Ivan Correia

Increased serum clearance of oligomannose species present on a human IgG1 molecule

The role of Fc glycans on clearance of IgG molecule has been examined by various groups in experiments where specific glycans have been enriched or the entire spectrum of glycans was studied after administration in pre-clinical or clinical pharmacokinetic (PK) studies. The overall conclusions from these studies are inconsistent, which may result from differences in antibody structure or experimental design. In the present study a well-characterized recombinant monoclonal IgG1 molecule (mAb-1) was analyzed from serum samples obtained from a human PK study. mAb-1 was recovered from serum using its ligand cross-linked to Sepharose beads. The overall purity and recovery of all isoforms were carefully evaluated using a variety of methods. Glycans were then enzymatically cleaved, labeled using 2-aminobenzamide and analyzed by normal phase high performance liquid chromatography. The assays for recovering mAb-1 from serum and subsequent glycan analysis were rigorously qualified at a lower limit of quantitation of 15 μg/mL, thus permitting analysis to day 14 of the clinical PK study. Eight glycans were monitored and classified into two groups: (1) the oligomannose type structures (M5, M6 and M7) and (2) fucosylated biantennary oligosaccharides (FBO) structures (NGA2F, NA1F, NA2F, NA1F-GlcNAc and NGA2F-GlcNAc). We observed that the oligomannose species were cleared at a much faster rate (40%) than FBOs and conclude that high mannose species should be carefully monitored and controlled as they may affect PK of the therapeutic; they should thus be considered an important quality attribute. These observations were only possible through the application of rigorous analytical methods that we believe will need to be employed when comparing innovator and biosimilar molecules.

Studies in serum support rapid formation of disulfide bond between unpaired cysteine residues in the VH domain of an immunoglobulin G1 molecule

Recombinant monoclonal antibodies undergo extensive posttranslational modifications. In this article, we characterize major modifications, separated by cation exchange chromatography, on an immunoglobulin G1 (IgG1) monoclonal antibody (mAb). We found that N-terminal cyclization of glutamine residues to pyroglutamate on the light and heavy chains are the major isoforms resolved during cation exchange chromatography. However, using CEX, we also separated and identified isoforms with unpaired cysteine residues in the V(H) domain of the molecule (Cys22-Cys96). Omalizumab, a therapeutic anti-IgE antibody, has unpaired cysteine residues in the V(H) domain between Cys22 and Cys96, and the Fab fragment, containing the unpaired cysteine residues, is reported to have reduced potency. Dynamic interchain disulfide rearrangement, with slow kinetics, was recently reported to take place in serum for an IgG2 molecule and resulted in predictable mature isoforms. Analytical evaluation of our mAb, after recovery from serum, revealed that the unpaired intrachain cysteine residues (Cys22-Cys96) reformed their disulfide bond. The significance of this study is that correct pairing occurred rapidly, and we speculate that thiol molecules such as cysteine, homocysteine, and glutathione in serum provide an environment, outside the endoplasmic reticulum, for correct linkage.

Comparison of the in vitro and in vivo stability of a succinimide intermediate observed on a therapeutic IgG1 molecule

Deamidation of asparagine residues, a post-translational modification observed in proteins, is a common degradation pathway in monoclonal antibodies (mAbs). The kinetics of deamidation is influenced by primary sequence as well as secondary and tertiary folding. Analytical hydrophobic interaction chromatography (HIC) is used to evaluate hydrophobicity of candidate mAbs and uncover post-translational modifications. Using HIC, we discovered atypical heterogeneity in a highly hydrophobic molecule (mAb-1). Characterization of the different HIC fractions using LC/MS/MS revealed a stable succinimide intermediate species localized to an asparagine-glycine motif in the heavy chain binding region. The succinimide intermediate was stable in vitro at pH 7 and below and increased on storage at 25°C and 40°C. Biacore evaluation showed a decrease in binding affinity of the succinimide intermediate compared with the native asparagine molecule. In vivo studies of mAb-1 recovered from a pharmacokinetic study in cynomolgus monkeys revealed an unstable succinimide species and rapid conversion to aspartic/iso-aspartic acid. Mutation from asparagine to aspartic acid led to little loss in affinity. This study illustrates the importance of evaluating modifications of therapeutic mAbs both in vitro and in serum, the intended environment of the molecule. Potential mechanisms that stabilize the succinimide intermediate in vitro are discussed.

Elevated cleavage of human immunoglobulin gamma molecules containing a lambda light chain mediated by iron and histidine

Monoclonal antibodies in liquid formulation undergo nonenzymatic hydrolysis when stored at 5 degrees C for extended periods. This hydrolysis is enhanced at extreme pH and high temperature. In this study we discover that iron in the presence of histidine also enhanced cleavage of human immunoglobulin gamma (IgG) molecules containing a lambda light chain when incubated at 40 degrees C. The level of cleavage was concentration dependent on both iron and histidine levels. Enhanced cleavage with iron and histidine was not observed on IgG molecules containing a kappa light chain. Using CE-SDS to quantify levels of Fab+Fc, the Fab arm, and free light chain (LC) and heavy chain (HC) fragments, we show that cleavage resulted in elevated levels of free light and heavy chain fragments. Using MS we find elevated scission between residues E/C on the LC resulting in LC fragment 1-215. We also observed enhanced cleavage between S/C residues of the HC resulting in HC fragment 1-217. The corresponding Fab+Fc fragment beginning with cys-218 was not found. Instead, we find elevation of a Fab+Fc fragment that began with aspartic acid (cleavage between C/D). Further studies to understand how iron and histidine enhance cleavage of lambda light chain IgG molecules are ongoing.

Identifying low‐level sequence variants via next generation sequencing to aid stable CHO cell line screening

Developing stable Chinese hamster ovary (CHO) cell lines for biotherapeutics is an irreversible process and therefore, key quality attributes, such as sequence variants, must be closely monitored during cell line development (CLD) to avoid delay in the developmental timeline, and more importantly, to assure product safety and efficacy. Sequence variants, defined as unintended amino acid substitution in recombinant protein primary structure, result from alteration at either the DNA or the protein level. Here, for the first time, we report the application of transcriptome sequencing (RNAseq) in an IgG1 monoclonal antibody (mAb) CLD campaign to detect, identify, and eliminate cell lines containing low‐level point mutations in recombinant coding sequence. Among the top eleven mAb producers chosen from transfectant, clone or subclone stages, three of the cell lines contained either missense or nonsense point mutations at a low level of less than 2%. Subsequent LC/MS/MS characterization detected ∼3% sequence variants with an amino acid change from Ser to Leu at residue 117 in the heavy chain of transfectants 11 and 27. This substitution is consistent with the RNAseq finding of a C/T mutation located at 407 base pair (TCA→TTA) in the heavy chain coding sequence. Here we demonstrate that RNAseq is a rapid and highly sensitive method to identify low‐level genetic mutation de novo corresponding to the amino acid substitution that elicits sequence variant(s). Its implementation in CLD constitutes an early and effective step in identifying desired CHO expression cell lines.

Cell culture media supplementation of uncommonly used sugars sucrose and tagatose for the targeted shifting of protein glycosylation profiles of recombinant protein therapeutics

Protein glycosylation is an important post‐translational modification toward the structure and function of recombinant therapeutics. The addition of oligosaccharides to recombinant proteins has been shown to greatly influence the overall physiochemical attributes of many proteins. It is for this reason that protein glycosylation is monitored by the developer of a recombinant protein therapeutic, and why protein glycosylation is typically considered a critical quality attribute. In this work, we highlight a systematic study toward the supplementation of sucrose and tagatose into cell culture media for the targeted modulation of protein glycosylation profiles on recombinant proteins. Both sugars were found to affect oligosaccharide maturation resulting in an increase in the percentage of high mannose N‐glycan species, as well as a concomitant reduction in fucosylation. The latter effect was demonstrated to increase antibody‐dependent cell‐mediated cytotoxicity for a recombinant antibody. These aforementioned results were found to be reproducible at different scales, and across different Chinese hamster ovary cell lines. Through the selective supplementation of these described sugars, the targeted modulation of protein glycosylation profiles is demonstrated, as well as yet another tool in the cell culture toolbox for ensuring product comparability.

A high throughput capillary electrophoresis method to obtain pharmacokinetics and quality attributes of a therapeutic molecule in circulation

Therapeutic proteins circulating in blood are in a highly crowded, redox environment at high temperatures of ~37°C. These molecules circulate in the presence of enzymes and other serum proteins making it difficult to predict from in vitro studies the stability, aggregation or pharmacokinetics of a therapeutic protein in vivo. Here, we describe use of a high throughput capillary electrophoresis based microfluidic device (LabChip GXII) to obtain pharmacokinetics (PK) of a fluorescently labeled human mAb directly from serum. The non-labeled and labeled mAbs were evaluated in single dose rat PK studies using a traditional ELISA method or LabChip GXII, respectively. The fluorescent dye did not significantly alter clearance of this particular mAb, and PK parameters were comparable for labeled and unlabeled molecules. Further, from the CE profile we concluded that the mAb was resistant to fragmentation or aggregation during circulation. In a follow-up experiment, dimers were generated from the mAb using photo-induced cross-linking of unmodified proteins (PICUP) and labeled with the same fluorophore. The extent of dimerization was incomplete and some monomer and higher molecular weight species were found in the preparation. In rat PK studies, the serum concentration-time profile of the three entities present in the dimer preparation could be followed simultaneously with the GXII technology. While further studies are warranted, we believe this method could be adapted to obtain PK of different forms of antibodies (oxidized, deamidated or various glycosylated species) and other proteins.

Engineering Elastic Properties into an Anti-TNFα Monoclonal Antibody

Abstract Injecting anti-tumor necrosis factor (TNF)α antibodies into patient joints at the site of inflammation, inter-articular (IA) delivery, has demonstrated modest success for treatment of Spondyloarthritis (SpA), Rheumatoid Arthritis (RA), and osteoarthritis. However, IA delivery is not the treatment route of choice due to rapid clearance from the site of administration. Elastin-like polypeptides (ELPs) are reported to undergo phase transition; forming reversible aggregates above their transition temperature, which when injected into IA space have a 25-fold longer half-life compared to the soluble form. Here, we fused an ELP repeat to the C-terminus of each heavy chain of an anti-TNFα monoclonal antibody (mAb) and provide detailed characterization of the fusion IgG molecule. Expression and purification yielded homogenous protein confirmed by gels, hydrophobic-interaction chromatography, Capilary Electrophoresis (CE), Mass Spectrometry (MS), and analytical ultracentrifugation. The ELPs altered hydrophobicity and pI of the parent mAb and new elastic properties were imparted to the molecule; forming large stable complexes with a hydrodynamic radius of 40 nm above 39°C that dissociated into soluble, active monomer below 37°C. The fusion mAb retained its affinity and ability to neutralize TNFα as determined by surface plasmon resonance and cell-based assay, respectively, with equal potency to unmodified anti-TNFα mAb. Differential-scanning calorimetry studies show stabilization of adjacent CH2 and CH3 domains in the fusion molecule and the aggregated molecule was found to be fully functional after 7 days at 37°C. For the first time, we reveal architecture of an ELP-fusion mAb and binding to antigen using single-particle-transmission-electron microscopy. Unstructured ELP was visualized at the C-terminus and binding to antigen was shown at the N-terminus. Collectively, these studies indicate that it is possible to impart elastic properties to a monoclonal antibody while retaining purity, stability, and ability to effectively bind and neutralize antigen.