Characterization of the higher-order structures in idursulfase (iduronate-2-sulfatase, I2S) has been accomplished through the use of hydrogen-deuterium exchange mass spectrometry (HDX-MS). The method has over 97% sequence coverage, including seven of the eight glycosylation sites, and has been used to study the impact of glycosylation on backbone proton exchange. In addition, the method adapted a well-used biophysical spectra comparison method (similarity scoring) to define quantitative acceptance criteria for analytical comparability of different batches of drug substance as well as samples with modulated glycans. Differences in the HDX profile were induced by enzymatic removal of terminal sialic and phosphate groups on negatively charged glycans. These differences were mapped to the crystal structure and demonstrated synergistic HDX changes focused around the N221 and N255 glycosylation sites, which contain mannose-6-phosphate motifs important for I2S uptake into cells.

Analytical methods must be qualified as part of the method development lifecycle for product characterization of biotherapeutics. For higher order structure characterization methods, such as near ultraviolet circular dichroism spectroscopy, qualification is performed to determine the expected variability of the method and to establish criteria for analytical product comparability, reference standard qualification, and analytical similarity evaluations. Typical method qualifications require a single product to be tested across several days with multiple replicates, essential to establish a quantitative limit for future product evaluation studies, which may be burdensome with respect to time, instrumentation, and material requirements. In this note, a methodology is proposed to expedite the qualification process for the near ultraviolet circular dichroism spectroscopy method, decreasing the number of required qualification runs, in many cases, to just one for each product. The significant reduction in the number of assays for qualification is achieved by utilizing historical data that applies universally across products of variable classification, size, and test date. Despite their differences, the products exhibit comparable method performance when compared to a product-specific reference standard, and a universal detection threshold is established for application to future product evaluations that meet pre-determined method suitability criteria following a single verification run.

Coagulation factor II (prothrombin; FII) is the pre-proteolyzed precursor to thrombin in the coagulation cascade. It has 10 sites of gamma-carboxylation, which are required for its bioactivity, and is N-glycosylated at three of four putative sites. Production of recombinant human FII (rhFII) using a platform fed-batch process designed for monoclonal antibody production resulted in low levels of gamma-carboxylation and sialylation. There have not been any prior reports of successful process development and clinical manufacture of rhFII with optimal, consistent gamma-carboxylation and sialylation. In order to develop such a fed-batch process, various process parameters were evaluated to determine their impact on product quality. Process temperature and temperature shift timing were important for both sialic acid level and gamma-carboxyglutamate (Gla) level. In addition, vitamin K concentration and the type of surfactant used for preparation of vitamin K stock solution were also important for gamma carboxylation. A fed-batch study performed with various medium additives known to be involved in the N-glycosylation pathway, such as N-acetyl-d-mannosamine (ManNAc), galactose (Gal), dexamethasone, and manganese sulfate, increased the level of sialylation and enabled the elucidation of some potential bottlenecks in the sialylation pathway. The optimized process based on these studies yielded a reduction in the level of missing Gla by 0.4 moles per mole of rhFII in cell culture and a nearly threefold increase in sialic acid level. The process was successfully implemented at the 2000 L scale where a high Gla level and sialylation levels were achieved in all GMP lots. Biotechnol. Bioeng. 2017;114: 1991-2000. © 2017 Wiley Periodicals, Inc.

ABSTRACT Glycosylation often plays a key role in the safety and efficacy of therapeutic proteins to patients, thus underlying the need for consistent control of this important post‐translational modification during biologics production. In this study, we profiled the site‐specific evolution of N‐glycans on a CTLA4‐Fc‐fusion protein, from the intracellular secretory pathway to the conditioned medium (CM) in fed‐batch cell culture. For this, we developed an approach that combined sub‐cellular fractionation with liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) analyses. The study revealed that there was a significant amount of heterogeneity in the glycans displayed amongst the three distinct N‐glycosylation sites. Furthermore, 54–60% of the intracellular protein was characterized by Man8 and Man9 glycans on day 10, when the cell density peaks, indicative of a significant bottleneck between the endoplasmic reticulum (ER) and the cis‐Golgi. At longer culture duration, the accumulation of intracellular protein with bi‐antennary‐fucosylated GlcNAc‐terminated residues identified the formation of another bottleneck in the medial and trans‐Golgi compartments, which subsequently led to a decrease in sialylated species in the secreted protein. Glucose deprivation caused a reduction in the Man8 and Man9 glycans in favor of Man5 glycans and bi‐antennary‐fucosylated GlcNAc‐terminated residues in the organellar pool of the Fc‐fusion protein. However, transient deprivation of glucose did not lead to major differences in the glycan profile of proteins secreted into the CM. The approach developed here allows us to probe the secretory pathway and sheds light on the site‐specific intracellular processing of glycans during fed‐batch cell culture, thus serving as an initial step towards their rational control. Biotechnol. Bioeng. 2017;114: 1550–1560. © 2017 Wiley Periodicals, Inc.

Affinity- and stability-engineered variants of CTLA4-Ig fusion molecules with enhanced pharmacokinetic profiles could yield improved therapies with the potential of higher efficacy and greater convenience to patients. In this study, to our knowledge, we have, for the first time, used in vitro evolution to simultaneously optimize CTLA4 affinity and stability. We selected for improved binding to both ligands, CD80 and CD86, and screened as dimeric Fc fusions directly in functional assays to identify variants with stronger suppression of in vitro T cell activation. The majority of CTLA4 molecules showing the largest potency gains in primary in vitro and ex vivo human cell assays, using PBMCs from type 1 diabetes patients, had significant improvements in CD80, but only modest gains in CD86 binding. We furthermore observed different potency rankings between our lead molecule MEDI5265, abatacept, and belatacept, depending on which type of APC was used, with MEDI5265 consistently being the most potent. We then created fusions of both stability- and potency-optimized CTLA4 moieties with human Fc variants conferring extended plasma t1/2 In a cynomolgus model of T cell-dependent Ab response, the CTLA4-Ig variant MEDI5265 could be formulated at >100 mg/ml for s.c. administration and showed superior efficacy and significantly prolonged serum t1/2 The combination of higher stability and potency with prolonged pharmacokinetics could be compatible with very infrequent, s.c. dosing while maintaining a similar level of immune suppression to more frequently and i.v. administered licensed therapies.

We employed top- and middle-down analyses with multiple fragmentation techniques including electron transfer dissociation (ETD), electron capture dissociation (ECD), and matrix-assisted laser desorption ionization in-source decay (MALDI-ISD) for characterization of a reference monoclonal antibody (mAb) IgG1 and a fusion IgG protein. Fourier transform ion cyclotron resonance (FT-ICR) or high performance liquid chromatography electrospray ionization (HPLC-ESI) on an Orbitrap was employed. These experiments provided a comprehensive view on the protein species; especially for different glycosylation level in these two proteins, which showed good agreement with oligosaccharide profiling. Top- and middle-down MS provided additional information regarding glycosylation sites and different combinational protein species that were not available from oligosaccharide mapping or conventional bottom-up analysis. Finally, incorporating a limited enzymatic digestion by immunoglobulin G-degrading enzyme of Streptococcus pyogene (IdeS) with MALDI-ISD analysis enabled extended sequence coverage of the internal region of protein without pre-fractionation.

Abstract Antibody-drug conjugates (ADCs) selectively deliver cytotoxic agents to tumors and provide the potential for increased clinical benefit to cancer patients. We have developed an ADC consisting of an antibody against a target expressed on the surface of tumor cells that is site-specifically conjugated with a pyrrolobenzodiazepine (PBD) dimer payload (PBD-ADC). The tumor-associated antigen is overexpressed in many carcinomas, on both bulk tumor cells and cancer stem cells (CSCs), while normal tissue expression is limited, thus making it an ideal ADC target. The PBD payload was selected due to its picomolar potency and because, unlike tubulin inhibitors, it could be cytotoxic to CSCs as well as bulk tumor cells. The PBD-ADC is significantly efficacious in target-positive in vivo tumor models of gastric, breast and lung carcinomas, inducing long-term tumor regressions and demonstrates greater potency compared to ADCs conjugated with either tubulysin or auristatin payloads, both tubulin inhibitors. Additionally, the PBD-ADC significantly reduced the CSC population of tumors in vivo, unlike ADCs bearing tubulin inhibitor payloads. The PBD-ADC appears to be efficacious against tumors that have acquired resistance to a tubulysin-based ADC, however possible mechanisms of resistance to this PBD-ADC are being investigated. Collectively, this novel ADC represents a promising therapy that may deliver durable clinical responses in indications with unmet medical need. Citation Format: Jay W. Harper, Chris Lloyd, Shenlan Mao, Xin Lin, Nazzareno Dimasi, Phil Howard, Ellen O'Connor, David Farkas, Christopher Barton, Jeff Smith, Helen Zhong, Tracy Chen, Mary Jane Hinrichs, Marlon Rebelatto, Xiang-Qing Yu, Susan Spitz, Andrew Pierce, Dominic Lai, Hurt Elaine, Lilian van Vlerken-Ysla, Jalla Sanjoo, Katy Miller, Karen McCullough, Priya Kannan, Simon Thompson, Parthiv Mahadevia, Chris Martin, Robert Hollingsworth, Adeela Kamal. A novel antibody-drug conjugate that induces long-term tumor regression and anticancer stem cell activity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 948. doi:10.1158/1538-7445.AM2015-948

A reference monoclonal antibody IgG1 and a fusion IgG protein were analyzed by top- and middle-down mass spectrometry with multiple fragmentation techniques including electron transfer dissociation (ETD) and matrix-assisted laser desorption ionization in-source decay (MALDI-ISD) to investigate heterogeneity of glycosylated protein species. Specifically, glycan structure, sites, relative abundance levels, and termini structural conformation were investigated by use of Fourier transform ion cyclotron resonance (FT-ICR) or high performance liquid chromatography electrospray ionization (HPLC-ESI) linked to an Orbitrap. Incorporating a limited enzymatic digestion by immunoglobulin G-degrading enzyme Streptococcus pyogenes (IdeS) with MALDI-ISD analysis extended sequence coverage of the internal region of the proteins without pre-fractionation. The data in this article is associated with the research article published in Journal of Proteomics (Tran et al., 2015) [1].

Vaccine prophylaxis with EBV glycoprotein 350 (gp350) subunit plus adjuvant has been demonstrated clinically to protect individuals against infectious mononucleosis (IM), but the specifications of the antigen required to elicit this protection has remained largely theoretical. Previous studies have shown that antibodies to gp350 comprise the principle component of EBV-neutralizing sera. Further, a murine monoclonal antibody against gp350 (clone 72A1) is able to prevent infection by the virus both in vitro and in vivo. In the present study, we identify the 72A1 epitope on recombinant gp350 antigen as the site required for binding to CD21 on human B cells. We also identify the need for conformational-dependence of the antigen to generate EBV-neutralizing antibodies in vivo. Further, we have characterized the glycosylation status and antigenicity profiles of both native and denatured CHO-produced soluble gp350 as well as non-glycosylated protein produced in Escherichia coli. Collectively our in vitro and in vivo data demonstrate the requirement for a conformationally accessible 72A1 epitope on gp350 to elicit EBV-neutralizing responses, and establish this as a critical attribute of this vaccine antigen. These data provide direction for commercial vaccine development, as the absence of this epitope on either E. coli-expressed or denatured gp350, may limit production and purification options for the antigen.

Detailed monoclonal antibody (mAb) characterization tools have enabled the discovery of structural variations, including many that compromise functionality or have other undesired properties. Size, charge, glycosylation, and disulfide bonding variants; oxidized amino acid residues; and polypeptide chain truncations, extensions, and cleavage points have been identified. Product quality attributes, including detection techniques, published knowledge about the process origins, and quality impacts of these variants are summarized.

Glycine-serine (GS) linkers are commonly used in recombinant proteins to connect domains. Here, we report the posttranslational O-glycosylation of a GS linker in a novel fusion protein. The structure of the O-glycan moiety is a xylose-based core substituted with hexose and sulfated hexauronic acid residues. The total level of O-xylosylation was approximately 30% in the material expressed in HEK-293 cell lines. There was an approximate 10-fold reduction in O-xylosylation levels when the material was expressed in Chinese hamster ovary cell lines. Similar O-glycan structures have been reported for human urinary thrombomodulin and represent the initial building block for proteoglycans such as chondroitin sulfate and heparin. The sites of attachment, determined by electron transfer dissociation mass spectrometry, were localized to serine in the linker regions of the recombinant fusion protein. This attachment could be attributed, in part, to the inherent xylosyltransferase motif present in GS linkers. Elimination of the O-glycan moiety was achieved with modified linkers containing only glycine residues. The aggregation and fragmentation behavior of the GGG construct were comparable to the GSG-linked material during thermal stress. The O-xylosylation reported has implications for the manufacturing consistency of recombinant proteins containing GS linkers.

Ghrelin, an endocrine hormone predominantly produced by the stomach, exists in acylated and unacylated forms in the circulation. Unacylated ghrelin (UAG), the more abundant form in blood, possesses similar, independent or opposite physiological actions as acylated ghrelin (AG). AZP502, a linear 8-amino acid peptide from the central region of UAG (UAG(6-13)), and its full (AZP531) and partially (AZP533) cyclised derivatives, exhibit the same pharmacological profile as UAG both in vitro and in vivo, independently of AG receptor binding. We investigated the stability of these three fragments in vitro in human blood samples and in vivo after subcutaneous and intravenous injection in rats and dogs using liquid chromatography-mass spectrometry. In both species, AZP502 is rapidly degraded generating two major metabolites. Partial cyclisation of AZP502 and acylation at its N-terminus (AZP533 peptide) improves its stability in human plasma in vitro. Full cyclisation of AZP502 (AZP531 peptide) also completely protects the peptide from peptidase degradation in vitro in human blood samples. Moreover this cyclisation strongly improves the stability and the bioavailability of this peptide in vivo in both dogs and rats (mean bioavailability of 10-15% and 85-95% for AZP502 and AZP531 respectively). Taken together these results support the rationale for developing AZP531 as a long-acting UAG analogue for subcutaneous injection for the treatment of type 2 diabetes mellitus and other metabolic disorders. (C) 2012 Elsevier B.V. All rights reserved.

Selected reaction monitoring (SRM) is a technique that applies tandem mass spectrometry to quantify specific proteins of biological interest. The key to SRM is finding the best peptide-to-product ion transitions to monitor. The MRMaid database (MRMaid-DB) is a new online database for capturing SRM transitions from published research papers to save practitioners time when searching for transitions that have been previously validated. It contains all the information needed to reproduce the transitions, such as information on the sample matrix, HPLC, and MS instrumentation used, and also includes details of the manuscript of origin. Transitions are submitted using simple Web-based data entry forms, meaning researchers have a simple way to increase access to their transitions, and in turn, may increase the citations for their research papers. MRMaid-DB is free to use, via the Web at www.mrmaid-db.info .

The development of protein biomarkers for the indirect detection of doping in horse is a potential solution to doping threats such as gene and protein doping. A method for biomarker candidate discovery in horse plasma is presented using targeted analysis of proteotypic peptides from horse proteins. These peptides were first identified in a novel list of the abundant proteins in horse plasma. To monitor these peptides, an LC-MS/MS method using multiple reaction monitoring was developed to study the quantity of 49 proteins in horse plasma in a single run. The method was optimised and validated, and then applied to a population of race-horses to study protein variance within a population. The method was finally applied to longitudinal time courses of horse plasma collected after administration of an anabolic steroid to demonstrate utility for hypothesis-driven discovery of doping biomarker candidates.

Multiple reaction monitoring (MRM) of peptides uses tandem mass spectrometry to quantify selected proteins of interest, such as those previously identified in differential studies. Using this technique, the specificity of precursor to product transitions is harnessed for quantitative analysis of multiple proteins in a single sample. The design of transitions is critical for the success of MRM experiments, but predicting signal intensity of peptides and fragmentation patterns ab initio is challenging given existing methods. The tool presented here, MRMaid (pronounced "mermaid") offers a novel alternative for rapid design of MRM transitions for the proteomics researcher. The program uses a combination of knowledge of the properties of optimal MRM transitions taken from expert practitioners and literature with MS/MS evidence derived from interrogation of a database of peptide identifications and their associated mass spectra. The tool also predicts retention time using a published model, allowing ordering of transition candidates. By exploiting available knowledge and resources to generate the most reliable transitions, this approach negates the need for theoretical prediction of fragmentation and the need to undertake prior "discovery" MS studies. MRMaid is a modular tool built around the Genome Annotating Proteomic Pipeline framework, providing a web-based solution with both descriptive and graphical visualizations of transitions. Predicted transition candidates are ranked based on a novel transition scoring system, and users may filter the results by selecting optional stringency criteria, such as omitting frequently modified residues, constraining the length of peptides, or omitting missed cleavages. Comparison with published transitions showed that MRMaid successfully predicted the peptide and product ion pairs in the majority of cases with appropriate retention time estimates. As the data content of the Genome Annotating Proteomic Pipeline repository increases, the coverage and reliability of MRMaid are set to increase further. MRMaid is freely available over the internet as an executable web-based service at www.mrmaid.info.

The fight against doping in sport using analytical chemistry is a mature area with a history of approximately 100 years in horse racing and at least 40 years in human sport. Over that period, the techniques used and the breadth of coverage have developed significantly. These improvements in the testing methods have been matched by the increased sophistication of the methods, drugs and therapies available to the cheat and, as a result, testing has been a reactive process constantly adapting to meet new threats. Following the inception of the World Anti-Doping Agency, research into the methods and technologies available for human doping control have received coordinated funding on an international basis. The area of biomarker research has been a major beneficiary of this funding. The aim of this article is to review recent developments in the application of biomarkers to doping control and to assess the impact this could make in the future.

Mannans are hemicellulosic polysaccharides that have previously been implicated as structural constituents of cell walls and as storage reserves but which may serve other functions during plant growth and development. Several members of the Arabidopsis cellulose synthase-like A (CSLA) family have previously been shown to synthesise mannan polysaccharides in vitro when heterologously expressed. It has also been found that CSLA7 is essential for embryogenesis, suggesting a role for the CSLA7 product in development. To determine whether the CSLA proteins are responsible for glucomannan synthesis in vivo, we characterised insertion mutants in each of the nine Arabidopsis CSLA genes and several double and triple mutant combinations. csla9 mutants showed substantially reduced glucomannan, and triple csla2csla3csla9 mutants lacked detectable glucomannan in stems. Nevertheless, these mutants showed no alteration in stem development or strength. Overexpression of CSLA2, CSLA7 and CSLA9 increased the glucomannan content in stems. Increased glucomannan synthesis also caused defective embryogenesis, leading to delayed development and occasional embryo death. The embryo lethality of csla7 was complemented by overexpression of CSLA9, suggesting that the glucomannan products are similar. We conclude that CSLA2, CSLA3 and CSLA9 are responsible for the synthesis of all detectable glucomannan in Arabidopsis stems, and that CSLA7 synthesises glucomannan in embryos. These results are inconsistent with a substantial role for glucomannan in wall strength in Arabidopsis stems, but indicate that glucomannan levels affect embryogenesis. Together with earlier heterologous expression studies, the glucomannan deficiency observed in csla mutant plants demonstrates that the CSLA family encodes glucomannan synthases.

The clinical application of targeted plasma protein analysis by selective reaction monitoring of peptides using LC-MS/MS requires the development of robust, inexpensive protein extraction techniques with the potential for high-throughput applications. We present the development of a novel mixed-mode solid phase extraction (SPE) technique for the removal of high abundance and high molecular weight proteins from plasma. This technique, coupled with fused-core HPLC-MS/MS analysis is compared to a previously developed extraction method to study a range of proteins in plasma, including routinely measured biomarkers of growth hormone action. To further validate this technique, it was used for the quantification of insulin-like growth factor I (IGF-I) levels and compared to a state-of-the-art immunoassay on a fully automated analyzer. Clinical reference materials were applied for method development to allow for further interlaboratory comparisons. The LC-MS/MS approach quantified IGF-I in plasma with an accuracy that is within the guidelines for macromolecular assays in a regulated laboratory environment. Furthermore, IGF-I levels determined using the SPE and ACN methods with LC-MS/MS analysis correlated well with the immunoassay results. This demonstrates the applicability of mixed-mode SPE coupled with fused-core HPLC-MS/MS to quantify plasma proteins with results suitable for clinical applications.