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CD24Fc is a homodimeric recombinant Fc-fusion protein comprised of human CD24 connected to immunoglobulin G1 (IgG1) Fc fragment. CD24 is heavily glycosylated, and its biological function is considered mainly mediated by its glycosylation. Identification of the O-glycosylation sites would facilitate an in-depth understanding of the functional role of O-glycans in CD24. However, the presence of clustered mucin-type O-glycans together with N-glycans makes the determination of O-glycosylation sites and abundance very challenging. In this study, two sets of liquid chromatography-mass spectrometry (LC-MS) workflows were developed for the comprehensive characterization of O-glycosylation in CD24: (1) Fractionation and collision-induced dissociation (CID) workflow involving multienzyme digestion, fractionation, OpeRATOR/SialEXO digestion, and CID analysis; (2) Direct OpeRATOR/SialEXO digestion followed by electron-transfer/higher-energy collision dissociation (EThcD) analysis. The precise O-glycosylation sites were identified in CD24 for the first time, and the site occupancy was assessed. A total of 12 O-glycosylation sites were identified. Seven glycosylation sites were identified by both workflows, and five additional sites were identified only by the EThcD workflow. The predominant O-glycosylation site in CD24 was Thr25 followed by Thr15. The CID workflow provided an overall relative quantitation of O-glycoforms at the CD24 level and site localization for singly O-glycosylated peptides. The EThcD workflow directly identified glycosylation sites by tandem mass spectrometry (MS/MS) for singly, doubly, and triply O-glycosylated peptides. Together, both workflows validated each other's results and can be applied to a complex mucin-type O-glycosylation site analysis of other glycoproteins and Fc-fusion therapeutics.

Host cell proteins (HCPs) are process‐related impurities derived from host organisms, which need to be controlled to ensure adequate product quality and safety. In this study, product quality attributes were tracked for several monoclonal antibodies (mAbs) under the intended storage and accelerated stability conditions. One product quality attribute not expected to be stability indicating is the N‐glycan heterogeneity profile. However, significant N‐glycan degradation was observed for one mAb under accelerated and stressed stability conditions. The root cause for this instability was attributed to hexosaminidase B (HEXB), an enzyme known to remove terminal N‐acetylglucosamine (GlcNAc). HEXB was identified by liquid chromatography–mass spectrometry (LC–MS)‐based proteomics approach to be enriched in the impacted stability batches from mAb‐1. Subsequently, enzymatic and targeted multiple reaction monitoring (MRM) MS assays were developed to support process and product characterization. A potential interaction between HEXB and mAb‐1 was initially observed from the analysis of process intermediates by proteomics among several mAbs and later supported by computational modeling. An improved bioprocess was developed to significantly reduce HEXB levels in the final drug substance. A risk assessment was conducted by evaluating the in silico immunogenicity risk and the impact on product quality. To the best of our knowledge, HEXB is the first residual HCP reported to have impact on the glycan profile of a formulated drug product. The combination of different analytical tools, mass spectrometry, and computational modeling provides a general strategy on how to study residual HCP for biotherapeutics development.

The development of comprehensive methods to characterize unpaired cysteines in monoclonal antibodies (mAbs) is very important for understanding structural heterogeneity, impurity, and stability. In this paper, unpaired cysteines observed in a therapeutic antibody (mAb1) were thoroughly studied by Liquid Chromatography-Mass Spectrometry (LC-MS) methods at the intact mAb, domain, and peptide levels. Three cysteine variants were observed at the intact mAb level with each variant containing two unpaired cysteines. Variants containing four or six unpaired cysteines were not observed. Domain analysis indicated that two Fab variants, each containing two unpaired cysteines, were present while the third variant contained two unpaired cysteines on the Fc region. Peptide mapping analysis localized the six unpaired cysteines to Cys22/Cys96, Cys146/Cys202, and Cys369/Cys427 in the heavy chain. No significant changes were observed for these unpaired cysteines in mAb1 under high pH and heat-stressed conditions. Structural analysis and molecular modeling revealed that these unpaired cysteines were buried inside the three-dimensional structure. The integrated LC-MS methods together with stress studies and structural analysis may potentially be applied to the analysis of unpaired cysteines in other mAbs.

Microsampling techniques have been employed as an alternative to traditional serum/plasma sampling because of their inherently proven and desirable advantages across the pharmaceutical industry. These include reduced animal usage in pre-clinical studies, as well as, permitting the collection of samples that would otherwise be inaccessible in clinical studies. The application of volumetric absorptive microsampling (VAMS®) technology, a second-generation dried microsampling method, coupled with LC-MS, has been extensively explored for small molecule drugs at various drug development stages. However, the potential of using VAMS technology and LC-MS analysis for biological therapeutic development has yet to be well-established. In this work, we describe the method development, validation, and a proof-of-concept non-human primate study of a LC-MS/MS method for VAMS utilized to obtain pharmacokinetic (PK) data for a therapeutic monoclonal antibody. A good correlation between VAMS data and data from conventional serum samples was established in rhesus monkeys and indicated the possibility of using of this novel sampling technology in clinical studies. However, during the initial clinical study, a significant difference in internal standard (IS) response between the patient fingerstick samples and the standard/QC samples was observed, which posed a question on the accuracy of the clinical results. A comprehensive investigation confirmed that the EDTA anticoagulant used in the standard/QC samples was the root cause of the observed anomalous IS responses. Special considerations and corresponding best practices during method development and validation are proposed to ensure early detection of potential issues and appropriate implementation of VAMS technology in clinical studies in the future.

Host cell proteins (HCPs) are process-related impurities derived from host organisms, which need to be controlled to ensure adequate product quality and safety. In this study, product quality attributes were tracked for several mAbs under the intended storage and accelerated stability conditions. One product quality attribute not expected to be stability indicating is the N-glycan heterogeneity profile. However, significant N-glycan degradation was observed for one mAbs under accelerated and stressed stability conditions. The root cause for this instability was attributed to Hexosaminidase B (HEXB), an enzyme known to remove terminal N-acetylglucosamine (GlcNAc). HEXB was identified by liquid chromatography–mass spectrometry (LC-MS) based proteomics approach to be enriched in the impacted stability batches from mAb-1. Subsequently, enzymatic and targeted multiple reaction monitoring (MRM) MS assays were developed to support process and product characterization. A potential interaction between HEXB and mAb-1 was initially observed from the analysis of process intermediates by proteomics among several mAbs and later supported by computational modeling. An improved bioprocess was developed to significantly reduce HEXB levels in the final drug substance. A risk assessment was conducted by evaluating the in silico immunogenicity risk and the impact on product quality. To the best of our knowledge, HEXB is the first residual HCP reported to have impact on the glycan profile of a formulated drug product. The combination of different analytical tools, mass spectrometry and computational modeling provide a general strategy on how to study residual HCP for biotherapeutics development.

Multiple reaction monitoring (MRM) is a liquid chromatography-mass spectrometry (LC-MS) based quantification platform with high sensitivity, specificity, and throughput. It is extensively used across the pharmaceutical industry for the quantitative analysis of therapeutic molecules. The potential of MRM analysis for the quantification of specific host cell proteins (HCPs) in bioprocess, however, has yet to be well established. In this work, we introduce a multiplex LC-MRM assay that simultaneously monitors two high risk lipases known to impact biologics product quality, Phospholipase B-like 2 protein (PLBL2) and Group XV lysosomal phospholipase A2 (LPLA2). Quantitative data generated from the LC-MRM assay were used to monitor the clearance of these lipases during biologics process development. The method is linear over a dynamic range of 1 to 500 ng/mg. To demonstrate the fitness for use and robustness of this assay, we evaluate a comprehensive method qualification package that includes intra- and inter-run precision and accuracy across all evaluated concentrations, selectivity, recovery and matrix effect, dilution linearity, and carryover. Additionally, we illustrate that this assay provides a rapid and accurate means of monitoring high risk HCP clearance for in-process support and can actively guide process improvement and optimization. Lastly, we compare direct digestion platforms and affinity depletion platforms to demonstrate the impact of HCP-mAb interaction on lipase quantification.

Aim: Factor P (Properdin), an endogenous glycoprotein, plays a key role in innate immune defense. Its quantification is important for understanding the pharmacodynamics (PD) of drug candidate(s). Results: In the present work, an immunoaffinity capturing LC–MS/MS method has been developed and validated for the first time for the quantification of factor P in monkey serum with a dynamic range of 125 to 25,000 ng/ml using the calibration standards and QCs prepared in factor P depleted monkey serum. The intra- and inter-run precision was ≤7.2% (CV) and accuracy within ±16.8% (%Bias) across all QC levels evaluated. Results of other evaluations (e.g., stability) all met the acceptance criteria. Conclusion: The validated method was robust and implemented in support of a preclinical PK/PD study.

Human milk proteins provide essential nutrition for growth and development, and support a number of vital developmental processes in the neonate. A complete understanding of the possible functions of human milk proteins has been limited by incomplete knowledge of the human milk proteome. In this report, we have analyzed the proteomes of whey from human transitional and mature milk using ion-exchange and SDS-PAGE based protein fractionation methods. With a larger-than-normal sample loading approach, we are able to largely extend human milk proteome to 976 proteins. Among them, 152 proteins are found to render significant regulatory changes between transitional milk and mature milk. We further found that immunoglobulins sIgA and IgM are more abundant in transitional milk, whereas IgG is more abundant in mature milk, suggesting a transformation in defense mechanism from newborns to young infants. Additionally, we report a more comprehensive view of a complement system and associated regulatory apparatus in human milk, demonstrating the presence and function of a system similar to that found in the circulation but prevailed by alternative pathway in complement activation. Proteins involved in various aspects of carbohydrate metabolism are also described, revealing either a transition in milk functionality to accommodate carbohydrate-rich secretions as lactation progresses, or a potentially novel way of looking at the metabolic state of the mammary tissue. Lately, a number of extracellular matrix (ECM) proteins are found to be in higher abundance in transitional milk and may be relevant to the development of infants' gastrointestinal tract in early life. In contrast, the ECM protein fibronectin and several of the actin cytoskeleton proteins that it regulates are more abundant in mature milk, which may indicate the important functional role for milk in regulating reactive oxygen species.

Bacterial electron transport pathways largely fall into two major categories: the light-driven photosynthetic electron transfer chain and the aerobic or anaerobic respiratory electron transfer chain. Despite the vast differences between photo- and oxidative phosphorylations, they both couple the chemical reactions between electron donors and electron accepters to the translocation of protons across the membrane, which then drives ATP formation and other energy-dependent processes (1). As a result, the common feature of all electron transport chains is the presence of a proton pump to create the transmembrane proton gradient. In respiratory electron transfer pathways, there may be as many as three types of proton pumping protein complexes reminiscent of mitochondria, depending on environmental factors (2). In contrast, the proton pump in all the photosynthetic electron transfer chains was until recently believed to involve a cytochrome bc1 or b6f complex, which resemble mitochondrial complex III in terms of overall structure and mechanism (3). In the species tree of bacteria based on 16S rRNA analysis (4), the phylum of filamentous anoxygenic phototrophs (FAPs) is not closely related to the other phyla that contain organisms that carry out chlorophyll-based photosynthesis; purple bacteria, cyanobacteria, heliobacteria, green sulfur bacteria and chloroacidobacteria. Instead, it exhibits a much deeper branching position to the other five bacterial phyla that contain phototrophic representatives (5, 6). Because of this distinctive feature, the study of FAPs may shed an interesting light on the evolutionary development of photosynthesis. The FAPs are a very diverse and unique phylum of bacteria including several genera: Chloroflexus (7), Oscillochloris (8), Chloronema (9), Heliothrix (10) and several Chloroflexus-like bacteria found in marine environment (11). Among them, Chloroflexus aurantiacus, a prominent microorganism of hot spring microbial mat communities, was the first described and is the most extensively studied representative of FAPs in terms of its photosynthetic and other metabolic pathways. The photosynthetic apparatus of Chloroflexus aurantiacus exhibits an interesting combination of characteristics found in very different and diverse groups of phototrophic prokaryotes. They have a type II photoreaction center and integral membrane antenna complex reminiscent of purple bacteria (12, 13). In addition, they have a peripheral chlorosome antenna complex (14) and a chlorophyll biosynthesis pathway that are both similar to those found in green sulfur bacteria (15, 16). Chloroflexus aurantiacus also contains a unique autotrophic carbon fixation pathway different from that found in any other phototrophs, the 3-hydroxypropinate cycle (17, 18). Therefore, the phylogenetic characterization and the versatile photosynthetic apparatus of Chloroflexus aurantiacus suggest that it occupies an important place in the origin and evolution of photosynthesis (19). An intriguing characteristic of Chloroflexus aurantiacus is its extraordinary electron transfer pathway. For all types of photosynthetic organisms, following the initial process where the light energy transforms into chemical energy, the electrons pass through a series of electron carriers and ultimately either return to the electron donor side of the photosystem via a cyclic electron transfer pathway, or reduce a terminal electron acceptor in a non-cyclic electron transfer process (1). The overall pattern of electron transfer depends critically on the type of the organism, the environment it occupies, whether aerobic or anaerobic metabolism takes place and what type of terminal oxidants and reductants are present. While the electron transfer pathways appear to be quite different in various groups of phototrophs, one component was until recently believed to be a constant constituent in all photosynthetic system: the cytochrome bc1 or b6f complex, which transfers electrons from quinol to soluble cytochrome c or plastocyanin and at the same time translocates protons across the membrane, creating a transmembrane proton motive force (20, 21). However, Chloroflexus aurantiacus, like other members of the FAP phylum, does not exhibit either biochemical or genomic evidence for the existence of a related cytochrome bc1 or b6f complex. The lack of a cytochrome bc1 or b6f complex suggests that this group of organisms contains an unusual photosynthetic electron transfer pathway. A multi-subunit protein complex containing c-type cytochromes but no characteristic features of a cytochrome bc1 complex was isolated from C. aurantiacus by Yanyushin (22). A similar complex from Rhodothermus marinus is now named alternative complex III (ACIII) (23, 24). These two complexes have been proposed to be the functional substitute of the cytochrome bc1 complex based on gene analysis of sequenced genomes of various species (25) and an enzymatic study of ACIII from R. marinus (23). Recent enzyme kinetic analysis showing that ACIII performs the function of a quinol:auracyanin oxidoreductase strongly supports the hypothesis that ACIII fulfills the functional role of cytochrome bc1 complex in the photosynthetic electron transfer chain in Chloroflexus aurantiacus (26). Figure 1 shows the proposed cyclic electron transfer pathway in Chloroflexus aurantiacus. Figure 1 The proposed photosynthetic cyclic electron transfer pathway in Chloroflexus aurantiacus. Based on the genome arrangement of ACIII genes and early fundamental structural studies, the organization of ACIII was revealed to be entirely different from that of cytochrome bc1 or b6f complexes. However, a challenging question emerges - how does ACIII, a complex with structure vastly different from the cytochrome bc complex, carry out the same function in the electron transfer pathway in photosynthesis? A complete picture of the structure and role of ACIII complex is still missing. The key to elucidating this system is therefore believed to reside in understanding the ACIII complex in terms of its substructure and how this relates to its function in photosynthesis and respiration. In this work, a schematic structural model of the photosynthetic ACIII complex is proposed based on chemical cross-linking of subunits in tandem with MALDI-TOF mass spectrometry. The size and type of each subunit was determined by gel electrophoresis including one and two dimensional SDS-PAGE and native PAGE. The type and number of cofactors existing in the ACIII complex was investigated using metal analysis, HPLC combined with ESI-MS and potentiometric titrations.

Monofunctional platinum(II) complexes represent a class of antitumor agents that do not obey the classical structure–activity relationships. We report herein two novel platinum(II) complexes [PtL1Cl] (L1 = l -valine-N-8-quinolylamide) and [PtL2Cl] (L2 = l -leucine-N-8-quinolylamide) in which Cl− acts as the only potential leaving group. These complexes showed comparable cytotoxicity to cisplatin against the murine leukemia cell line (P-388) and the human non-small-cell lung cancer cell line (A-549), and could potentially form monofunctional adducts with DNA.

The filamentous anoxygenic phototrophic bacterium Chloroflexus aurantiacus possesses an unusual electron transfer complex called Alternative Complex III instead of the cytochrome bc or bf type complex found in nearly all other known groups of phototrophs. Earlier work has confirmed that Alternative Complex III behaves as a menaquinol:auracyanin oxidoreductase in the photosynthetic electron transfer chain. In this work, we focus on elucidating the contribution of individual subunits to the overall function of Alternative Complex III. The monoheme subunit ActE has been expressed and characterized in Escherichia coli . A partially dissociated Alternative Complex III missing subunit ActE and subunit ActG was obtained by treatment with the chaotropic agent KSCN, and was then reconstituted with the expressed ActE. Enzymatic activity of the partially dissociated Alternative Complex III was greatly reduced and was largely restored in the reconstituted complex. The redox potential of the heme in the recombinant ActE was +385 mV vs. NHE, similar to the highest potential heme in the intact complex. The results strongly suggest that the monoheme subunit, ActE, is the terminal electron carrier for Alternative Complex III.

Heliobacterium modesticaldum is a Gram-positive, anaerobic, anoxygenic photoheterotrophic bacterium. Its cytochrome bc complex (Rieske/cyt b complex) has some similarities to cytochrome b(6)f complexes from cyanobacteria and chloroplasts, and also shares some characteristics of typical bacterial cytochrome bc(1) complexes. One of the unique factors of the heliobacterial cytochrome bc complex is the presence of a diheme cytochrome c instead of the monoheme cytochrome f in the cytochrome b(6)f complex or the monoheme cytochrome c(1) in the bc(1) complex. To understand the structure and function of this diheme cytochrome c protein, we expressed the N-terminal transmembrane-helix-truncated soluble H. modesticaldum diheme cytochrome c in Escherichia coli. This 25kDa recombinant protein possesses two c-type hemes, confirmed by mass spectrometry and a variety of biochemical techniques. Sequence analysis of the H. modesticaldum diheme cytochrome c indicates that it may have originated from gene duplication and subsequent gene fusion, as in cytochrome c(4) proteins. The recombinant protein exhibits a single redox midpoint potential of +71mV versus NHE, which indicates that the two hemes have very similar protein environments.

The surprising lack of the cytochrome bc1 complex in the filamentous anoxygenic phototrophic bacterium Chloroflexus aurantiacus suggests that a functional replacement exists to link the cyclic electron transfer chain. Earlier work identified the alternative complex III (ACIII) as a substitute of cytochrome bc1 complex. Herein, the enzymatic activity of ACIII is studied. The results strongly support the view that the ACIII functions as menaquinol:auracyanin oxidoreductase in the C. aurantiacus electron transfer chain. Among all the substrates tested, auracyanin is the most efficient electron acceptor of ACIII, suggesting that ACIII directly transfers the electron to auracyanin instead of cytochrome c-554. The lack of sensitivity to common inhibitors of the cytochrome bc1 complex indicates a different catalytic mechanism for the ACIII complex.