Himanshu S. Gadgil

Elucidation of Acid-induced Unfolding and Aggregation of Human Immunoglobulin IgG1 and IgG2 Fc

Background: Monoclonal antibodies and Fc fusion proteins contain an IgG Fc moiety, which is associated with various degradation processes, including aggregation. Results: Fc unfolding is triggered by the protonation of acidic residues and depends on the IgG subclass and CH2 domain glycosylation. Conclusion: Fc aggregation in acidic conditions is determined by CH2 stability. Significance: Understanding Fc aggregation is important for improving the quality of Fc-based therapeutics. Understanding the underlying mechanisms of Fc aggregation is an important prerequisite for developing stable and efficacious antibody-based therapeutics. In our study, high resolution two-dimensional nuclear magnetic resonance (NMR) was employed to probe structural changes in the IgG1 Fc. A series of 1H-15N heteronuclear single-quantum correlation NMR spectra were collected between pH 2.5 and 4.7 to assess whether unfolding of CH2 domains precedes that of CH3 domains. The same pH range was subsequently screened in Fc aggregation experiments that utilized molecules of IgG1 and IgG2 subclasses with varying levels of CH2 glycosylation. In addition, differential scanning calorimetry data were collected over a pH range of 3–7 to assess changes in CH2 and CH3 thermostability. As a result, compelling evidence was gathered that emphasizes the importance of CH2 stability in determining the rate and extent of Fc aggregation. In particular, we found that Fc domains of the IgG1 subclass have a lower propensity to aggregate compared with those of the IgG2 subclass. Our data for glycosylated, partially deglycosylated, and fully deglycosylated molecules further revealed the criticality of CH2 glycans in modulating Fc aggregation. These findings provide important insights into the stability of Fc-based therapeutics and promote better understanding of their acid-induced aggregation process.

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

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

Characterization of Site-Specific Glycation During Process Development of a Human Therapeutic Monoclonal Antibody

A therapeutic recombinant monoclonal antibody (mAb1) was found to be highly susceptible to glycation during production. Up to 42% glycation was observed in mAb1, which was significantly greater than the glycation observed in 17 other monoclonal antibodies (mAbs). The majority of the glycation was localized to lysine 98 of a unique sequence in the heavy chain complementarity determining region 3. Upon incubation with 5% glucose at 37 °C for 5 days, the level of glycation rose to 80% of the total protein where the majority of the additional glycation was on the lysine 98 residue. These data suggested that the lysine 98 residue was highly susceptible to glycation. However, three other mAbs with a lysine residue in the same position did not show high rates of glycation in the forced glycation assay, suggesting that primary and perhaps secondary structural constraints could contribute to the rate of glycation at that lysine. Interestingly, a portion of the glycation in mAb1 was found to be reversible and upon incubation in phosphate buffer (pH 7) at 37 °C for 5 days, the glycation dropped from starting levels of 42% to 20%. Variation was observed in the total glycation levels between different lots of mAb1. The variability in glycation introduced charge heterogeneity in the form of an acidic peak on cation exchange chromatography and lead to product inconsistency. Mutation of lysine 98 to arginine reduced the starting level of glycation without any impact on potency.

Elucidation of Two Major Aggregation Pathways in an IgG2 Antibody

Two major aggregation pathways observed in an IgG2 molecule are described. Different aggregate species generated by long-term incubation of the antibody at 37 degrees C were collected by a semi-preparative size exclusion chromatography method. These purified species were analyzed extensively by denaturing size-exclusion chromatography methods. The major aggregation pathway at low pH (4.0) resulted in the formation of both dimers and high molecular weight (HMW) aggregates. It was found that these dimers and HMW aggregates contain antibody molecules that have a peptide bond cleavage between an aspartic acid and proline residue in the CH2 domain. Evidence that unfolding of the CH2 domain may be driving the aggregation at low pH is presented. At higher pH (pH - 6.0), formation of a dimer having approximately 75% covalent character was the major aggregation pathway while formation of higher molecular weight aggregates were largely suppressed. The covalent dimer consisted of both disulfide linked antibody molecules and another species (approximately 26%) that was formed due to nondisulfide covalent bonds between two heavy chains. At pH - 5.0, both dimer and higher molecular weight aggregates were formed and the aggregation pathway was a combination of the major pathways observed at pH - 4.0 and 6.0. The dimer species formed at pH - 5.0 had a larger contribution from covalent species-both disulfide and nondisulfide linked, while the HMW aggregate contained a higher percentage of molecules that had the peptide bond cleavage in the CH2 domain. The dimer formed at pH - 6.0 was found to have identical secondary and tertiary structure as the intact antibody molecule. However, the dimer and higher molecular weight aggregate formed at pH - 4.0 have altered secondary and tertiary structure.

Comparison of LC and LC/MS Methods for Quantifying N-Glycosylation in Recombinant IgGs

High-performance liquid chromatography (LC) and liquid chromatography/electrospray ionization time-of-flight mass spectrometry (LC/ESI-MS) methods with various sample preparation schemes were compared for their ability to identify and quantify glycoforms in two different production lots of a recombinant monoclonal IgG1 antibody. IgG1s contain a conserved N-glycosylation site in the fragment crystallizable (Fc) subunit. Six methods were compared: (1) LC/ESI-MS analysis of intact IgG, (2) LC/ESI-MS analysis of the Fc fragment produced by limited proteolysis with Lys-C, (3) LC/ESI-MS analysis of the IgG heavy chain produced by reduction, (4) LC/ESI-MS analysis of Fc/2 fragment produced by limited proteolysis and reduction, (5) LC/MS analysis of the glycosylated tryptic fragment (293EEQYNSTYR301) using extracted ion chromatograms, and (6) normal phase HPLC analysis of N-glycans cleaved from the IgG using PNGase F. The results suggest that MS quantitation based on the analysis of Fc/2 (4) is accurate and gives results that are comparable to normal phase HPLC analysis of N-glycans (6).

Improving Mass Accuracy of High Performance Liquid Chromatography/ Electrospray Ionization Time-of-Flight Mass Spectrometry of Intact Antibodies

The glycosylation profile of intact antibody due to the galactose and fucose heterogeneity in the N-linked sugars was determined with instrument resolution of 5000 and 10,000. After deconvolution of electrospray ionization mass spectra to complete convergence, several extra peaks appeared in addition to the peaks observed in the original mass spectra. The artificial peaks were avoided if deconvolution was stopped after a smaller number of iterations. A standard antibody was used as an external calibrant to minimize mass measurement errors during long-period experiments. Precision of four consecutive LC/MS measurements of the same antibody was 10 ppm (±1.5 Da). By using this approach, the masses of 11 intact antibodies were measured. All antibodies containing N-terminal glutamines had a negative mass shift due to the formation of pyroglutamate (−17 Da). Although the pyroglutamate variant of intact antibody was not resolved from the unmodified variant, this modification led to a mass shift proportional to the percentage of N-terminal pyroglutamate. By accurately measuring the mass shift we were able to quantify the abundance of pyroglutamic acid on intact antibodies. Mass accuracy in measuring different antibodies was below 30 ppm (±4 Da). The accurate mass measurement can be an effective tool for monitoring chemical degradations in therapeutic antibodies.

The effect of sucrose hydrolysis on the stability of protein therapeutics during accelerated formulation studies

Stability studies of protein therapeutics are often accelerated by storing potential formulations at elevated temperatures where the rates of various chemical and physical degradation pathways are increased. An often overlooked caveat of using these studies is the potential degradation of the formulation components themselves. In this report, we show that the monoclonal antibody MAB001 aggregated at a faster rate when formulated with sucrose compared to samples that contained sorbitol or no excipient during accelerated stability studies following an initial lag phase where the rates of aggregate formation were similar in all formulations. The duration of the lag phase was both pH and temperature dependent and a significant increase of protein glycation was noticed during this time. These observations indicate that the enhanced rate of antibody aggregation in sucrose containing formulations is likely due to protein glycation following sucrose hydrolysis under accelerated conditions. This hypothesis was confirmed by demonstrating that antibody directly glycated with glucose aggregated at a faster rate than nonglycated antibody stored in the identical formulation. These findings question the utility of using accelerated stability data for predicting protein stability in sucrose containing formulations stored at 2-8 degrees C, where no glycation or change in aggregation rate was observed.

Effect of Sugar Molecules on the Viscosity of High Concentration Monoclonal Antibody Solutions

ABSTRACTPurposeTo assess the effect of sugar molecules on solution viscosity at high protein concentrations.MethodsA high throughput dynamic light scattering method was used to measure the viscosity of monoclonal antibody solutions. The effects of protein concentration, type of sugar molecule (trehalose, sucrose, sorbitol, glucose, fructose, xylose and galactose), temperature and ionic strength were evaluated. Differential scanning fluorimetry was used to reveal the effect of the same sugars on protein stability and to provide insight into the mechanism by which sugars increase viscosity.ResultsThe addition of all seven types of sugar molecules studied result in a significant increase in viscosity of high concentration monoclonal antibody solutions. Similar effects of sugars were observed in the two mAbs examined; viscosity could be reduced by increasing the ionic strength or temperature. The effect by sugars was enhanced at higher protein concentrations.ConclusionsDisaccharides have a greater effect on the solution viscosity at high protein concentrations compared to monosaccharides. The effect may be explained by commonly accepted mechanisms of interactions between sugar and protein molecules in solution.

Top-down N-terminal sequencing of Immunoglobulin subunits with electrospray ionization time of flight mass spectrometry

An N-terminal top-down sequencing approach was developed for IgG characterization, using high-resolution HPLC separation and collisionally activated dissociation (CAD) on a single-stage LCT Premier time of flight (TOF) mass spectrometer. Fragmentation of the IgG chains on the LCT Premier was optimized by varying the ion guide voltage values. Ion guide 1 voltage had the most significant effect on the fragmentation of the IgG chains. An ion guide 1 voltage value of 100 V was found to be optimum for the N-terminal fragmentation of IgG heavy and light chains, which are approximately 50 and 25 kDa, respectively. The most prominent ion series in this CAD experiment was the terminal b-ion series which allows N-terminal sequencing. Using this technique, we were able to confirm the sequence of up to seven N-terminal residues. Applications of this method for the identification of N-terminal pyroglutamic acid formation will be discussed. The method described could be used as a high-throughput method for the rapid N-terminal sequencing of IgG chains and for the detection of chemical modifications in the terminal residues.

Detection and quantitation of IgG 1 hinge aspartate isomerization: a rapid degradation in stressed stability studies.

In biopharmaceutical process development, it is desirable to identify sites of covalent degradations to ensure product consistency. One characterization method used for therapeutic immunoglobulin gamma (IgG) 1 antibodies is limited LysC proteolysis followed by reversed-phase LC/MS. Limited LysC proteolysis leads to high efficiency cleavage at the C-terminal side of the hinge lysine 222 residue, generating Fab and Fc fragments. In this report, we show that IgG 1 samples incubated under mildly acidic conditions at elevated temperatures were partially resistant to LysC cleavage at the hinge and resulted in a species where one of the Fab arms remained connected to the Fc region (Fab-Fc). The growth of the Fab-Fc species was proportional to the duration and storage temperature of the incubation period and correlated with the amount of isomerization of the aspartic acid residue preceding lysine 222, determined by peptide mapping. The isomerization rates of samples stored for up to one year at 4 degrees C, 6 months at 29 or 37 degrees C, or 3 months at 45 degrees C were determined, and the activation energy for this conversion was calculated to be approximately 33 kJ mol(-1). The apparent isomerization rate constant was only 0.02 week(-1) for samples stored at 4 degrees C, which resulted in a modest increase from 5.1 to 6.0% isoD after twenty four weeks of storage and, hence, is not a significant concern under normal storage conditions typically used for monoclonal antibodies. However, when stored at 29 degrees C, the apparent rate constant of this reaction was found to be 0.06 week(-1) and resulted in an increase from 5.1 to 21.1% isoD after twenty four weeks of storage and is a major degradant in stressed IgG 1 antibodies.