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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.

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.

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.

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.