Characterization of N-Terminal Glutamate Cyclization in Monoclonal Antibody and Bispecific Antibody Using Charge Heterogeneity Assays and Hydrophobic Interaction Chromatography.

N-terminal glutamate (E) cyclization to form pyroglutamate (pE) generates charge heterogeneities for mAbs and proteins. Thus far, pE formation rate in lyophilized formulation as compared to in liquid formulation has not been reported. Impact of pE on antibody biological activity has only been predicted or assessed using stressed samples that may contain other confounding degradations besides pE. Additionally, application of hydrophobic interaction chromatography (HIC) to separate pE has not been reported. In our study, N-terminal E cyclization was identified as the major degradation pathway in lyophilized formulation at elevated temperature for both monoclonal antibody (mAb-A) and IgG-like bispecific antibody (bsAb-A). pE was enriched in salt-gradient ion exchange chromatography (IEC) as pre-peak and in HIC as post-peak for both mAb-A and bsAb-A. Structure-function studies with pE-enriched IEC and HIC fractions confirmed that pE did not affect binding activities for mAb-A and bsAb-A. In vitro incubation of bsAb-A in serum and PBS revealed that the serum matrix may play a role in pE conversion in human serum, in contrast to the chemical reaction mechanism reported. These techniques can help in characterization of N-terminal E-to-pE cyclization and quality attribute severity assessment during therapeutic protein product development.

Eliminating protein oxidation artifacts during High Performance Liquid Chromatography peak fractionation processes.

Therapeutic monoclonal antibodies (mAbs) are inherently heterogeneous and hence generally studied and controlled by an array of orthogonal separation methods. During drug candidate development, fractionation by HPLC is regularly employed to assist peak identification and product understanding. One overlooked challenge is the protein oxidation introduced by the fractionation process. In this study, we report the extent of fractionation-induced protein oxidation, which tends to complicate data interpretation and peak assignments. Higher-energy detectors such as fluorescence detectors and lower fraction concentration were found to exacerbate the oxidation artifacts. Other contributing factors than the detector-induced photostress were also found to contribute significantly to protein oxidation. Furthermore, our study showed that collecting fractions into a solution with oxidation scavengers, such as histidine and methionine, was effective in eliminating the oxidation artifacts introduced by detector exposure and fraction processing steps. Through an example, we demonstrate that the modified fractionation workflow improves the accuracy of peak assignments.

Identification of a CE-SDS shoulder peak as disulfide-linked fragments from common CH2 cleavages in IgGs and IgG-like bispecific antibodies.

Fragmentation is a well-characterized degradation pathway of therapeutic antibodies and is usually monitored by capillary electrophoresis-sodium dodecyl sulfate (CE-SDS). Although fragments due to cleavage in CH2 domains linked by intrachain disulfide bonds are common and can be detected by reduced reversed-phase - liquid chromatography mass spectrometry (RP-LCMS) and reduced CE-SDS methods, their separation in nonreduced CE-SDS (nrCE-SDS) has not been reported but speculated as comigrating with intact IgG. A shoulder peak in nrCE-SDS was observed in the stability samples of an IgG-like bispecific antibody and was determined to be mainly caused by fragments from clipping at the C-terminus of leucine (L)306 or L309 (EU numbering) in the CH2 domain of both heavy chains (HCs) and, to a lesser degree, at the C-terminus of L182 in the CH1 domain of the knob HC. Subunit LCMS analysis verified that the crystallizable fragment contained variants with one or multiple mass additions of ~18 Da due to clipping. Further investigation revealed that CH2 clippings at L306 and L309 were largely due to proteolytic activity, and cleavages were present at various levels in all in-house IgG1 and IgG4 molecules studied. Our study shows that CH2 domain cleavages, with complementary fragments still linked by intrachain disulfide, can be electrophoretically resolved as a front shoulder of the main peak in nrCE-SDS. Given the high occurrence of CH2 cleavages in antibodies, these findings will have broad applicability and could help manufacturers of therapeutic antibodies in process improvement, product characterization, investigations, formulation stability, and stability comparability studies.

Analytical band centrifugation for the separation and quantification of empty and full AAV particles.

Analytical band centrifugation (ABC) was first developed for the separation of macromolecules in centrifugation cells ~60 years ago. Since its development, ABC has been predominantly utilized to study macromolecular interactions or chemical reactions between two solutions in situ upon mixing. In this current study, we evaluated ABC separations on modern analytical ultracentrifugation (AUC) instruments for therapeutic adeno-associated viruses (AAVs). ABC provided sufficient separation between the genome-containing full AAV particle and the empty AAV capsid, which need to be controlled during the manufacturing process. Because ABC produces a physical separation, no complex algorithm or sophisticated software is needed to process the experimental raw data. ABC profiles, dubbed "centrifugrams", can be analyzed with a similar approach as typically used for electrophoretic separations to produce relative percent area. Sedimentation coefficients (s) of analytes can also be determined from ABC. The relative area percent and s value obtained in ABC experiments were shown to be consistent with those determined by conventional sedimentation velocity AUC (SV-AUC). Additionally, the separation and quantification by ABC were found to be reproducible and did not appear to be sensitive to experimental variations of initial rotor temperature or cell misalignment. The robustness of the separation, ease of data processing, and universal applicability for analysis of different AAV serotypes make ABC a promising technique for routine analysis of empty and full AAV particle composition in therapeutic products.

Characterization and Monitoring of a Novel Light-heavy-light Chain Mispair in a Therapeutic Bispecific Antibody.

Site-specific cysteine engineering, along with other genetic mutations, is broadly implemented in bispecific antibodies (bsAb). Thus far, homodimer, half hole antibody, one-light chain mispaired and light chain swapped variants have been reported as chain-pairing variants for the asymmetric IgG-like bispecific antibodies. Here we report a novel mispair in which the CH3 engineered cysteine on the hole heavy chain (HC) of a knob-into-hole (KiH) bsAb is linked to the engineered cysteine in CL through a disulfide bond, forming a LHL species in a bsAb construct. Due to its impact on bioactivity, it is critical to implement an analytical strategy to monitor this CQA and mitigate risk for the future products. A set of orthogonal physicochemical assays that include hydrophobic interaction chromatography (HIC), capillary electrophoresis sodium dodecyl sulfate (CE-SDS), reverse phase liquid chromatography ultra-performance chromatography mass spectrometry (RP-UPLC MS) and disulfide bond mapping have been utilized to monitor and characterize this chain-pairing impurity for manufacturing process control and product release. Our data shows the LHL mispair in condition medium (CM) is approximately 1.3 - 1.9%. LambdaFabSelect affinity chromatography removes two major chain-pairing variants in CM - i.e. the hole-hole homodimer and hole half-antibody, while retaining the LHL species. Process improvement in Capto Q (anion exchange) and HS50 (cation exchange) chromatography steps removes LHL to as low as 0.2% in the final product. We have demonstrated an orthogonal analytical methodology that is capable of characterizing and monitoring bsAb mispairing, suitable for use in manufacturing process control and product release, and can be potentially implemented for similar bsAb constructs with engineered disulfide bonds.

Sensitive, Rapid, Robust, and Reproducible Workflow for Host Cell Protein Profiling in Biopharmaceutical Process Development.

There is a growing industry and regulatory need to detect host cell protein (HCP) impurities in the production of protein biopharmaceuticals, as certain HCPs can impact product stability, safety, and efficacy, even at low levels. In some cases, regulatory agencies require the identification and the quantification of HCPs in drug products (DPs) for risk assessment, and this is an active and growing topic of conversation in the industry and amongst regulators. In this study, we developed a sensitive, robust, and reproducible workflow for HCP detection and quantification in a significantly shorter turnaround time than that previously reported using an Evosep ONE LC system coupled to an Orbitrap Fusion Lumos mass spectrometer. Because of its fast turnaround time, this HCP workflow can be integrated into process development for the high-throughput (60 samples analyzed per day) identification of HCPs. The ability to rapidly measure HCPs and follow their clearance throughout the downstream process can be used to pinpoint sources of HCP contamination, which can be used to optimize biopharmaceutical production to minimize HCP levels. Analysis of the NIST monoclonal antibody reference material using the rapid HCP profiling workflow detected the largest number of HCPs reported to date, underscoring an improvement in performance along with an increased throughput. The HCP workflow can be readily implemented and adapted for different purposes to guide biopharmaceutical process development and enable better risk assessment of HCPs in drug substances and DPs.

A novel method for removing polyethyleneimine from biopharmaceutical samples: improving assay sensitivity of residual DNA qPCR.

Polyethyleneimine (PEI) is a flocculent that is widely used in the downstream purification of monoclonal antibodies. It is an in-process residual that is carried through the drug purification process and strongly inhibits residual DNA quantitation by real-time quantitative PCR assay. Very high sample dilutions (e.g., 1:10,000) can overcome the interference of PEI, but at the cost of DNA assay sensitivity. Diluting samples poses a significant risk to the assay sensitivity needed to satisfy regulatory requirements on the quantitation of residual genomic DNA present per dose (i.e., 10 ng/dose). Removing PEI while retaining DNA, by the use of sodium dodecyl sulfate, heparin and/or sarkosyl can overcome the interference of PEI and allow a more accurate quantitation of residual DNA.

Combination of Eribulin and Aurora A Inhibitor MLN8237 Prevents Metastatic Colonization and Induces Cytotoxic Autophagy in Breast Cancer.

Recent findings suggest that the inhibition of Aurora A (AURKA) kinase may offer a novel treatment strategy against metastatic cancers. In the current study, we determined the effects of AURKA inhibition by the small molecule inhibitor MLN8237 both as a monotherapy and in combination with the microtubule-targeting drug eribulin on different stages of metastasis in triple-negative breast cancer (TNBC) and defined the potential mechanism of its action. MLN8237 as a single agent and in combination with eribulin affected multiple steps in the metastatic process, including migration, attachment, and proliferation in distant organs, resulting in suppression of metastatic colonization and recurrence of cancer. Eribulin application induces accumulation of active AURKA in TNBC cells, providing foundation for the combination therapy. Mechanistically, AURKA inhibition induces cytotoxic autophagy via activation of the LC3B/p62 axis and inhibition of pAKT, leading to eradication of metastases, but has no effect on growth of mammary tumor. Combination of MLN8237 with eribulin leads to a synergistic increase in apoptosis in mammary tumors, as well as cytotoxic autophagy in metastases. These preclinical data provide a new understanding of the mechanisms by which MLN8237 mediates its antimetastatic effects and advocates for its combination with eribulin in future clinical trials for metastatic breast cancer and early-stage solid tumors. Mol Cancer Ther; 15(8); 1809-22. ©2016 AACR.

High performance liquid chromatographic evaluation of artemisinin, raw material in the synthesis of artesunate and artemether.

A barrier to the development of artemisinin derivative based combination treatment of malaria is the lack of defined specifications and purity test methods for the raw material artemisinin. An HPLC method previously published in the International Pharmacopoeia to evaluate purity of artemisinin as an active pharmaceutical ingredient is adapted for use. Excellent method precision and linearity are demonstrated along with observations of robustness. In support of the development of specifications major impurities are identified using high resolution HPLC-MS, isolation via preparative HPLC followed by NMR. The identified impurities differ from those previously claimed.

Large-scale evaluation of quantitative reproducibility and proteome coverage using acid cleavable isotope coded affinity tag mass spectrometry for proteomic profiling.

Strategies employing non-gel based methods for quantitative proteomic profiling such as isotope coded affinity tags coupled with mass spectrometry (ICAT-MS) are gaining attention as alternatives to two-dimensional gel electrophoresis (2-DE). We have conducted a large-scale investigation to determine the degree of reproducibility and depth of proteome coverage of a typical ICAT-MS experiment by measuring protein changes in Escherichia coli treated with triclosan, an inhibitor of fatty acid biosynthesis. The entire ICAT-MS experiment was conducted on four independent occasions where more than 24 000 peptides were quantitated using an ion-trap mass spectrometer. Our results demonstrated that quantitatively, the technique provided good reproducibility (median coefficient of variation of ratios was 18.6%), and on average identified more than 450 unique proteins per experiment. However, the method was strongly biased to detect acidic proteins (pI < 7), under-represented small proteins (<10 kDa) and failed to show clear superiority over 2-DE methods in monitoring hydrophobic proteins from cell lysates.

Proteomic analysis of hyperdynamic mouse hearts with enhanced sarcoplasmic reticulum calcium cycling.

Depressed sarcoplasmic reticulum (SR) Ca-cycling is a hallmark of human and experimental heart failure. Strategies to improve this impairment by either increasing SERCA2a levels or decreasing phospholamban (PLN) activity have been suggested as promising therapeutic targets. Indeed, ablation of PLN gene in mice was associated with greatly enhanced cardiac Ca-cycling and performance. Intriguingly, this hyperdynamic cardiac function was maintained throughout the lifetime of the mouse without observable pathological consequences. To determine the cellular alterations in the expression or modification of myocardial proteins, which are associated with the enhanced cardiac contractility, we performed a proteomics-based analysis of PLN knockout (PLN-KO) hearts in comparison to isogenic wild-types. By use of 2-dimensional gel electrophoresis (2-DE), approximately 3300 distinct protein spots were detected in either wild-type or PLN-KO ventricles. Protein spots observed to be altered between PLN-KO and wild-type hearts were subjected to tryptic peptide mass fingerprinting for identification by MALDI-TOF mass spectrometry in combination with LC/MS/MS analysis. In addition, two-dimensional 32P-autoradiography was performed to analyze the phosphorylation profiles of PLN-KO cardiomyocytes. We identified alterations in the expression level of more than 100 ventricular proteins, along with changes in phosphorylation status of important regulatory proteins in the PLN-KO. These protein changes were observed mainly in two subcellular compartments: the cardiac contractile apparatus, and metabolism/energetics. Our findings suggest that numerous alterations in protein expression and phosphorylation state occurred upon ablation of PLN and that a complex functional relationship among proteins involved in calcium handling, myofibrils, and energy production may exist to coordinately maintain the hyperdynamic cardiac contractile performance of the PLN-KO mouse in the long term.