, amino acid salts (histidine–HCl, arginine–HCl), and polyols (mannitol and glycerol). DSC and TSML showed that Fab, rather than C_H3, unfolding corresponded with the onset of aggregation for each condition. Isothermal incubation at 56.5°C, 40°C, and 2°C–8°C resulted in aggregation, while fragmentation occurred readily at only 40°C. The primary effect of the different excipients appeared to be preferential accumulation/exclusion, affecting the concentrations of partially unfolded monomer key intermediates. In addition, aggregation rates were clearly non-Arrhenius, causing aggregation to dominate over fragmentation at high and low T, and making long-term stability predictions problematic based on commonly employed 40°C conditions. Possible reasons for non-Arrhenius behavior include a strong T-dependence of the Fab unfolding enthalpy and/or a switch from Fab-mediated to Fc-mediated aggregation as one moves from high to low T.     

Evaluation of a Dual-Wavelength Size Exclusion HPLC Method With Improved Sensitivity to Detect Protein Aggregates and Its Use to Better Characterize Degradation Pathways of an IgG1 Monoclonal Antibody

The evaluation of a dual wavelength size exclusion high performance liquid chromatography (DW-SE-HPLC) method with improved sensitivity to detect aggregates in a high concentration IgG1 monoclonal antibody formulation is presented. This technique utilizes ultraviolet detection at two different wavelengths to monitor the levels of monomer, aggregate, and fragments and was shown to have improved sensitivity for the detection aggregates and fragments compared to light scattering (LS) detection. After assay optimization including the use of column conditioning, the limit of quantitation for aggregates was determined to be 0.04% with essentially complete recovery of aggregates from the column (> 99.5%). The DW-SE-HPLC method was used to evaluate the level of protein aggregates generated by different environmental conditions such as exposure to elevated temperatures/acidic pH or intense light. The detection and characterization of protein aggregates by DW-SE-HPLC was compared with an orthogonal biophysical technique (sedimentation velocity analytical ultracentrifugation, SV-AUC). A good overall correlation was observed for levels of monomer, aggregates (dimer and multimers), and fragments as measured by the two analytical techniques (e.g., 6.0% vs. 5.3% and 14% vs. 11% for dimeric aggregates generated by elevated temperature/acidic pH and light exposure, respectively). The stability profile of a high concentration IgG1 monoclonal antibody formulation was investigated under stressed storage conditions (40 °C over 3 months) using the DW-SE-HPLC method including the loss of monomeric species with the concomitant accumulation of both aggregates and fragments. The nature and composition of the aggregates (primarily noncovalent dimers) and fragments (primarily loss of Fab from an intact IgG1) formed during storage were further characterized by a combination of LS measurements and mass spectroscopy analysis of deglycosylated IgG1 samples isolated by preparative SE-HPLC. The combination of DW-SE-HPLC, SV-AUC, LS, and mass spectroscopy results provided a detailed overall understanding the monomer, aggregate, fragment degradation pathway(s) for a high concentration IgG1 monoclonal antibody formulation during storage.     

Monoclonal Antibodies Follow Distinct Aggregation Pathways During Production-Relevant Acidic Incubation and Neutralization

Purpose

Aggregation aspects of therapeutic monoclonal antibodies (mAbs) are of common concern to the pharmaceutical industry. Low pH treatment is applied during affinity purification and to inactivate endogenous retroviruses, directing interest to the mechanisms of acid-induced antibody aggregation.

Methods

We characterized the oligomerization kinetics at pH 3.3, as well as the reversibility upon neutralization, of three model mAbs with identical variable regions, representative of IgG1, IgG2 and IgG4 respectively. We applied size-exclusion high performance liquid chromatography and orthogonal analytical methods, including small-angle X-ray scattering and dynamic light scattering and supplemented the experimental data with crystal structure-based spatial aggregation propensity (SAP) calculations.

Results

We revealed distinct solution behaviors between the three mAb models: At acidic pH IgG1 retained monomeric, whereas IgG2 and IgG4 exhibited two-phase oligomerization processes. After neutralization, IgG2 oligomers partially reverted to the monomeric state, while on the contrary, IgG4 oligomers tended to aggregate. Subclass-specific aggregation-prone motifs on the Fc fragments were identified, which may lead to two distinct pathways of reversible and irreversible aggregation, respectively.

Conclusions

We conclude that subtle variations in mAb sequence greatly affect responses towards low-pH incubation and subsequent neutralization, and demonstrate how orthogonal biophysical methods distinguish between reversible and irreversible mAb aggregation pathways at early stages of acidic treatment.

     

High-Throughput Biophysical Analysis and Data Visualization of Conformational Stability of an IgG1 Monoclonal Antibody After Deglycosylation

The structural integrity and conformational stability of an IgG1 monoclonal antibody (mAb), after partial or complete enzymatic removal of the N-linked Fc glycan, were compared with the untreated mAb over a wide range of temperature (10 °C-90 °C) and solution pH (3-8) using circular dichroism, fluorescence spectroscopy, and static light scattering combined with data visualization employing empirical phase diagrams. Subtle-to-larger stability differences between the different glycoforms were observed. Improved detection of physical stability differences was then demonstrated over narrower pH range (4.0-6.0) using smaller temperature increments, especially when combined with an alternative data visualization method (radar plots). Differential scanning calorimetry and differential scanning fluorimetry were then utilized and also showed an improved ability to detect differences in the physical stability of a mAb glycoform. On the basis of these results, a two-step methodology was used in which conformational stability of a mAb glycoform is first screened with a wide variety of instruments and environmental stresses, followed by a second evaluation with optimally sensitive experimental conditions, analytical techniques, and data visualization methods. With this approach, a high-throughput biophysical analysis to assess relatively subtle conformational stability differences in protein glycoforms is demonstrated.     

Evaluation of Effects of Fc Domain High-Mannose Glycan on Antibody Stability

Elevated levels of CH2 domain N-linked high-mannose (HM) glycans are commonly observed in therapeutic monoclonal antibodies at various stages of the development. The effect of HM glycans on antibody stability was evaluated by using two approaches. In the first approach, immunoglobulin G (IgG) 1 material containing 21% HM was incubated at 29°C for 6 weeks and fractionated into monomeric and aggregate species by using size-exclusion chromatography (SEC). These fractions were analyzed for the levels of HM. No significant difference was observed in the amount of HM in aggregate and monomer fractions indicating that the HM-containing fractions did not have a greater tendency to form aggregates. In the second approach, both IgG1 material and IgG2 material were separated by Concanavalin-A affinity chromatography into a HM-enriched fraction and a HM-depleted fraction, respectively. Real-time and accelerated stability studies were carried out with these fractions together with untreated samples under standard formulation conditions. The stability of these fractions over time was monitored using SEC and cation-exchange chromatography. No significant difference was observed in rates of aggregation or charge variant formation. These data indicate that HM glycans had no effect on the IgG1 and IgG2 product's stability under the formulation conditions studied.     

Biophysical Properties and Heating-Induced Aggregation of Lysine-Conjugated Antibody-Drug Conjugates

The commercially available antibody-drug conjugate (ADC) product, Kadcyla^® is synthesized using a 2-step reaction, wherein the linker is conjugated to native lysines on the mAb in step 1, followed by drug conjugation to the linker-modified antibody in step 2. In our study, we synthesized a lysine-conjugated ADC (Syn-ADC) on the same trastuzumab scaffold as Kadcyla^® using a 1-step reaction. Mass spectrometry of both products revealed a subpopulation of Kadcyla^® containing free linkers conjugated to the mAb, but not conjugated to the drug, which were absent in the 1-step reaction ADC product. Differential scanning calorimetry thermograms showed that the drug and linker conjugation significantly reduced the thermal stability and energies of activation for the denaturation of the C_H2 domain of the ADCs. The heating induced aggregation events started as early as ～57°C and ～45°C for Kadcyla^® and Syn-ADC, respectively, compared with 71°C for Herceptin^®. The colloidal stability measurements clearly showed that the hydrophobic drug payload on ADCs significantly reduced the repulsive interprotein interactions when compared to the unconjugated antibody under formulation buffer conditions (pH 6.0). Attaching hydrophobic drug and linker moieties onto the antibody lowered the thermal and colloidal stabilities and increased the aggregation propensity of the ADCs.     

Quantitative in vivo comparisons of the Fc gamma receptor-dependent agonist activities of different fucosylation variants of an immunoglobulin G antibody

Although it has been shown that functions of immunoglobulin G (IgG) antibodies (Abs) that depend on binding to certain Fc gamma receptors (Fc gamma R) can be influenced by Fc glycan fucosylation, quantitative in vivo analyses comparing the effects of different levels of fucose are still lacking. We used a simple mouse model to compare Fc gamma R-dependent T cell activation induced by different fucosylation variants of a hamster/human IgG1 chimeric version of anti-mouse CD3 monoclonal Ab, 145-2C11 (2C11). Initial studies supported the expectation that this agonist activity by 2C11 was a reflection of Fc gamma R binding, including comparisons of human IgG1 and IgG4 variants of 2C11 that showed the IgG4 to be dramatically less active at inducing T cell activation. Dose-response analyses in mice then showed that a sample of the human IgG1 version of 2C11 Ab in which 40% of the Fc glycans in the population of Ab molecules were fucosylated was 3-5 times more potent than a sample with 90% of its Fc glycans fucosylated. A sample with 10% fucosylation showed the same activity as the 40% fucosylated sample, revealing that complete absence of fucose was not necessary to achieve maximal Fc function in this model. In vitro binding to recombinant mouse Fc gamma Rs by the 2C11 variants revealed interesting relationships between fucose content and receptor affinity, and suggested the involvement of Fc gamma RIV in mediating 2C11 activity in vivo. These analyses showed that low-fucose human IgG1 Abs indeed show greater Fc gamma R-dependent activities in mice, but that Abs with moderate levels of fucose may be just as potent as Abs with very low or no fucose.     

Equilibrium studies of protein aggregates and homogeneous nucleation in protein formulation

Shaking or heat stress may induce protein aggregates. Aggregation behavior of an IgG1 stressed by shaking or heat following static storage at 5 and 25°C was investigated to determine whether protein aggregates exist in equilibrium. Aggregates were detected using different analytical methods including visual inspection, turbidity, light obscuration, size exclusion chromatography, and dynamic light scattering. Significant differences were evident between shaken and heated samples upon storage. Visible and subvisible particles (insoluble aggregates), turbidity and z-average diameter decreased whilst soluble aggregate content increased in shaken samples over time. Insoluble aggregates were considered to be reversible and dissociate into soluble aggregates and both aggregate types existed in equilibrium. Heat-induced aggregates had a denatured protein structure and upon static storage, no significant change in insoluble aggregates content was shown, whilst changes in soluble aggregates content occurred. This suggested that heat-induced insoluble aggregates were irreversible and not in equilibrium with soluble aggregates. Additionally, the aggregation behavior of unstressed IgG1 after spiking with heavily aggregated material (shaken or heat stressed) was studied. The aggregation behavior was not significantly altered, independent of the spiking concentration over time. Thus, neither mechanically stressed native nor temperature-induced denatured aggregates were involved in nucleating or propagating aggregation. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99:632–644, 2010     

Stability of Monoclonal Antibodies at High-Concentration: Head-to-Head Comparison of the IgG1 and IgG4 Subclass

Few studies have so far directly compared the impact of antibody subclass on protein stability. This case study investigates two mAbs (one IgG₁ and one IgG₄) with identical variable region. Investigations of mAbs that recognize similar epitopes are necessary to identify possible differences between the IgG subclasses. Both physical and chemical stability were evaluated by applying a range of methods to measure formation of protein aggregates [sizeexclusion chromatography (SEC)–HPLC and UV340 nm], structural integrity (circular dichroism and FTIR), thermodynamic stability (differential scanning calorimetry), colloidal interactions (dynamic light scattering), and fragmentation and deamidation (SEC–HPLC and capillary isoelectric focusing). The impact of pH (4–9) and ionic strength (10 and 150 mm) was investigated using highlyconcentrated (150 mg/mL) mAb formulations. Lower conformational stability was identified for the IgG₄ resulting in increased levels of soluble aggregates. The IgG₁ was chemically less stable as compared with the IgG₄, presumably because of the higher flexibility in the IgG₁ hinge region. The thermodynamic stability of individual mAb domains was also addressed in detail. The stability of our mAb molecules is clearly affected by the IgG framework, and this study suggests that subclass switching may alter aggregation propensity and aggregation pathway and thus potentially improve the overall formulation stability while retaining antigen specificity. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 103:115–127, 2014     

High Throughput Prediction Approach for Monoclonal Antibody Aggregation at High Concentration

Purpose

Characterization of the monoclonal antibody aggregation process and identification of stability factors that could be used as indicators of aggregation propensity with an emphasis on a large number of samples and low protein material consumption.

Methods

Differential scanning calorimetry, dynamic light scattering and size exclusion chromatography were used as the main methodological approaches. Conformational stability, colloidal stability and aggregation kinetics were assessed for two different IgG monoclonal antibody (mAbs) subclasses. Aggregation was induced by exposing the mAbs to 55°C for 3 weeks. mAb samples were prepared in different formulations and concentrations from 1 mg/mL to 50 mg/mL.

Results

High temperature stress of mAb samples revealed that monoclonal antibodies followed first order aggregation kinetics, which suggests that the rate-limiting step of monomer loss was unimolecular. Conformational stability of mAbs was estimated with denaturation temperature measurements. Colloidal stability was assessed with dynamic interaction parameter k _D . The correlation between aggregation kinetics and colloidal and conformational stability factors was evaluated and the dynamic interaction parameter was found to be a promising predictor of aggregation propensity of monoclonal antibodies. The meaning of using an intermolecular interaction parameter for prediction of what is essentially a unimolecular process is also discussed.

Conclusions

This work estimates the significance of different predictors of aggregation propensity at high concentrations as a part of a high throughput, low resource screening method and is a contribution towards determining protein aggregation phenomena in actual systems used for the development and production of biopharmaceuticals.

     

Comparison of high-throughput biophysical methods to identify stabilizing excipients for a model IgG2 monoclonal antibody: conformational stability and kinetic aggregation measurements

The overall conformational stability of a model IgG2 monoclonal antibody (mAb) was examined as a function of temperature and pH using an empirical phase diagram approach. Stabilizing excipients were then identified based on high-throughput methods including (1) kinetic studies measuring aggregation via increases in optical density and (2) thermally induced structural transitions as measured by differential scanning calorimetry (DSC) and fluorescence spectroscopy. The kinetic profiles of antibody aggregation at 65 °C were pH dependent and correlated well with pH effects on secondary and tertiary structural transitions due to heat stress. For the screening of stabilizing excipients, the inhibition of the rate of protein aggregation at pH 4.5 at 65°C, as represented by changes in optical density, was shown to have a clear trend with a modest correlation coefficient compared with the stabilizing effect of the same excipients on the conformational stability of the antibody as measured by DSC and tryptophan fluorescence spectroscopy. These results demonstrate the utility of combining high-throughput data from protein aggregation kinetic experiments and conformational stability studies to identify stabilizing excipients that minimize the physical degradation of an IgG2 mAb.     

Nonnative aggregation of an IgG1 antibody in acidic conditions: part 1. Unfolding, colloidal interactions, and formation of high-molecular-weight aggregates

Monomeric and aggregated states of an IgG1 antibody were characterized under acidic conditions as a function of solution pH (3.5-5.5). A combination of intrinsic/extrinsic fluorescence (FL), circular dichroism, calorimetry, chromatography, capillary electrophoresis, and laser light scattering were used to characterize unfolding, refolding, native colloidal interactions, aggregate structure and morphology, and aggregate dissociation. Lower pH led to larger net repulsive colloidal interactions, decreased thermal stability of Fc and Fab regions, and increased solubility of thermally accelerated aggregates. Unfolding of the Fab domains, and possibly the CH3 domain, was inferred as a key step in the formation of aggregation-prone monomers. High-molecular-weight soluble aggregates displayed nonnative secondary structure, had a semi-rigid chain morphology, and bound thioflavin T (ThT), consistent with at least a portion of the monomer forming amyloid-like structures. Soluble aggregates also formed during monomer refolding under conditions moving from high to low denaturant concentrations. Both thermally and chemically induced aggregates showed similar ThT binding and secondary structural changes, and were noncovalent based on dissociation in concentrated guanidine hydrochloride solutions. Changes in intrinsic FL during chemical versus thermal unfolding suggest a greater degree of structural change during chemical unfolding, although aggregation proceeded through partially unfolded monomers in both cases.