A high-throughput UPLC method for the characterization of chemical modifications in monoclonal antibody molecules.

Development of high-throughput release and characterization assays is critical for the effective support of the rapidly growing biologics pipeline for biotherapeutics. Clipping of polypeptide chains is commonly monitored during process optimization, formulation development, and stability studies. A reduced capillary electrophoresis-sodium dodecyl sulfate (rCE -SDS) method is often used as a purity release assay for monitoring clips in monoclonal antibodies (mAbs); however, it has a cycle time of approximately 40 min, which is not suited for high-throughput screening. Additionally, the characterization of clips and variants from electropherograms is not straightforward and takes significant time. Reduced reversed-phase (RP) chromatography has been a popular assay for the characterization and identification of clips and variants because it can be directly coupled with online mass spectrometric analysis. However, the high-column temperature and low pH required for RP assays can induce on-column cleavage and therefore skew the results. To minimize on-column degradation, we have developed a high-throughput method with a significantly shorter cycle time of 5 min. The short cycle time was achieved using an ultra-high-pressure liquid chromatography (UPLC) system with a 1.7 µm phenyl column. This UPLC method allowed quantitation of hinge clipping in an IgG1 molecule and acid induced aspartic acid/proline (D/P) clip in an IgG2 molecule. The results from the UPLC method were comparable to those obtained with rCE-SDS. Additionally, the phenyl column offered partial resolution of oxidation and other chemical modifications, making this technique an attractive assay for high-throughput process characterization and formulation screens.

Effect of sugar molecules on the viscosity of high concentration monoclonal antibody solutions.

PURPOSE: To assess the effect of sugar molecules on solution viscosity at high protein concentrations. METHODS: A 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. RESULTS: The 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. CONCLUSIONS: Disaccharides 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.

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.

Middle-down fragmentation for the identification and quantitation of site-specific methionine oxidation in an IgG1 molecule.

A middle-down LC/MS approach, for the rapid quantitation and characterization of site-specific methionine oxidation in a recombinant monoclonal IgG1 molecule, is described. An IgG1 antibody was digested with endoprotease LysC under limited proteolytic conditions to produce two major components; an antigen binding fragment (Fab) and a crystallizable fraction (Fc). These fractions were then reduced to produce three major species; light chain (LC), Fc/2 which is the C terminal region of the heavy chain (HC) and the N-terminal heavy chain region (Fd). These three fragments were separated by reversed-phase HPLC using a diphenyl column. The diphenyl column resolved site-specific methionine oxidation in all three subunits. Middle-down N-terminal sequencing with a LCT premier mass spectrometer was used to identify the sites of oxidation in the LC. Sites of oxidation in the Fc/2 were identified using middle-down collision-induced dissociation (CID) on a Qtof premier. This method allowed for the rapid quantitation and identification of oxidation on each methionine residue in an IgG1 molecule.

LC/MS analysis of complex multiglycosylated human alpha(1)-acid glycoprotein as a model for developing identification and quantitation methods for intact glycopeptide analysis.

The site-specific characterization of the complex glycans in multiglycosylated proteins requires developing methods where the carbohydrates remain covalently bound to the protein. The complexity in the carbohydrate composition of alpha(1)-acid glycoprotein (AAG) makes it an ideal model protein for such development. AAG has five N-asparaginyl-linked glycosylation sites, each varying in its bi-, tri-, and tetraantennary glycan content. We present an on-line liquid chromatography/mass spectrometry (LC/MS) method that uses high-low cone voltage switching for in-source fragmentation to determine the structures of the complex glycans present on each site for the two gene products of AAG. High cone voltage caused carbohydrate fragmentation, leading to the generation of signature carbohydrate ions that we used as markers to identify the glycopeptides. Low cone voltage produced minimal carbohydrate fragmentation and enabled the identification and quantification of the intact oligosaccharide structures on each glycopeptide based on its monoisotopic mass and intensity. Quantitation was accomplished by using the intensities of peaks from deconvoluted and deisotoped mass spectra or from the areas of the extracted ion chromatograms from the tryptic peptide maps. The combined results from the two methods can be used to better characterize and quantitate site heterogeneity in multiglycosylated proteins.

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.

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.

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.

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).

Removal of cysteinylation from an unpaired sulfhydryl in the variable region of a recombinant monoclonal IgG1 antibody improves homogeneity, stability, and biological activity.

The antibody MAB007 was recently shown to be cysteinylated on an unpaired cysteine residue in the CDR3 variable region. Cysteinylation at this position was not complete and resulted in heterogeneous lots of MAB007 with respect to this posttranslational modification. In this report, a mild redox step was used that effectively removed cysteinylation while keeping native inter and intra-molecular disulfide bonds intact. Biophysical methods were employed to determine what consequences cysteinylation of the variable region had by directly comparing cysteinylated and de-cysteinylated MAB007 antibodies. No differences were detected in secondary structure; however, several pieces of evidence indicated that cysteinylation may result in tertiary or quaternary structural perturbations. These included differences in the cation-exchange chromatography and fluorescence-emission spectra of the cysteinylated and de-cysteinylated antibodies as well as differences in the solvent accessibility of the unpaired cysteine residue determined by labeling experiments. Such structural changes induced by cysteinylation were shown to increase the rate of MAB007 aggregation and to decrease the melting temperature of the Fab region by as much as 6 degrees C. The bioactivity of MAB007 was also shown to be adversely affected by cysteinylation and a direct correlation was made between the percent cysteinylation and biological activity.

Reversed-phase liquid chromatography-mass spectrometry of site-specific chemical modifications in intact immunoglobulin molecules and their fragments.

The employment of a diphenyl column for the separation of intact monoclonal antibodies (mAbs) and their fragments by reversed-phase HPLC is discussed as a novel approach for the characterization of chemical modifications in a site-specific manner. Chromatographic separation of the intact mAb07 on the diphenyl support resulted in the separation of the cysteinylated from the non-cysteinylated mAb. A detected mass increase of 119 Da by mass spectrometric sequence analysis confirmed the cysteinylation. Furthermore, the diphenyl column resolved site-specific oxidation of five different methionine residues in the heavy chain (HC) of mAb03. Oxidized mAb03 HC eluted as five distinct peaks with shorter retention times than the corresponding peak representing unoxidized HC. Analysis of these peaks by in-line mass spectrometric analysis confirmed the site-specific oxidation of five different methionine residues. In another application, the diphenyl column was able to resolve free sulfhydryl groups containing Fc and Fab fragments, which were generated by limited proteolysis with endoproteinase Lys-C. The free sulfhydryl groups were responsible for a mass shift of approximately 2 Da. Their identity was further confirmed by N-ethylmaleimide labeling, which caused a shift in their chromatographic retention and led to a mass increase of 250 Da. This is the first report about chromatographic resolution on a reversed-phase column that results in site-specific separation of chemical modifications in intact mAb and mAb fragments.

Reversed-phase liquid chromatography of immunoglobulin G molecules and their fragments with the diphenyl column.

A diphenyl column was able to resolve two closely related monoclonal IgG2 molecules, while a C8 column failed to separate these IgGs under identical chromatographic conditions. The diphenyl column also showed a better separation of a mixture of two light and two heavy chains than the C8 column. The influence of amino acid side chains from protein sequences in binding to the diphenyl and C8 stationary phases was studied by using a set of synthetic peptides with the sequence GXXLLLKK, where X represents substitution with all of the 20 amino acids. Peptides containing aromatic amino acids showed a greater binding on the diphenyl column than on the C8 column. This increase in retention was attributed to pi-pi interactions between the aromatic amino acid side chains and the diphenyl ligand. Based on the retention of peptides on the diphenyl column, new retention coefficients were assigned for the separation of proteins. A good correlation was observed between the sum of retention coefficients (SigmaRc) for IgGs and their retention time on the diphenyl column. On-column hydrogen-deuterium exchange showed that the diphenyl column had a larger surface of interaction with protein than the C8 column. pi-pi interactions and the large contact surface resulted in improved resolution of IgGs and their fragments on the diphenyl column.

The LC/MS analysis of glycation of IgG molecules in sucrose containing formulations.

Glycation of a recombinant monoclonal IgG2 molecule, in sucrose containing liquid formulations, was studied using reversed-phase LC/MS analysis of the intact IgG, the F(ab')2 fragments and after complete tryptic digestion. The extent of glycation in sucrose containing formulations was monitored at different temperatures over a period of 21 months using the Hexose index (Hex(I)). Hex(I) represents the average number of hexose molecules per molecule of IgG and was calculated by using the intensity values of peaks corresponding to hexose isoforms in the deconvoluted mass spectra. The rate of glycation in mildly acidic sucrose containing formulations was proportional to the incubation temperature. No glycation was observed in sucrose containing formulations incubated at 4 degrees C even after 18 months. However, when the same formulations were incubated at 37 degrees C glycation was observed after just 1 month. The glycation sites were mapped to 10 lysine residues distributed throughout the molecule. The amino terminal end of the light chain was also shown to contain glycation. The surface accessibility of the lysine side chain could influence its susceptibility to glycation.

Screening and sequencing of glycated proteins by neutral loss scan LC/MS/MS method.

Nonenzymatic protein glycation is caused by a Schiff's base reaction between the aldehyde groups of reducing sugars and the primary amines of proteins. A reversed-phase liquid chromatography method followed by a neutral loss scan mass spectrometric method was developed for the screening of glycation in proteins. The neutral loss scan was based on a unique sugar moiety neutral loss (-162 Da) that we observed in the fragmentation spectra of glycated peptides on Q-Tof type mass spectrometers. The collision energy was optimized for this neutral loss using a glycated synthetic peptide, and 20 eV was found to be the optimum collision energy. The neutral loss scan experiment was composed of two segments. In the first segment, the glycated peptides were identified based on the signature neutral loss of 162 Da when the collision energy was elevated to 20 eV. In the second segment, the glycated peptides were selected as the parent ions and fragmented at higher collision energy to break the peptide bonds. The fragmentation spectra of the selected glycated peptides revealed both the amino acid sequences and the sites of glycation. This neutral loss scan method was used to study the glycation in human serum albumin (HSA). The glycation sites in HSA were identified based on the retention time shift of glycated peptides, the mass accuracy from the MS scan, the signature neutral loss, and MS/MS information. Using this method, we were able to identify that 31 lysine residues were partially glycated from the glycated HSA sample, which has a total of 59 lysine residues.

Identification of cysteinylation of a free cysteine in the Fab region of a recombinant monoclonal IgG1 antibody using Lys-C limited proteolysis coupled with LC/MS analysis.

MAB007, an IgG1 monoclonal antibody, is unique because of the presence of a free cysteine residue in the Fab region at position 104 on the heavy chain in the CDR3 region. Mass spectrometric analysis of intact MAB007 showed multiple peaks varying in mass by 120-140 Da that cannot be fully attributed to glycosylation isoforms typically present in IgG molecules. Limited proteolysis of MAB007 with Lys-C led to a single cleavage at the C-terminus of a lysine residue in the hinge region of the heavy chain at position 222, generating free Fab and Fc fragments. Reversed-phase liquid chromatography/mass spectrometry analysis of the Fab and Fc fragments revealed several modifications. The Fab fraction showed cysteinylation of a free cysteine in the CDR3 region resulting in a mass shift of 119 Da. Using limited proteolysis, we also identified modifications resulting in a mass increase of 127 Da in the Fc region, corresponding to C-terminal lysine variants in the heavy chain. Other modifications, such as oxidation (+16 Da) and succinimide formation (-17 Da), were also detected in the Fab fragment. The cysteinylation observed after limited proteolysis was confirmed by peptide mapping coupled with tandem mass spectrometry analysis.

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.

Paraffin-wax-coated plates as matrix-assisted laser desorption/ionization sample support for high-throughput identification of proteins by peptide mass fingerprinting.

We compared trysin-digested protein samples desalted by ZipTip(C18) reverse-phase microcolumns with on-plate washing of peptides deposited either on paraffin-coated plates (PCP), Teflon-based AnchorChip plates, or stainless steel plates, before analysis by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). Trypsinized bovine serum albumin and ovalbumin and 16 protein spots extracted from silver-stained two-dimensional gels of murine C(2)C(12) myoblasts or human leukocytes, prepared by the above two methods, were subjected to MALDI on PCP, AnchorChip plates, or uncoated stainless steel plates. Although most peptide mass peaks were identical regardless of the method of desalting and concentrating of protein samples, samples washed and concentrated by the PCP-based method had peptide peaks that were not seen in the samples prepared using the ZipTip(C18) columns. The mass spectra of peptides desalted and washed on uncoated stainless steel MALDI plates were consistently inferior due to loss of peptides. Some peptides of large molecular masses were apparently lost from samples desalted by ZipTip(C18) microcolumns, thus diminishing the quality of the fingerprint needed for protein identification. We demonstrate that the method of washing of protein samples on paraffin-coated plates provides an easy, reproducible, inexpensive, and high-throughput alternative to ZipTip(C18)-based purification of protein prior to MALDI-TOF-MS analysis.

Proteome of monocytes primed with lipopolysaccharide: analysis of the abundant proteins.

We performed a proteomic analysis of monocytes primed by lipopolysaccharide (LPS) in vitro, using two-dimensional gels stained with Coomassie blue. We found 16 proteins of approximately 500 detected that either increased or decreased in abundance as a result of priming by LPS (14 with P