Monoclonal Antibody Aggregates Research Articles

Monitoring of Flow‐Induced Aggregation and Conformational Change of Monoclonal Antibodies

AbstractThe aggregation process and conformational change of two monoclonal antibodies, monitored by in situ measuring techniques during stressing in a well‐defined flow field, was experimentally studied. The increase in turbidity, which was detected by in‐situ UV‐vis spectroscopy, was attributed to the formation of aggregates. A stronger aggregate formation could be related to the higher exposure of hydrophobic groups, which was monitored by in situ fluorescence spectroscopy. Both presented techniques are powerful methods to monitor the aggregation process of monoclonal antibodies directly in flow fields.

Water Proton NMR: A Tool for Protein Aggregation Characterization.

Formulation stability is a critical attribute of any protein-based biopharmaceutical drug due to a protein's inherent tendency to aggregate. Advanced analytical techniques currently used for characterization of protein aggregates are prone to a number of limitations and usually require additional manipulations with the sample, such as dilution, separation, labeling, and use of special cuvettes. In the present work, we compared conventional techniques for the analysis of protein aggregates with a novel approach that employs the water proton transverse relaxation rate R2(1H2O). We explored differences in the sensitivity of conventional techniques, size-exclusion chromatography (SEC), microflow imaging (MFI), and dynamic light scattering (DLS), and water NMR (wNMR) toward the presence of monoclonal antibody aggregates generated by different stresses. We demonstrate that wNMR outperformed SEC, DLS, and MFI in that it was most consistently sensitive to increases in both soluble and insoluble aggregates, including subvisible particles. The simplicity of wNMR, its sensitivity, and possibility of noninvasive measurements are unique advantages that would permit its application for more efficient and higher throughput optimization of protein formulations.

Utility of High Throughput Screening Techniques to Predict Stability of Monoclonal Antibody Formulations During Early Stage Development

Selecting optimal formulation conditions for monoclonal antibodies for first time in human clinical trials is challenging due to short timelines and reliance on predictive assays to ensure product quality and adequate long-term stability. Accelerated stability studies are considered to be the gold standard for excipient screening, but they are relatively low throughput and time consuming. High throughput screening (HTS) techniques allow for large amounts of data to be collected quickly and easily, and can be used to screen solution conditions for early formulation development. The utility of using accelerated stability compared to HTS techniques (differential scanning light scattering and differential scanning fluorescence) for early formulation screening was evaluated along with the impact of excipients of various types on aggregation of monoclonal antibodies from multiple IgG subtypes. The excipient rank order using quantitative HTS measures was found to correlate with accelerated stability aggregation rate ranking for only 33% (by differential scanning fluorescence) to 42% (by differential scanning light scattering) of the antibodies tested, due to the high intrinsic stability and minimal impact of excipients on aggregation rates and HTS data. Also explored was a case study of employing a platform formulation instead of broader formulation screening for early formulation development.

Preparative separation of monoclonal antibody aggregates by cation-exchange laterally-fed membrane chromatography.

Cation exchange (CEX) chromatography is widely used for large-scale separation of monoclonal antibody (mAb) aggregates. The aggregates bind more strongly to CEX media and hence elute after the monomeric mAb in a salt gradient. However, monomer-aggregate resolution that is typically obtained is poor, which results in low product recovery. In the current study we address this challenge through the use of cation-exchange laterally-fed membrane chromatography (LFMC). Three different LFMC devices, each containing a bed of strong cation-exchange (S) membranes were used for preparative-scale removal of mAb aggregates. Trastuzumab (IgG1) biosimilar derived from human embryonic kidney 293 (293) cells was used as the primary model mAb in our study. The other mAbs investigated were Chinese hamster ovary (CHO) cell line derived Alemtuzumab (Campath-1H) and a heavy chain chimeric mAb EG2-hFc. In each of these case-studies, aggregates were well-resolved from the respective monomer. The separated and collected monomer and aggregate fractions were analyzed using techniques such as hydrophobic interaction membrane chromatography (HIMC), native polyacrylamide gel electrophoresis (or PAGE), and size-exclusion high-performance liquid chromatography (SE-HPLC). The high efficiency of separation obtained in each case was due to a combination of the small membrane pore size (3-5μm), and the use of LFMC technology, which has been shown to be suitable for high-resolution, multi-component protein separations. Also, the LFMC based separation processes reported in this study were more than an order of magnitude faster than equivalent resin-based, cation exchange chromatography.

Ultrafast Separation and Analysis of Monoclonal Antibody Aggregates Using Membrane Chromatography

We discuss a method for rapid and cost-effective analysis of monoclonal antibody (mAb) aggregates. Hydrophobic interaction membrane chromatography, which was previously shown to be highly suitable for such separation and analysis, was used in a recently developed format referred to as laterally fed membrane chromatography (or LFMC). A stack of rectangular polyvinylidene fluoride (or PVDF) membranes having 0.22 μm pores housed within a modified analytical-scale LFMC device was used for analyzing aggregate types and content in different monoclonal antibody samples. High-resolution separations could be achieved in less than 1.5 min, this being faster than other currently available techniques such as size exclusion ultraperformance liquid chromatography (SE-UPLC). Moreover, the operating pressure was less than 200 kPa, which eliminated the need for an expensive high-pressure pump and chromatography system. The resolution obtained using the LFMC was comparable to that obtained using SE-UPLC. The effect of design variations such as change in dead volume and pillar size within the lateral channels within the LFMC device was also examined.

Prediction and Reduction of the Aggregation of Monoclonal Antibodies

Protein aggregation remains a major area of focus in the production of monoclonal antibodies. Improving the intrinsic properties of antibodies can improve manufacturability, attrition rates, safety, formulation, titers, immunogenicity, and solubility. Here, we explore the potential of predicting and reducing the aggregation propensity of monoclonal antibodies, based on the identification of aggregation-prone regions and their contribution to the thermodynamic stability of the protein. Although aggregation-prone regions are thought to occur in the antigen binding region to drive hydrophobic binding with antigen, we were able to rationally design variants that display a marked decrease in aggregation propensity while retaining antigen binding through the introduction of artificial aggregation gatekeeper residues. The reduction in aggregation propensity was accompanied by an increase in expression titer, showing that reducing protein aggregation is beneficial throughout the development process. The data presented show that this approach can significantly reduce liabilities in novel therapeutic antibodies and proteins, leading to a more efficient path to clinical studies.

Pre-study and in-study validation of a size-exclusion chromatography method with different detection modes for the analysis of monoclonal antibody aggregates.

Size exclusion chromatography (SEC) with different detection modes was assessed as a means to characterize the type of bevacizumab aggregate that forms under thermal stress, quantitatively monitoring the aggregation kinetics. The combination of SEC with light-scattering (SEC/LS) detection was validated using in-study validation process. This was performed by applying a strategy based on a control chart to monitor the process parameters and by inserting quality control samples in routine runs. The SEC coupled with a differential refractive-index detector (SEC/RI) was validated using a pre-study validation process in accordance with the ICH-Q2 (R1) guidelines and in-study monitoring in accordance with the Analytical Target Profile (ATP) criteria. The total error and β-expectation tolerance interval rules were used to assess method suitability and control the risk of incorrectly accepting unsuitable analytical methods. The aggregation kinetics data were interpreted using a modified Lumry-Eyring model. The true order of the reaction was determined using the initial-rate approach. All the kinetic data show a linear Arrhenius dependence within the studied temperature range. The Arrhenius approach over-predicted the aggregation rate for 5°C, but provides an idea of the aggregation process and amount of aggregate formed. In any case, real-time stability data are necessary to establish the product shelf-life.

Kinetics of Monoclonal Antibody Aggregation from Dilute toward Concentrated Conditions.

Gaining understanding on the aggregation behavior of proteins under concentrated conditions is of both fundamental and industrial relevance. Here, we study the aggregation kinetics of a model monoclonal antibody (mAb) under thermal stress over a wide range of protein concentrations in various buffer solutions. We follow experimentally the monomer depletion and the aggregate growth by size exclusion chromatography with inline light scattering. We describe the experimental results in the frame of a kinetic model based on population balance equations, which allows one to discriminate the contributions of the conformational and of the colloidal stabilities to the global aggregation rate. Finally, we propose an expression for the aggregation rate constant, which accounts for solution viscosity, protein-protein interactions, as well as aggregate compactness. All these effects can be quantified by light scattering techniques. It is found that the model describes well the experimental data under dilute conditions. Under concentrated conditions, good model predictions are obtained when the solution pH is far below the isoelectric point (pI) of the mAb. However, peculiar effects arise when the solution pH is increased toward the mAb pI, and possible explanations are discussed.

Experimental Model System to Study pH Shift-Induced Aggregation of Monoclonal Antibodies Under Controlled Conditions.

To present a convenient screening method for evaluating additive effects on the renaturation of an acid-exposed monoclonal antibody (mAb). The assay involves brief incubation of a mAb at acidic pH and subsequent neutralization in the absence or presence of additive to induce mainly aggregation. An increase in absorbance depicted aggregation. The recorded aggregation data traces were fitted with a nucleation-autocatalytic growth model for the extraction of kinetic parameters. All kinetic data traces were fitted successfully with the selected model and the adjusted R square values were greater than 0.99. Trehalose had strongly stabilizing, proline mildly stabilizing and trimethylamine oxide had destabilizing effects on both the nucleation and growth phase of the reaction. Histidine was strongly stabilizing but was limited by its poor solubility. The results demonstrate the suitability of the experimental mAb aggregation system and the nucleation-autocatalytic growth fit in the screening and quantification of additive effects on the renaturation of an acid-exposed mAb respectively. This will aid the investigation and derivation of quantitative structure-activity relationships of additive effects on mAb solubility.

Kinetic plots in aqueous size exclusion chromatography of monoclonal antibodies and virus particles

The growing importance of monoclonal antibodies and virus particles has led to a pressure for faster size exclusion chromatography. In recent years, numerous small particle columns for size exclusion chromatography of biologicals have been introduced. Small particles are a strategy to reduce analysis time. In the following study, opportunities of small particles in size exclusion chromatography of large biomolecules are investigated. Poppe plots reveal that the lower particle size limit depends on the size of the sample molecule. Hydrodynamic radii of monoclonal antibody monomer, aggregates and H1N1 as well as the diffusion coefficients were determined. Considering this sample compound dependency, kinetic plots referring to the resolution of a distinct compound pair instead of the plate number of a single analyte are more meaningful. Plate times were found to be equivalent with 4 and 2μm particles for a monoclonal antibody aggregate separation at resolutions smaller than 1.8. Quantification of a H1N1 in clarified cell culture can be accomplished with 17μm and 13μm particles at equal plate times at resolutions smaller than 2.5. Virus polydispersity is likely to be affected by run times of several hours at room temperature and shear forces resulting from particles smaller than 10μm. Comparatively high flow rates should be applied in size exclusion chromatography of the 100nm H1N1 virions.

Protein A chromatography increases monoclonal antibody aggregation rate during subsequent low pH virus inactivation hold

Protein A chromatography is a near-ubiquitous method of mAb capture in bioprocesses. The use of low pH buffer for elution from protein A is known to contribute to product aggregation. Yet, a more limited set of evidence suggests that low pH may not be the sole cause of aggregation in protein A chromatography, rather, other facets of the process may contribute significantly. This paper presents a well-defined method for investigating this problem. An IgG4 was incubated in elution buffer after protein A chromatography (typical of the viral inactivation hold) and the quantity of monomer in neutralised samples was determined by size exclusion chromatography; elution buffers of different pH values predetermined to induce aggregation of the IgG4 were used. Rate constants for monomer decay over time were determined by fitting exponential decay functions to the data. Similar experiments were implemented in the absence of a chromatography step, i.e. IgG4 aggregation at low pH. Rate constants for aggregation after protein A chromatography were considerably higher than those from low pH exposure alone; a distinct shift in aggregation rates was apparent across the pH range tested.

Predicting the Agitation-Induced Aggregation of Monoclonal Antibodies Using Surface Tensiometry.

Adsorption of antibody therapeutics to air-liquid interfaces can enhance aggregation, particularly when the solution does not contain protective surfactant or when the surfactant is diluted as occurs during preparation of intravenous infusion bags. The ability to predict an antibody's propensity for interfacially mediated aggregation is particularly useful during product development to ensure the quality, potency, and safety of the therapeutic. To develop a predictive tool, we investigated the surface pressure and surface excess of a panel of 16 antibodies as well as determined their aggregation propensity at the air-liquid interface in an agitation stress model. Our data demonstrated that the initial rate of surface pressure increase upon antibody adsorption to the air-liquid interface strongly predicted the extent of agitation-induced aggregation. Other factors, including the hydrophobicity, equilibrium surface pressure, and interfacial concentration of an antibody, were not adequate predictors of its susceptibility to aggregation. In addition to developing a predictive tool, we extended the interfacial characterization to better understand the mechanisms of antibody aggregation at an air-liquid interface during agitation stress. We believe that the kinetics of antibody rearrangement and conformational change after adsorbing to the interface, leading to the development of attractive antibody-antibody interactions, dictated the extent of aggregation. Overall, our results demonstrate how surface pressure measurements can be implemented as a rapid screening tool for the identification of antibodies with a high propensity to aggregate upon adsorption to an air-liquid interface while also furthering our understanding of interfacially mediated protein aggregation.

Intrinsic fluorescence-based at situ soft sensor for monitoring monoclonal antibody aggregation.

Intrinsic fluorescence spectroscopy, in conjunction with partial least squares regression (PLSR), was investigated as a potential technique for online quality control and quantitative monitoring of Immunoglobulin G (IgG) aggregation that occurs following exposure to conditions that emulate those that can occur during protein downstream processing. Initially, the impact of three stress factors (temperature, pH, and protein concentration) on the degree of aggregation determined using size exclusion chromatography data, was investigated by performing a central composite designexperiment and applying a fitting response surface model. This investigation identified the influence of the factors as well as the operating regions with minimum propensity to induce protein aggregation. Spectral changes pertinent to the stressed samples were also investigated and found to corroborate the high sensitivity of the intrinsic fluorescence to conformational changes of the proteins under study. Ultimately, partial least squares regression was implemented to formulate two fluorescence-based soft sensors for quality control--product classification--and quantitative monitoring--concentration of monomer. The resulting regression models exhibited accurate prediction ability and good potential for in situ monitoring of monoclonal antibody downstream purification processes.

High-throughput assay for measuring monoclonal antibody self-association and aggregation in serum.

Subcutaneous delivery is one of the preferred administration routes for therapeutic monoclonal antibodies (mAbs). High antibody dosing requirements and small injection volumes necessitate formulation and delivery of highly concentrated mAb solutions. Such elevated antibody concentrations can lead to undesirable solution behaviors such as mAb self-association and aggregation, which are relatively straightforward to detect using various biophysical methods because of the high purity and concentration of antibody formulations. However, the biophysical properties of mAbs in serum can also impact antibody activity, but these properties are less well understood because of the difficulty characterizing mAbs in such a complex environment. Here we report a high-throughput assay for directly evaluating mAb self-association and aggregation in serum. Our approach involves immobilizing polyclonal antibodies specific for human mAbs on gold nanoparticles, and then using these conjugates to capture human antibodies at a range of subsaturating to saturating mAb concentrations in serum. Antibody aggregation is detected at subsaturating mAb concentrations via blue-shifted plasmon wavelengths due to the reduced efficiency of capturing mAb aggregates relative to monomers, which reduces affinity cross-capture of mAbs by multiple conjugates. In contrast, antibody self-association is detected at saturating mAb concentrations via red-shifted plasmon wavelengths due to attractive interparticle interactions between immobilized mAbs. The high-throughput nature of this assay along with its compatibility with unusually dilute mAb solutions (0.1-10 μg per mL) should make it useful for identifying antibody candidates with high serum stability during early antibody discovery.

Unfolding and aggregation of monoclonal antibodies on cation exchange columns: Effects of resin type, load buffer, and protein stability

The chromatographic behavior of a monoclonal antibody (mAb) that exhibits a pronounced two-peak elution behavior is studied for a range of strong cation exchange resins and with varying load buffer pH and composition. Six stationary phases are considered, including two tentacle-type resins (Fractogel EMD SO3-(M) and Eshmuno S), a resin with grafted polymeric surface extenders (Nuvia S), a resin with a bimodal pore size distribution (POROS HS 50), and two macroporous resins without polymer grafts (Source 30S and UNOsphere Rapid S). The two-peak elution behavior is very pronounced for the tentacle and polymer-grafted resins and for POROS HS 50, but is essentially absent for the two macroporous resins. The extent of this behavior decreases as the buffer pH and concentration increase and, consequently, mAb binding becomes weaker. Replacing sodium with arginine as the buffer counterion, which is expected to decrease the mAb binding strength, nearly completely eliminates the two-peak behavior, while replacing sodium with tetra-n-butylammonium hydroxide, which is expected to increase the mAb binding strength, dramatically exacerbate the effect. As shown by hydrogen–deuterium exchange mass spectrometry (HX-MS), the two-peak elution behavior is related to conformational changes that occur when the mAb binds. These changes result in increased solvent exposure of specific peptides in the Fc-region for either the Fractogel or the Nuvia resin. No significant conformational changes were seen by HX-MS when the mAb was bound to the UNOsphere resin or on the Fractogel resin when arginine was used in lieu of sodium as the load buffer counterion. Experiments with two additional mAbs on the Fractogel resin show that the two-peak elution behavior is dependent on the particular antibody. Circular dichroism suggests that the propensity of different mAbs to either precipitate directly or to form stabilizing intermolecular structures upon exposure to thermal stress can be related to their chromatographic behaviors.

Effect of polyol sugars on the stabilization of monoclonal antibodies

We investigate the impact of sugars and polyols on the heat-induced aggregation of a model monoclonal antibody whose monomer depletion is rate-limited by protein unfolding. We follow the kinetics of monomer consumption by size exclusion chromatography, and we interpret the results in the frame of two mechanistic schemes describing the enhanced protein stability in the presence of polyols. It is found that the stabilization effect increases with increasing polyol concentration with a comparable trend for all of the tested polyols. However, the stabilization effect at a given polyol concentration is polyol specific. In particular, the stabilization effect increases as a function of polyol size until a plateau is reached above a critical polyol size corresponding to six carbon atoms. Our results show that the stabilization by polyols does not depend solely on the volume fraction filled by the polyol molecules, but is also affected by the polyol chemistry.

Circular dichroism spectroscopy as a tool for monitoring aggregation in monoclonal antibody therapeutics.

Aggregation continues to be a critical quality attribute for a monoclonal antibody therapeutic product due to its perceived significant impact on immunogenicity. This paper aims to establish the versatility of circular dichorism (CD) spectroscopy toward understanding aggregation of monoclonal antibody (mAb) therapeutics. The first application involves the use of far-UV CD as a complementary analytical technique to size exclusion chromatography (SEC) for understanding protein aggregation. The second application uses thermal scanning CD as a high throughput screening tool for examining stability of a mAb therapeutic in various formulation and downstream buffers. For establishing far-UV CD as an orthogonal technique, a mAb was incubated in different downstream processing buffers and another mAb in formulation buffers, and they were analyzed by SEC and far-UV CD for aggregate content and conformational stability, respectively. To examine thermal scanning as a high throughput screening tool, ellipticity as a function of the temperature was measured at 218 nm from 20 to 90 °C. Far-UV CD was found to display high sensitivity toward early detection of conformational changes in mAb. CD measurements were also able to elucidate the different aggregation mechanisms. Furthermore, thermal stability scan allowed us to estimate T(onset) which has been found to correlate with aggregation induced by salt, low pH, and buffer species. T(onset) temperature from thermal scanning at 218 nm using CD was correlated successfully to aggregate content measured by SEC. Results from both the studies demonstrate the usefulness of CD for assessing stability of therapeutic proteins during process development, formulation development, and product characterization.

Aggregation Kinetics for Monoclonal Antibody Products

Monoclonal antibodies have emerged as a valuable class of therapeutic products. However, the industry still faces pertinent challenges with respect to product stability, particularly product aggregation that leads to toxicity and immunogenicity. The present project is an attempt to propose a kinetic model for the aggregation of monoclonal antibodies using the analysis of experiments that were performed for several different combinations of buffer type, temperature, pH and salt concentration pertaining to three types of chromatography - protein A chromatography, cation exchange chromatography and anion exchange chromatography. Modified Lumry Eyring model has been employed by taking into account the reversibility of each step in the aggregation process. MATLAB R2011b has been used to find the optimum values of the kinetic rate constants for the different cases. The model can go a long way in reducing the extent of aggregation by helping to accurately choose the experimental conditions with the minimum number of experiments.

Analytical QbD: Development of a native gel electrophoresis method for measurement of monoclonal antibody aggregates

This paper presents a quality by design (QbD) based development of a novel native PAGE (N-PAGE) method as a low-cost analytical tool for analysis of aggregates of monoclonal antibodies. Comparability to the present gold standard of SEC has been established. The motivation is the fact that SEC requires relatively expensive equipment and consumables, thus making N-PAGE relevant to those academicians and other small companies involved in early-stage development of biotherapeutics that do not have access to SEC, especially in developing countries. Furthermore, SEC suffers from certain disadvantages including the possibility of secondary interactions between the stationary phase and analyte resulting in higher elution time and therefore underestimation of the analyte size. The proposed N-PAGE method can also serve as an orthogonal analytical method for aggregate analysis. A QbD-based approach has been used for development and optimization of the protocol. First, initial screening studies were carried out with parameters including the running buffer pH, running buffer molarity, gel buffer pH, loading dye, sample concentration, and running voltage. Next, optimization of operating parameters was performed using principles of design of experiments. The final optimized protocol was compared to the traditional SEC method and the results were found to be comparable. While N-PAGE has been in use for protein analysis for several decades, use of N-PAGE for analysis of mAb aggregates with data comparable to SEC such as the case presented here is novel.

Prediction of IgG1 aggregation in solution.

Interest in monoclonal antibody aggregation is increasing as aggregates of biopharmaceuticals can cause an immunogenic response when injected into the body. In this work, a stoichiometric reaction model from concentration-time data is developed to predict the dimer ratio in stored antibody solutions over time. IgG1 was incubated at pH from 4.5 to 5.5, salt concentrations from 100 to 600 mmol/kg and protein concentrations of 10.6-26.3 g/L; samples were taken at intervals of 20 min to 5 h over time periods from 4 h to 7.6 days, and analyzed with size-exclusion chromatography. The experiments showed the formation of dimers from monomers, but no higher order aggregates. Dilution of samples containing dimers led to the reversal of the dimerization reaction. Measurements of the concentrations of each component were made by fitting exponentially modified Gaussian peaks to the chromatograms used to measure the concentrations of the different forms of protein. This stoichiometric reaction model was able to predict the formation of dimers by the antibody studied. The equilibrium constant was found to be dependent on the salt concentration, and the kinetic constant showed a dependence on the pH of the solution. The prediction of the aggregation leads to a possibility of optimizing the conditions in order to prevent the dimer formation and to maximize the monomer concentration.