Alison Wallace

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.

Glutamine-linked and Non-consensus Asparagine-linked Oligosaccharides Present in Human Recombinant Antibodies Define Novel Protein Glycosylation Motifs

We report the presence of oligosaccharide structures on a glutamine residue present in the VL domain sequence of a recombinant human IgG2 molecule. Residue Gln-106, present in the QGT sequence following the rule of an asparagine-linked consensus motif, was modified with biantennary fucosylated oligosaccharide structures. In addition to the glycosylated glutamine, analysis of a lectin-enriched antibody population showed that 4 asparagine residues: heavy chain Asn-162, Asn-360, and light chain Asn-164, both of which are present in the IgG1 and IgG2 constant domain sequences, and Asn-35, which was present in CDRL1, were also modified with oligosaccharide structures at low levels. The primary sequences around these modified residues do not adhere to the N-linked consensus sequon, NX(S/T). Modeling of these residues from known antibody crystal structures and sequence homology comparison indicates that non-consensus glycosylation occurs on Asn residues in the context of a reverse consensus motif (S/T)XN located on highly flexile turns within 3 residues of a conformational change. Taken together our results indicate that protein glycosylation is governed by more diversified requirements than previously appreciated.

Separation of populations of antibody variants by fine tuning of hydrophobic-interaction chromatography operating conditions

The following report describes the use of hydrophobic-interaction chromatography (HIC) to separate and characterize populations of monoclonal antibodies resulting from variable N- and C-terminal processing, stressed-induced covalent modifications and conformationally altered populations present in the drug product. We investigated the use of HIC to characterize heterogeneity in the intact molecule and the Fab and Fc sub-domains resulting from papain cleavage. We found that certain classes of covalent modifications to antibodies are highly amenable to HIC separation. Specific covalent modifications occurring on antibodies could be separated into pure fractions which contained unmodified, singly modified (on 1 heavy or light chain) and doubly modified (on both heavy or light chains) molecules. This report demonstrates the utility of HIC for assessing the heterogeneity, stability and, in some cases, potency of monoclonal antibodies.

Isolation and Isotope Labeling of Cysteine- and Methionine-containing Tryptic Peptides Application to the Study of Cell Surface Proteolysis

Inexpensive methods were developed for isolating and isotopically labeling tryptic peptides that contain either cysteine or methionine. After covalently capturing cysteine-containing peptides with pyridyl disulfide reactive groups on agarose beads, extensive wash steps were applied, and the attached peptides were released using a reducing agent. This approach results in less nonspecifically bound peptides and eliminates the possibility of generating avidin peptide background ions that can arise when using methods based on biotin and avidin (e.g. isotope-coded affinity tag). The thiols were alkylated using either N-ethyl- or N-D5-ethyl-iodoacetamide, both of which can be synthesized in a single step using inexpensive reagents. This isotopic labeling does not greatly increase the peptide mass, nor does it affect the peptide ion charge state in electrospray ionization. In addition, methionine-containing peptides were captured using commercially available methionine-reactive beads, and relative quantitation of peptides was achieved by isotopic labeling of amino groups using activated esters of either nicotinic acid or D4-nicotinic acid. These methods were used to study the metalloprotease-mediated shedding of cell surface proteins from a mouse monocyte cell line that had been treated with a phorbol ester and lipopolysaccharide. In addition to the identification of proteins previously determined to be inducibly shed, three new shed proteins were identified: CD18, ICOS ligand, and tumor endothelial marker 7-related protein.

Asparagine-linked Oligosaccharides Present on a Non-consensus Amino Acid Sequence in the CH1 Domain of Human Antibodies

We report that N-linked oligosaccharide structures can be present on an asparagine residue not adhering to the consensus site motif NX(S/T), where X is not proline, described in the literature. We have observed oligosaccharides on a non-consensus asparaginyl residue in the CH1 constant domain of IgG1 and IgG2 antibodies. The initial findings were obtained from characterization of charge variant populations evident in a recombinant human antibody of the IgG2 subclass. HPLC-MS results indicated that cation-exchange chromatography acidic variant populations were enriched in antibody with a second glycosylation site, in addition to the well documented canonical glycosylation site located in the CH2 domain. Subsequent tryptic and chymotryptic peptide map data indicated that the second glycosylation site was associated with the amino acid sequence TVSWN162SGAL in the CH1 domain of the antibody. This highly atypical modification is present at levels of 0.5–2.0% on most of the recombinant antibodies that have been tested and has also been observed in IgG1 antibodies derived from human donors. Site-directed mutagenesis of the CH1 domain sequence in a recombinant-human IgG1 antibody resulted in an increase in non-consensus glycosylation to 3.15%, a greater than 4-fold increase over the level observed in the wild type, by changing the −1 and +1 amino acids relative to the asparagine residue at position 162. We believe that further understanding of the phenomenon of non-consensus glycosylation can be used to gain fundamental insights into the fidelity of the cellular glycosylation machinery.

Separation and characterization of an IgG2 antibody containing a cyclic imide in CDR1 of light chain by hydrophobic interaction chromatography and mass spectrometry.

Hydrophobic interaction chromatography (HIC) was used to separate populations of recombinant IgG2 antibody that were created as a result of prolonged incubation at 40 degrees C. Antibody was separated by HIC into three major and seven minor fractions. All but one fraction was composed of antibody with distinct chemical modifications that resulted from exposure to elevated temperature. The results of intact and reduced mass analysis as well as peptide map data derived from the three major HIC fractions indicated that the antibody was being chromatographically separated into populations containing a succinimidyl intermediate in complementarity determining region 1 (CDR1) on zero, one, and two light chain arms. Lower level species purified by HIC were analyzed by intact and reduced mass analysis and laser-induced fluorescence capillary electrophoresis (CE-LIF) and consisted of an antibody that was clipped in four different places in the heavy chain as well as misfolded and aggregated antibody. The potency of the recombinant antibody containing a succinimidyl intermediate on zero, one, and two light chain arms was analyzed by LANCE binding assay and a cell based in vitro bioassay, and the occurrence of this modification on one or both light chain arms was associated with a reduction in the binding affinity of the molecule to the target by approximately 10%. We show that HIC has the unique ability as a first step purification method to separate populations of antibody which are covalently modified under stability programs. The method conditions that have been developed for the HIC assay are ideal for purifying antibodies with labile modifications for the purpose of further characterization.

O-Fucosylation of an antibody light chain: Characterization of a modification occurring on an IgG1 molecule

We describe the characterization of an O-fucosyl modification to a serine residue on the light chain of a recombinant, human IgG1 molecule expressed in Chinese hamster ovary (CHO) cells. Cation exchange chromatography (CEX) and hydrophobic interaction chromatography (HIC) were used to isolate a Fab population which was 146 Da heavier than the expected mass. Isolated Fab fragments were treated with a reducing agent to facilitate mass spectrometric analysis of the reduced light chain (LC) and fragment difficult (Fd). An antibody light chain with a net addition of 146 Da was detected by mass spectrometric analysis of the modified Fab. A light chain tryptic peptide in complementarity determining region-1 (CDR-1) was subsequently identified with a net addition of 146 Da by a peptide map. Results from a nanospray infusion of the modified peptide into a linear ion trap mass spectrometer with electron transfer dissociation (ETD) functionality indicated that the modified residue was a serine at position 30 in the light chain. Acid hydrolysis of the modified tryptic peptide followed by fluorescent labeling with 2-aminoanthranilic acid (2AA) and HPLC comparison with monosaccharide standards confirmed the presence of fucose on the light chain peptide. The presence of O-fucose on an antibody has not been previously reported. Currently, O-fucose has been described as occurring on mammalian proteins with amino acid sequence motifs associated with epidermal growth factor (EGF)-like repeats or thrombospondin type 1 repeats (TSRs). The amino acid sequence around the modified Ser in the IgG1 molecule does not conform to any known O-fucosylation sequence motif and thus is the first description of this type of modification on a nonconsensus sequence in a mammalian protein.

Assessing analytical methods to monitor isoAsp formation in monoclonal antibodies

A ubiquitous post-translational modification observed in proteins is isomerization of aspartic acid to isoaspartic acid (isoAsp). This non-enzymatic post-translational modification occurs spontaneously in proteins and plays a role in aging, autoimmune response, cancer, neurodegeneration, and other diseases. Formation of isoAsp is also a significant issue for recombinant monoclonal antibody based protein therapeutics particularly when isomerization occurs in a complementarity-determining region due to potential impact to the clinical efficacy. Here, we present and compare three analytical methods to monitor and/or quantify isoAsp formation in a monoclonal antibody. The methods include two peptide map based technologies with quantitation from either UV integration or total ion peak areas, as well as an alternative approach using IdeS digestion to generate Fc/2 and Fab’2 regions, followed by hydrophobic interaction chromatography (HIC) to separate the population of Fab’2 containing an isoAsp. The level of isoAsp detected by the peptide map and the digested-HIC methods presented here show similar trends although sample throughput varies by method.

A chemical and computational approach to comprehensive glycation characterization on antibodies

Non-enzymatic glycation is a challenging post-translational modification to characterize due to the structural heterogeneity it generates in proteins. Glycation has become increasingly recognized as an important product quality attribute to monitor, particularly for the biotechnology sector, which produces recombinant proteins under conditions that are amenable to protein glycation. The elucidation of sites of glycation can be problematic using conventional collision-induced dissociation (CID)-based mass spectrometry because of the predominance of neutral loss ions. A method to characterize glycation using an IgG1 monoclonal antibody (mAb) as a model is reported here. The sugars present on this mAb were derivatized using sodium borohydride chemistry to stabilize the linkage and identified using CID-based MS2 mass spectrometry and spectral search engines. Quantification of specific glycation sites was then done using a targeted MS1 based approach, which allowed the identification of a glycation hot spot in the heavy chain complementarity-determining region 3 of the mAb. This targeted approach provided a path forward to developing a structural understanding of the propensity of sites to become glycated on mAbs. Through structural analysis we propose a model in which the number and 3-dimensional distances of carboxylic acid amino acyl residues create a favorable environment for glycation to occur.

Photochemical degradation of citrate buffers leads to covalent acetonation of recombinant protein therapeutics

Novel acetone and aldimine covalent adducts were identified on the N‐termini and lysine side chains of recombinant monoclonal antibodies. Photochemical degradation of citrate buffers, in the presence of trace levels of iron, is demonstrated as the source of these modifications. The link between degradation of citrate and the observed protein modifications was conclusively established by tracking the citrate decomposition products and protein adducts resulting from photochemical degradation of isotope labeled 13C citrate by mass spectrometry. The structure of the acetone modification was determined by nuclear magnetic resonance (NMR) spectroscopy on modified–free glycine and found to correspond to acetone linked to the N‐terminus of the amino acid through a methyl carbon. Results from mass spectrometric fragmentation of glycine modified with an acetone adduct derived from 13C labeled citrate indicated that the three central carbons of citrate are incorporated onto protein amines in the presence of iron and light. While citrate is known to stoichiometrically decompose to acetone and CO2 through various intermediates in photochemical systems, it has never been shown to be a causative agent in protein carbonylation. Our results point to a previously unknown source for the generation of reactive carbonyl species. This work also highlights the potential deleterious impact of trace metals on recombinant protein therapeutics formulated in citrate buffers.