Evaluation of Hydrogen Exchange Mass Spectrometry as a Stability-Indicating Method for Formulation Excipient Screening for an IgG4 Monoclonal Antibody

Antibodies are molecules that exhibit diverse conformational changes on different timescales, and there is ongoing interest to better understand the relationship between antibody conformational dynamics and storage stability. Physical stability data for an IgG4 monoclonal antibody (mAb-D) were gathered through traditional forced degradation (temperature and stirring stresses) and accelerated stability studies, in the presence of different additives and solution conditions, as measured by differential scanning calorimetry, size exclusion chromatography, and microflow imaging. The results were correlated with hydrogen exchange mass spectrometry (HX-MS) data gathered for mAb-D in the same formulations. Certain parameters of the HX-MS data, including hydrogen exchange in specific peptide segments in the C_H2 domain, were found to correlate with stabilization and destabilization of additives on mAb-D during thermal stress. No such correlations between mAb physical stability and HX-MS readouts were observed under agitation stress. These results demonstrate that HX-MS can be set up as a streamlined methodology (using minimal material and focusing on key peptide segments at key time points) to screen excipients for their ability to physically stabilize mAbs. However, useful correlations between HX-MS and either accelerated or real-time stability studies will be dependent on a particular mAb's degradation pathway(s) and the type of stresses used.     

Impact of Glycosylation on the Local Backbone Flexibility of Well-Defined IgG1-Fc Glycoforms Using Hydrogen Exchange-Mass Spectrometry

We have used hydrogen exchange–mass spectrometry to characterize local backbone flexibility of 4 well-defined IgG1-Fc glycoforms expressed and purified from Pichia pastoris, 2 of which were prepared using subsequent in vitro enzymatic treatments. Progressively decreasing the size of the N-linked N297 oligosaccharide from high mannose (Man8-Man12), to Man5, to GlcNAc, to nonglycosylated N297Q resulted in progressive increases in backbone flexibility. Comparison of these results with recently published physicochemical stability and Fcγ receptor binding data with the same set of glycoproteins provide improved insights into correlations between glycan structure and these pharmaceutical properties. Flexibility significantly increased upon glycan truncation in 2 potential aggregation-prone regions. In addition, a correlation was established between increased local backbone flexibility and increased deamidation at asparagine 315. Interestingly, the opposite trend was observed for oxidation of tryptophan 277 where faster oxidation correlated with decreased local backbone flexibility. Finally, a trend of increasing C'E glycopeptide loop flexibility with decreasing glycan size was observed that correlates with their FcγRIIIa receptor binding properties. These well-defined IgG1-Fc glycoforms serve as a useful model system to identify physicochemical stability and local backbone flexibility data sets potentially discriminating between various IgG glycoforms for potential applicability to future comparability or biosimilarity assessments.     

The Influence of In Vivo Metabolic Modifications on ADMET Properties of Green Tea Catechins–In Silico Analysis

The health effects of green tea are associated with catechins: (?)-epigallocatechin-3-O-gallate (EGCG), (?)-epigallocatechin, (?)-epicatechin-3-O-gallate, and (?)-epicatechin. An understanding of compound absorption, distribution, metabolism, excretion, and toxicity characteristics is essential for explaining its biological activities. Herein, absorption, distribution, metabolism, excretion, and toxicity properties of in vivo detected metabolites of green tea catechins (GTCs) have been analyzed in silico. The influence of metabolic transformations on absorption, distribution, metabolism, and excretion profiles of GTCs corresponds to the effects of size, charge, and lipophilicity, as already observed for other small molecules. Mutagenic, carcinogenic, or liver toxic effects were predicted only for a few metabolites. Similar to galloylated GTCs EGCG and (--)-epicatechin-3-O-gallate, the sulfo-conjugates were predicted to bind at the warfarin binding site. The low free plasma concentration of these derivatives may be consequential to their serum albumin binding. The activity cliff detected for methylated conjugates of EGCG indicates that GTCs' pro-oxidative activity in bound state comes primarily from free hydroxyl groups of the pyrogallol ring B.     

HPLC 法测定硫酸氢氯吡格雷片有关物质的不确定度评定

目的：对高效液相色谱法(HPLC)测定硫酸氢氯吡格雷片有关物质的不确定度进行评估,找出影响不确定度因素。方法：建立数学模型,对有关物质测定过程中各影响因素进行分析评估。结果：通过计算各变量的不确定度,合成不确定度,最终得出测量结果的扩展不确定度。结论：分析产生不确定度的主要来源,为有效控制该有关物质测定方法的准确性提供可靠的理论依据。     

柚皮苷对H_2O_2诱导H9c2心肌细胞损伤保护作用

目的探讨柚皮苷对H_2O_2诱导的H9c2心肌细胞凋亡的保护作用及机制。方法体外培养H9c2心肌细胞,用H_2O_2诱导建立细胞凋亡模型。实验设对照组、模型组、柚皮苷低、中、高剂量组(10、20、40μmol/L),噻唑蓝法检测细胞活力并用显微镜观察各组细胞形态;原位末端标记法检测H9c2心肌细胞凋亡情况;RT-PCR及Western blot法检测凋亡相关因子Bcl-2、Bax、caspase-3 m RNA及蛋白表达。结果与对照组比较,模型组细胞凋亡率[(17.2±2.1)%]明显升高(P<0.01);与模型组比较,10、20、40μmol/L柚皮苷组细胞凋亡率[分别为(10.7±1.9)%、(5.7±1.2)%、(6.4±1.5)%]均下降(均P<0.05)。与对照组比较,模型组H9c2心肌细胞Bcl-2蛋白表达水平(0.76±0.16)明显下调,Bax、caspase-3蛋白表达水平[分别为(5.42±0.52)、(1.09±0.11)]均上调(均P<0.01);与模型组比较,10、20、40μmol/L柚皮苷组H9c2心肌细胞Bcl-2蛋白表达水平[分别为(1.37±0.11)、(1.65±0.09)、(1.65±0.15)]均上调,Bax、caspase-3蛋白表达水平[分别为(2.78±0.55)、(3.43±0.15)、(2.69±0.26)和(0.59±0.08)、(0.77±0.06)、(0.82±0.05)]均下调(均P<0.05)。结论柚皮苷对H_2O_2诱导的H9c2心肌细胞损伤具有一定保护作用,其机制可能与其对凋亡信号途径的抑制作用有关。     

Synthesis,Polymorphism and Thermal Decomposition Process of (n-C4H9)4NRE(BH4)4 for RE = Ho,Tm and Yb

In total, three novel organic derivatives of lanthanide borohydrides, n-But₄NRE(BH₄)₄ (TBAREB), RE = Ho, Tm, Yb, have been prepared utilizing mechanochemical synthesis and purified via solvent extraction. Studies by single crystal and powder X-ray diffraction (SC-XRD and PXRD) revealed that they crystalize in two polymorphic forms, α- and β-TBAREB, adopting monoclinic (P2₁/c) and orthorhombic (Pnna) unit cells, previously found in TBAYB and TBAScB, respectively. Thermal decomposition of these compounds has been investigated using thermogravimetric analysis and differential scanning calorimetry (TGA/DSC) measurements, along with the analysis of the gaseous products with mass spectrometry (MS) and with analysis of the solid decomposition products with PXRD. TBAHoB and TBAYbB melt around 75 °C, which renders them new ionic liquids with relatively low melting points among borohydrides.     

Water oxidation by photosystem II is the primary source of electrons for sustained H2 photoproduction in nutrient-replete green algae

The unicellular green alga Chlamydomonas reinhardtii is capable of photosynthetic H₂ production. H₂ evolution occurs under anaerobic conditions and is difficult to sustain due to 1) competition between [FeFe]-hydrogenase (H₂ase), the key enzyme responsible for H₂ metabolism in algae, and the Calvin–Benson–Bassham (CBB) cycle for photosynthetic reductants and 2) inactivation of H₂ase by O₂ coevolved in photosynthesis. Recently, we achieved sustainable H₂ photoproduction by shifting algae from continuous illumination to a train of short (1 s) light pulses, interrupted by longer (9 s) dark periods. This illumination regime prevents activation of the CBB cycle and redirects photosynthetic electrons to H₂ase. Employing membrane-inlet mass spectrometry and H218O, we now present clear evidence that efficient H₂ photoproduction in pulse-illuminated algae depends primarily on direct water biophotolysis, where water oxidation at the donor side of photosystem II (PSII) provides electrons for the reduction of protons by H₂ase downstream of photosystem I. This occurs exclusively in the absence of CO₂ fixation, while with the activation of the CBB cycle by longer (8 s) light pulses the H₂ photoproduction ceases and instead a slow overall H₂ uptake is observed. We also demonstrate that the loss of PSII activity in DCMU-treated algae or in PSII-deficient mutant cells can be partly compensated for by the indirect (PSII-independent) H₂ photoproduction pathway, but only for a short (<1 h) period. Thus, PSII activity is indispensable for a sustained process, where it is responsible for more than 92% of the final H₂ yield.

Many species of green algae have [FeFe]-hydrogenases (H₂ases) (1) that catalyze the reversible reduction of protons to molecular hydrogen:2H++2e−⇄H2.[1]Since [FeFe]-H₂ases are extremely O₂-sensitive (2), reaction 1 typically proceeds under anoxic conditions. With respect to H₂ metabolism, Chlamydomonas reinhardtii is the most studied alga. This alga possesses two [FeFe]-H₂ases in the chloroplast, HYDA1 and HYDA2 (3, 4). In the light, they accept electrons from photosynthetically reduced ferredoxin (FDX1) (5), while in the dark electrons come from the activity of pyruvate ferredoxin oxidoreductase (PFR1) (6). PFR1 catalyzes the oxidation of pyruvate to acetyl-CoA, and its activity is linked to H₂ase via FDX1 (7). Since [FeFe]-H₂ases interact with the photosynthetic electron transport chain at the level of ferredoxin, they may accept electrons originating both from water oxidation via the photosystem II (PSII)-dependent pathway (“direct water biophotolysis”) and from the degradation of organic substrates via a PSII-independent mechanism (“indirect water biophotolysis” or “indirect pathway”) (8). In the latter case, the reductants are supplied to the plastoquinone (PQ) pool by the type II NADPH dehydrogenase (NDA2), thus bypassing PSII (9, 10).The release of H₂ leads to a loss of metabolic energy. In healthy, actively growing C. reinhardtii cultures, H₂ production is therefore only a temporal phenomenon observed during dark anoxia and upon subsequent onset of illumination (11). In contrast to dark fermentation, H₂ photoproduction is a very efficient process that proceeds for only a short period of time (from a few seconds to a few minutes). Two theories have been developed to explain the short duration. The first is based on the oxygen sensitivity of H₂ases (12, 13). In the light, algae accumulate O₂ that is produced by water oxidation at PSII (14). As a result, H₂ photoproduction may cease over time (14, 15), and the duration of this process is reported to shorten with increased light intensity (16). Because of the negative correlation between the rates of H₂ photoproduction and O₂ evolution, the inhibition of H₂ases by O₂ is frequently quoted as the primary reason for the rapid loss in H₂ photoproduction after the onset of illumination (17).Alternatively, the loss in the H₂ photoproduction efficiency during illumination could be explained by the light-induced induction of competitive pathways, which may drain reducing equivalents away from the [FeFe]-H₂ase enzyme (18, 19). Candidates for this role are the Mehler-like reaction driven by flavodiiron proteins (FDPs) (15, 20, 21) and the Calvin–Benson–Bassham (CBB) cycle (22). Compelling evidence for the competition between these two pathways and H₂ production has been accumulated in recent studies (23–25). As CO₂ fixation provides the strongest sink for photosynthetic reductants, it should play a major role in the cessation of H₂ photoproduction in algae when the CBB cycle is active (19, 22).For preventing competition between the [FeFe]-H₂ases and the CBB cycle, we recently devised a pulse-illumination protocol that allows H₂ production in nutrient-replete algal cultures for up to 3 d (23). To achieve this, we specifically selected the duration of light pulses in the light/dark sequence to avoid activation of the CBB cycle, thus allowing for the redirection of photosynthetic electrons toward the [FeFe]-H₂ases. Typically, a train of 1- to 6-s light pulses interrupted by 9-s dark periods is sufficient for sustained H₂ photoproduction in C. reinhardtii cultures (23, 25). Our protocol thus differs from earlier pulse-illumination approaches that aimed at preventing the accumulation of O₂ in the cultures (26).While we could demonstrate competition of [FeFe]-H₂ase with FDPs (25), the origin of reductants for H₂ photoproduction in the pulse-illuminated algae remained unclear. The relatively high efficiency of the process suggests the involvement of water oxidation by PSII, and consequently the simultaneous production of H₂ and O₂. Although widely proposed in the current literature (8, 24), the presence of the direct water biophotolysis in H₂-producing green algae has not yet been proven by direct experimental data.In the present study, we provide clear evidence for the presence of PSII-dependent oxidation of ¹⁸O-labeled water H218O with concomitant evolution of ¹⁶O₂ and ^16,18O₂ during H₂ photoproduction in the pulse-illuminated green alga C. reinhardtii under anoxic conditions. O₂ evolution is balanced by light-dependent and light-independent respiration that sustains the anoxic condition. We also demonstrate that the loss of PSII activity in algae can be partly compensated by the PSII-independent H₂ photoproduction pathway. Nevertheless, the activity of PSII is indispensable for the sustained process, where it contributes to more than 92% of the final H₂ yield.     

Structural insight into the molecular mechanism of allosteric activation of human cystathionine β-synthase by S-adenosylmethionine

Cystathionine β-synthase (CBS) is a heme-dependent and pyridoxal-5′-phosphate–dependent protein that controls the flux of sulfur from methionine to cysteine, a precursor of glutathione, taurine, and H₂S. Deficiency of CBS activity causes homocystinuria, the most frequent disorder of sulfur amino acid metabolism. In contrast to CBSs from lower organisms, human CBS (hCBS) is allosterically activated by S-adenosylmethionine (AdoMet), which binds to the regulatory domain and triggers a conformational change that allows the protein to progress from the basal toward the activated state. The structural basis of the underlying molecular mechanism has remained elusive so far. Here, we present the structure of hCBS with bound AdoMet, revealing the activated conformation of the human enzyme. Binding of AdoMet triggers a conformational change in the Bateman module of the regulatory domain that favors its association with a Bateman module of the complementary subunit to form an antiparallel CBS module. Such an arrangement is very similar to that found in the constitutively activated insect CBS. In the presence of AdoMet, the autoinhibition exerted by the regulatory region is eliminated, allowing for improved access of substrates to the catalytic pocket. Based on the availability of both the basal and the activated structures, we discuss the mechanism of hCBS activation by AdoMet and the properties of the AdoMet binding site, as well as the responsiveness of the enzyme to its allosteric regulator. The structure described herein paves the way for the rational design of compounds modulating hCBS activity and thus transsulfuration, redox status, and H₂S biogenesis.Cystathionine β-synthase (CBS; EC 4.2.1.22) is a pyridoxal-5′-phosphate (PLP)-dependent enzyme that catalyzes the β-replacement of the hydroxyl group of l-serine (Ser) by l-homocysteine (Hcy), yielding cystathionine (Cth) (1). A deficient activity of human CBS (hCBS) is the cause of classical homocystinuria [CBS-deficient homocystinuria (CBSDH); Online Mendelian Inheritance in Man (OMIM) no. 236200], an autosomal, recessive inborn error of sulfur amino acid metabolism, characterized by increased levels of Hcy in plasma and urine. CBSDH manifests as a combination of connective tissue defects, skeletal deformities, vascular thrombosis, and mental retardation (2).The hCBS is a homotetrameric enzyme whose subunits are organized into three structural domains. The N-terminal region binds heme and is thought to function in redox sensing and/or enzyme folding (3, 4). The central catalytic core shows the fold of the type II family PLP-dependent enzymes (5, 6). Finally, the C-terminal region consists of a tandem pair of CBS motifs (7–9) that bind S-adenosylmethionine (AdoMet) and lead to an increase in catalytic activity by up to fivefold (10, 11). The CBS motif pair, commonly known as a “Bateman module” (12, 13), is responsible for CBS subunit tetramerization (14, 15). The presence of pathogenic missense mutations in this region often does not impair enzyme activity but typically interferes with binding of AdoMet and/or the enzyme’s activation by AdoMet (15–17). Removal of the regulatory region leads to a dimer with much increased activity (14, 15). Recently, we showed that removal of residues 516–525, forming a flexible loop of the CBS2 motif of hCBS, yields dimeric species (hCBSΔ516–525) with intact AdoMet binding capacity and activity responsiveness to AdoMet similar to a native hCBS WT (18).hCBS is regulated by a complex molecular mechanism that remains poorly understood. More than a decade ago, we and others hypothesized that hCBS might exist in two different conformations: a “basal” state with low activity, where the C-terminal regulatory domain would restrict the access of substrates into the catalytic site, and an AdoMet-bound “activated” state, where the AdoMet-induced conformational change would allow for enzyme activation (16, 19). Recently, we have unveiled the relative orientations of the regulatory and catalytic domains in hCBS (18), which were in a striking contrast to those of both the previous in silico models (20, 21) and the Drosophila melanogaster (dCBS) structure (22). Our data showed that, although the pairing mode and the orientation of catalytic cores are similar in both insect dCBS and hCBS, the position of their regulatory domains is markedly different (18). In the basal state, the Bateman modules from each hCBS unit are far apart and do not interact with each other, being placed just above the entrance of the catalytic site of the complementary subunit, thus hampering the access of substrates into this cavity. Our hCBSΔ516–525 structure additionally revealed the presence of two major cavities in the Bateman module, S1 and S2, one of which (S2) is solvent-exposed and probably represents the primary binding site for AdoMet (18). These findings are in agreement with the much higher basal activity of dCBS and its inability to bind or to be regulated by AdoMet (23, 24) and suggest that the structural basis underlying the regulation of the human enzyme markedly differs from CBS regulation in insects or yeast (24). Taken together, the available data indicate that binding of AdoMet to the Bateman module weakens the interaction between the regulatory domain and the catalytic core although the mechanism and the magnitude of the underlying structural effect are still under debate (16, 19, 25–27).To solve the molecular mechanism of hCBS regulation by AdoMet, we have analyzed the crystals of an engineered hCBSΔ516–525 protein that bears the mutation E201S, which potentially weakens and/or disrupts the interaction between the Bateman module and the catalytic core (Fig. 1A), thus favoring the activation of the enzyme. The data presented here fill a long-sought structural gap by unraveling the crystal structure of AdoMet-bound hCBS, thus providing the overall fold of the enzyme in its activated conformation and the identity of the AdoMet binding sites. Comparison with the structures of hCBS in basal conformation and constitutively activated dCBS was instrumental in the understanding of the regulatory role played by the C-terminal domain as well as the effect of some of the pathogenic mutations in the activation and/or inhibition of this key molecule of transsulfuration.Open in a separate windowFig. 1.Interactions between protein domains in basal hCBS. (A) In hCBSΔ516–525, residues Y484, N463, and S466 anchor the Bateman module (blue) to the protein core (gray) through H-bonds with the residues E201 and D198 from the loop L191–202, thus occluding the entrance to the catalytic pocket. (B) The CBS-specific activity of selected hCBS variants in the absence (blue bars) and the presence (red bars) of 300 µM AdoMet. hCBS enzyme species marked with “Δ” lack residues 516–525 and form dimers.