Effect of Glass Transition Temperature on the Stability of Lyophilized Formulations Containing a Chimeric Therapeutic Monoclonal Antibody

Purpose. The purpose of this study is to highlight the importance of knowing the glass transition temperature, T_g, of a lyophilized amorphous solid composed primarily of a sugar and a protein in the interpretation of accelerated stability data. Methods. Glass transition temperatures were measured using DSC and dielectric relaxation spectroscopy. Aggregation of protein in the solid state was monitored using size-exclusion chromatography. Results. Sucrose formulation (T_g ~ 59°C) when stored at 60°C was found to undergo significant aggregation, while the trehalose formulation (T_g ~ 80°C) was stable at 60°C. The instability observed with sucrose formulation at 60°C can be attributed to its T_g (~59°C) being close to the testing temperature. Increase in the protein/sugar ratio was found to increase the T_gs of the formulations containing sucrose or trehalose, but to different degrees. Conclusions. Since the formulations exist in glassy state during their shelf-life, accelerated stability data generated in the glassy state (40°C) is perhaps a better predictor of the relative stability of formulations than the data generated at a higher temperature (60°C) where one formulation is in the glassy state while the other is near or above its T_g.     

The Effect of Formulation Excipients on Protein Stability and Aerosol Performance of Spray-Dried Powders of a Recombinant Humanized Anti-IgE Monoclonal Antibody1

Purpose. To study the effect of trehalose, lactose, and mannitol on the biochemical stability and aerosol performance of spray-dried powders of an anti-IgE humanized monoclonal antibody. Methods. Protein aggregation of spray-dried powders stored at various temperature and relative humidity conditions was assayed by size exclusion chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis. Protein glycation was determined by isoelectric focusing and affinity chromatography. Crystallization was examined by X-ray powder diffraction. Aerosol performance was assessed as the fine particle fraction (FPF) of the powders blended with coarse carrier lactose, and was determined using a multiple stage liquid impinger. Results. Soluble protein aggregation consisting of non-covalent and disulfide-linked covalent dimers and trimers occurred during storage. Aggregate was minimized by formulation with trehalose at or above a molar ratio in the range of 300:1 to 500:1 (excipient:protein). However, the powders were excessively cohesive and unsuitable for aerosol administration. Lactose had a similar stabilizing effect, and the powders exhibited acceptable aerosol performance, but protein glycation was observed during storage. The addition of mannitol also reduced aggregation, while maintaining the FPF, but only up to a molar ratio of 200:1. Further increased mannitol resulted in crystallization, which had a detrimental effect on protein stability and aerosol performance. Conclusions. Protein stability was improved by formulation with carbohydrate. However, a balance must be achieved between the addition of enough stabilizer to improve protein biochemical stability without compromising blended powder aerosol performance.     

Effect of Product Temperature During Primary Drying on the Long-Term Stability of Lyophilized Proteins

Our objective was to investigate the effect of performing primary drying at product temperatures below and above Tg′ (glass transition temperature of the freeze-concentrated phase) on the long-term stability of lyophilized proteins. Two protective media differing in the nature of the bulking agent used (amorphous or crystalline) were selected. Several lyophilization cycles were performed by using various combinations of shelf temperature and chamber pressure to obtain different values of product temperature during primary drying. The antigenic activity of the proteins was measured after lyophilization and after 6 months of storage at 4°C and 25°C. After 6 months of storage and regardless of the protective medium, the losses of antigenic activity of both toxins increased from 0% when primary drying was performed at a product temperature lower than Tg′ and to 25% when the product temperature was higher than Tg′. The use of partially crystalline systems makes it possible to withstand high primary drying temperatures (above Tg′). However, the shelf life of lyophilized proteins may be decreased when the amorphous phase including the protein and the stabilizing molecule changes to the viscous state.     

The Relationship Between Protein Aggregation and Molecular Mobility Below the Glass Transition Temperature of Lyophilized Formulations Containing a Monoclonal Antibody

Purpose. To find out if the physical instability of a lyophilized dosage form is related to molecular mobility below the glass transition temperature. Further, to explore if the stability data generated at temperatures below the glass transition temperature can be used to predict the stability of a lyophilized solid under recommended storage conditions. Methods. The temperature dependence of relaxation time constant, , was obtained for sucrose and trehalose formulations of the monoclonal antibody (5 mg protein/vial) from enthalpy relaxation studies using differential scanning calorimetry. The non-exponentiality parameter, , in the relaxation behavior was also obtained using dielectric relaxation spectroscopy. Results. For both sucrose and trehalose formulations, the variation in with temperature could be fitted Vogel-Tammann-Fulcher (VTF) equation. The two formulations exhibited difference sensitivities to temperature. Sucrose formulation was more fragile and exhibited a stronger non-Arrhenius behavior compared to trehalose formulation below glass transition. Both formulations exhibited <2% aggregation at t values <10, where t is the time of storage. Conclusions. Since the relaxation times for sucrose and trehalose formulations at 5°C are on the order of 10⁸ and 10⁶ hrs, it is likely that both formulations would undergo very little (<2%) aggregation in a practical time scale under refrigerated conditions.     

Enhancing Stability and Reducing Viscosity of a Monoclonal Antibody With Cosolutes by Weakening Protein-Protein Interactions

An understanding of how cosolutes affect the viscosity and storage stability of highly concentrated mAbs as a function of protein-protein interactions (PPIs) would be desirable for improving processing and administration of protein therapeutics. The effects of inorganic and organic cosolutes on the viscosity and stability of mAb5 were determined for concentrations up to 250 mg/mL. Organic electrolytes Arg(HCl) and His(HCl) produced the largest viscosity reductions, indicating screening of local anisotropic short-ranged attractive and hydrophobic interactions. These cosolutes significantly reduced mAb5 aggregate concentration as measured by size-exclusion chromatography after 4 weeks of 40°C storage at 200 mg/mL, with the largest reduction for Arg(Glu). The effects of the cosolutes on storage stability and viscosity are related to their ability to reduce attractive PPIs at high concentration (200 mg/mL), as shown by comparing measurements of structure factor (by small-angle X-ray scattering) and collective diffusion (by dynamic light scattering) with models of hard and attractive spheres. The improved stability of Arg(Glu) over Arg(HCl) despite similar PPI by small-angle X-ray scattering at high concentration is consistent with higher protein conformational stability as determined by differential scanning fluorimetry and differential scanning light scattering.     

Chemical and Physical Stability of Chimeric L6, a Mouse−Human Monoclonal Antibody

Chimeric L6 is a mouse–human monoclonal antibody specific for tumor cell-associated antigens. The factors affecting the physical and chemical stability of chimeric L6 were assessed at elevated temperatures (30–60°C) and by multiple freezing and thawing. Three routes of degradation were observed: chemical degradation to smaller molecular weight species, irreversible aggregation, and formation of a reversible dimer. The specific pathway depended on the stress condition applied and the pH, with maximal overall stability to both thermal stress and multiple freezing/thawing observed at about pH 5.5. Other factors including antibody concentration, buffer concentration, NaCl concentration, and agitation had minimal influence on the stability. Commonly used sugars, polyhydric alcohols, and amino acids effectively prevented freeze/thaw-induced aggregation.     

The Stabilization of a Human IgM Monoclonal Antibody with Poly(vinylpyrrolidone)

An IgM anti-group B Streptococcus monoclonal antibody (4B9) was found to undergo irreversible heat-induced aggregation at 50°C. A variety of excipients was tested for their ability to inhibit antibody aggregation. The amount of 4B9 aggregation, which was determined by analysis on a size-exclusion HPLC, was significantly reduced in the presence of low concentrations [between 0.1 and 1.0% (w/v)] of poly(vinylpyrrolidone) (PVP) molecules ranging in molecular weight from 10 to 40 kDa. When the PVP concentration was greater than 1.0%, antibody aggregation was enhanced, and with the highest molecular weight PVP, antibody precipitation occurred. HPLC was used to show that more PVP was associated with the 4B9 at 50°C than at 25°C. Differential scanning calorimetry revealed that PVP concentrations greater than 2.0% decreased the antibody thermal transition temperature. Enzyme-linked immunosorbent assays were used to assess the effects of PVP on the antigen binding capacity of 4B9 and on 4B9 quantitation. At 4°C, PVP solutions of up to 5.0% had no effect on either 4B9 quantitation or antigen binding. At 50°C, however, less 4B9 was detected in the 5.0% PVP solution. The heat stabilization of the 4B9 antibody by low concentrations of PVP can be explained by a weak binding of PVP to the native protein. The PVP may sterically interfere with protein–protein interactions, thus reducing aggregation. Higher concentrations of PVP lead to protein aggregation and precipitation, probably by a volume-exclusion mechanism. Low concentrations of less than 1.0% PVP can be used to stabilize proteins against heat-induced aggregation, but care should be exercised, since even slightly higher concentrations of PVP can also lead to protein destabilization.     

A Quantitative Assessment of the Significance of Molecular Mobility as a Determinant for the Stability of Lyophilized Insulin Formulations

Purpose The purpose was to explore a method for quantitatively assessing the contribution of molecular mobility to the chemical reactivity of amorphous solids. Degradation of insulin in lyophilized formulations containing trehalose and poly(vinylpyrrolidone)(PVP) was chosen as a model system, and the temperature- and glass transition temperature (T_g)-dependence of the degradation rate was analyzed to obtain the relative contributions of molecular mobility and that of the chemical activational barrier reflected in the energy of activation.Methods Insulin degradation and dimerization in lyophilized trehalose and PVP formulations were monitored at various relative humidities (6–60% RH) and temperatures (10–60°C) by reverse-phase high-performance liquid chromatography (HPLC) and high-performance size-exclusion chromatography (HP-SEC), respectively. The T_g and fragility parameter of the lyophilized insulin formulations were determined by differential scanning calorimetry (DSC).Results Insulin degradation in the initial stage was describable with first-order kinetics for both of the trehalose and PVP formulations. The temperature- and T_g-dependence of the degradation rate indicated that the reactivity of insulin in the trehalose formulation is affected by molecular mobility at low humidity (12% RH), such that the ratio of the observed rate constant (k′) to the rate constant governed only by the activational barrier (k) was 0.051 at the T_g. At higher humidities, in contrast, the value of k′/k was much higher (0.914, 0.978, and 0.994 for 23% RH, 33% RH, and 43% RH, respectively), indicating that insulin degradation rate is determined predominantly by the activational barrier. For insulin degradation in the PVP formulation at temperatures below T_g, the contribution of molecular mobility to the degradation rate appeared to be negligible, as the extrapolated value of t₉₀ at the T_g exhibited a large difference between the formulations with differing T_g values (because of differing water contents).Conclusions The reactivity of insulin in the trehalose and PVP formulations can be described by an equation including factors reflecting the activational barrier (activation energy and frequency coefficient) and factors reflecting the molecular mobility (T_g, fragility parameter and a constant representing the relationship between the molecular mobility and the reaction rate). Thus, analysis of temperature dependence based on the proposed equation allows quantitative assessment of the significance of molecular mobility as a factor affecting chemical reactivity.     

A New Approach to Study the Physical Stability of Monoclonal Antibody Formulations—Dilution From a Denaturant

The early-stage assessment of the physical stability of new monoclonal antibodies in different formulations is often based on high-throughput techniques that suffer from various drawbacks. Accordingly, new approaches that facilitate the protein formulation development can be of high value to the industry. In this study, a dynamic light scattering plate reader is used to measure the aggregation (by means of the increase in the hydrodynamic radius [R_h]) of monoclonal antibody samples that were subject to incubation and subsequent dilution from different concentrations of a denaturing agent, that is, guanidine hydrochloride. The increase in the R_h of the protein samples is dependent not only on the denaturant concentration used but also on the buffer in which the incubation/dilution was performed. We also compare the aggregation after dilution from a denaturant with other high-throughput stability-indicating methods and find good agreement between the techniques. The proposed approach to probe the physical stability of monoclonal antibodies in different formulation conditions offers a unique combination of features—it is isothermal, probes both the resistance to denaturant-induced unfolding and the colloidal protein stability, it is entirely label-free, does not rely on complex data evaluation, and requires very short instrument measurement time on standard equipment.     

Analytical Approaches to the Study of Monoclonal Antibody Stability

The stability of two purified monoclonal antibodies, MN12 and WT31, was investigated. The monoclonal antibodies were incubated for 32 days at different pH values (ranging from 3.0 to 10.0) at 4 and 37°C. Various analytical methods were used to assess changes in physicochemical properties of the proteins. The monoclonal antibodies were more susceptible to degradation at 37°C than at 4°C. At low pH irreversible precipitation occurred. Decomposition of the proteins was enhanced at increasing pH values in the alkaline range. This was concluded from mouse IgG-specific and antigen-specific enzyme-linked immunosorbent assays, flow cytometry, analytical gel permeation chromatography, sodium dodecyl sulfate–polyacrylamide gel electrophoresis, isoelectric focusing, and immunoblotting. No substantial change in the apparent affinity constant of MN12 was observed, as determined by an affinity enzyme-linked immunosorbent assay. Fluorescence spectra, fluorescence polarization values, and fluorescence quenching parameters of MN12 and WT31 were not substantially affected, indicating that no major irreversible conformational changes had occurred. It was concluded that each of the techniques used has only limited value for stability assessment of monoclonal antibodies and, hence, that the application of several analytical techniques is essential to gain insight into monoclonal antibody stability.     

A New Strategy for Enhancing the Stability of Lyophilized Protein: The Effect of the Reconstitution Medium on Keratinocyte Growth Factor

Purpose. Protein stabilization during lyophilization has previously focused on optimization of the formulation as well as the freezing and dehydration process parameters. However, the effect of the reconstitution medium has been largely neglected. We have investigated its effect on aggregate formation using recombinant keratinocyte growth factor (KGF). Methods. The protein was lyophilized under suboptimal conditions to induce aggregation and precipitation upon reconstitution with water. A series of additives were examined by UV spectrophotometry and size exclusion chromatography (SEC-HPLC) for their effects on decreasing the degree of KGF aggregation and precipitation by the increase in recovery of soluble monomer. Results. Several additives resulted in a significant reduction of aggregation, including sulfated polysaccharides, surfactants, polyphosphates, and amino acids. A similar effect was achieved by adjusting the ionic strength of the reconstitution medium. SEC-HPLC indicated that the amount of soluble monomer was also increased by these additives suggesting that the recovery of the soluble protein correlates with the native, monomeric protein. Conclusions. These results suggest that optimization of reconstitution conditions will be a useful methodology for increasing the recovery of soluble, active proteins and that for KGF, the recovery of the soluble protein correlates with the native, monomeric form.     

Protein Structural Conformation and Not Second Virial Coefficient Relates to Long-Term Irreversible Aggregation of a Monoclonal Antibody and Ovalbumin in Solution

Purpose The purpose of the study was to investigate the relationship of the second virial coefficient, B₂₂, to the extent of irreversible protein aggregation upon storage. Methods A monoclonal antibody and ovalbumin were incubated at 37°C (3 months) under various solution conditions to monitor the extent of aggregation. The B₂₂ values of these proteins were determined under similar solution conditions by a modified method of flow-mode static light scattering. The conformation of these proteins was studied using circular dichroism (CD) spectroscopy and second-derivative Fourier transform infrared spectroscopy. Results Both proteins readily aggregated at pH 4.0 (no aggregation observed at pH 7.4); the extent of aggregation varied with the ionic strength and the presence of cosolutes (sucrose, glycine, and Tween 80). Debye plots of the monoclonal antibody showed moderate attractive interactions at pH 7.4, whereas, at pH 4.0, nonlinear plots were obtained, indicating self-association. CD studies showed partially unfolded structure of antibody at pH 4.0 compared with that at pH 7.4. In the case of ovalbumin, similar B₂₂ values were obtained in all solution conditions irrespective of whether the protein aggregated or not. CD studies of ovalbumin indicated the presence of a fraction of completely unfolded as well as partially unfolded species at pH 4.0 compared with that at pH 7.4. Conclusions The formation of a structurally altered state is a must for irreversible aggregation to proceed. Because this aggregation-prone species could be an unfolded species present in a small fraction compared with that of the native state or it could be a partially unfolded state whose net interactions are not significantly different compared with those of the native state, yet the structural changes are sufficient to lead to long-term aggregation, it is unlikely that B₂₂ will correlate with long-term aggregation.     

An Unexpected pH Effect on the Stability of Moexipril Lyophilized Powder

Because of the limited stability of moexipril (RS-10085; 1) in aqueous solution, lyophilized parenteral formulations were evaluated as a function of pH in this study. In general, the lyophilized powder of 1 showed about two orders of magnitude less reactivity at 50°C than in aqueous solution at pH values below 3 or above 6. At pH 5.1, however, the lyophilized powder had maximum reactivity, with the rate actually comparable to that observed in aqueous solution. When the distribution of the two major products, diketopiperazine (DKP) 2 and ester hydrolysis analogue 3, was compared to the observed kinetics as a function of pH, it was clear that removal of water via lyophilization suppressed the spontaneous k ₁ cyclization process, the spontaneous k ₃ hydrolysis process, and the specific base-catalyzed k ₄ hydrolysis process. The overall spontaneous k ₂ cyclization process, however, was not affected by lyophilization. The latter result is accounted for by the increased equilibrium constant for the formation of the tetrahedral intermediate, To, as a result of lyophilization. This study demonstrates that stability data in solution can not be used for predicting the stability of moexipril in lyophilized powder form.     

Effect of Freezing Rate on the Stability of Liposomes During Freeze-Drying and Rehydration

Purpose. In the present study we examined the effect of the freezing protocol on carboxyfluorescein (CF) retention in liposomes after freeze-drying and rehydration. Methods. Liposomes were frozen slowly at 0.5°C/min, or quickly by submerging the samples in boiling nitrogen before freeze-drying. The thermal behaviour of the frozen dispersions was analysed by Modulated Temperature Differential Scanning Calorimetry (MTDSC). The dried cakes were analysed by SEM, MTDSC and FTIR. The % encapsulated CF of the (re)hydrated liposomes was determined by fluorimetry after GPC, their vesicle size was measured by the Dynamic Light scattering Technique and their bilayer transition was studied by DSC. Results. Slow freezing resulted in a markedly higher CF retention after freeze-drying and rehydration as compared to quick freezing. The effect of the freezing rate depended on the lipid composition and was most pronounced for rigid liposomes. The damage caused by quick freezing did not occur after a freezing/thawing cycle. The freezing protocol did not influence the interaction between the phospholipids and the lyoprotectants (sucrose, trehalose or glucose) in the freeze-dried state. However, analysis by DSC of dipalmitoylphosphatidylcholine (DPPC): dipalmitoylphosphatidylglycerol (DPPG) =10:1 and DPPC liposome dispersions showed that the freezing protocol affected the bilayer melting characteristics of these liposomes after freeze-drying and rehydration. Conclusions. A proper design of the freezing protocol is essential to achieve optimal stability of rigid liposomes during a freeze-drying and rehydration cycle.     

Effect of Sucrose/Raffinose Mass Ratios on the Stability of Co-Lyophilized Protein During Storage Above the Tg

Purpose. To examine the potential of raffinose as an excipient in stabilizing protein and to study the effect of sucrose/raffinose mass ratios on the stability of co-lyophilized protein and amorphous solids during storage at an elevated temperature. Methods. Glucose-6-phosphate dehydrogenase (G6PDH) was co-lyophilized with sucrose and raffinose mixed at different mass ratios. The activity of dried G6PDH was monitored during storage at 44°C. Thermal properties of sucrose/raffinose matrices were determined by differential scanning calorimetry (DSC). Results. Mass ratios of sucrose to raffinose did not affect the recovery of G6PDH activity after freeze-drying, but significantly affected the stability of freeze-dried G6PDH during storage. The sucrose-alone formulation offered the best enzyme stabilization during storage. With increasing fraction of raffinose, the G6PDH stability decreased, sugar crystallization inhibited, and crystal-melting temperature increased. Conclusions. Despite the higher T_g of the formulations with higher fraction of raffinose, they provided less protection for G6PDH than did sucrose alone during storage. Our data do not support the prediction from recent thermophysical studies that raffinose should be superior to sucrose and trehalose as a potential excipient or stabilizer.     

Process-Induced Crystallinity Changes in Albuterol Sulfate and Its Effect on Powder Physical Stability

Pharmaceutical powders are often milled to achieve the optimum particle size. These size reduction processes can introduce dislocations and/or defects onto particle surfaces affecting the overall crystallinity of the powder. If enough energy is imparted, amorphous regions on the particle surfaces may be produced. These amorphous regions have the propensity to absorb significant quantities of water. In this study the effect of sorbed water on the physical characteristics of albuterol sulfate is investigated. Physical properties of this compound are studied in both micronized and unmicronized states using scanning electron microscopy, differential scanning calorimetry, powder x-ray diffraction, solution microcalorimetry, laser diffraction particle size analysis and water vapor sorption analysis. Subtle differences in crystallinity induced by air jet micronization are detected by several analytical methods. Amorphous to crystalline conversions are observed, the kinetics of which are found to be both temperature and relative humidity dependent. These experiments show the dynamic nature of micronized albuterol sulfate and aid in the determination of the actual physical state of this pharmaceutical powder.     

The Effect of the Reconstitution Medium on Aggregation of Lyophilized Recombinant Interleukin-2 and Ribonuclease A

Pharmaceutical Research -     

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.     

New Insight Into Thermodynamical Stability of Carbamazepine

Carbamazepine (CBZ)—an antiepileptic drug—belongs to Biopharmaceutics Classification System II Class. It has low solubility and consequently limited bioavailability. One of the ways to improve drugs solubility is amorphization of their structure. Herein, cooling CBZ—at different cooling rates—was investigated as a way to obtain glassy, better soluble form. During preliminary differential scanning calorimetry experiments, some peculiar behavior of the examined material, different from those stated in the literature, was observed. Further investigations using differential scanning calorimetry, thermogravimetric analysis, and polarizing optical microscope revealed that decomposition temperature of CBZ is about 30°C lower than previously assumed. Moreover, high-resolution thermogravimetric measurements indicate that some decomposition processes could start even below the temperature reported as the melting point of the form I of CBZ.     

Effect of Peroxide- Versus Alkoxyl-Induced Chemical Oxidation on the Structure,Stability, Aggregation,and Function of a Therapeutic Monoclonal Antibody

Current guidelines indicate that the effects of oxidation should be included as part of forced degradation studies on protein drugs. We probed the effect of 3 commonly used oxidants, hydrogen peroxide, tert-butyl hydroperoxide, and 2,2'-Azobis(2-amidinopropane) dihydrochloride (AAPH), on a therapeutic monoclonal IgG1 antibody (mAb8). Upon oxidation, mAb8 did not show noticeable changes in its secondary structure but showed minor changes in tertiary structure. Significant decrease in conformational stability was observed for all the 3 oxidized forms. Both hydrogen peroxide and tert-butyl hydroperoxide destabilized mainly the C_H2 domain, whereas AAPH destabilized the variable domain in addition to C_H2. Increased aggregation was found for AAPH-oxidized mAb8. In addition, a significant decrease in Fc receptor binding was observed for all 3 oxidized forms. Antibody dependent cell-mediated cytotoxicity, binding to target protein receptor, and cell proliferation activity were significantly reduced in the case of AAPH-oxidized mAb8. The presence of free methionine in the formulation buffer seems to alleviate the effect of oxidation. The results of this study show that the 3 oxidants differ in terms of their effects on the structure and function of mAb8 because of chemical modification of different sets of residues located in Fab versus Fc.