Rapid Glutathione Analysis with SERS Microneedles for Deep Glioblastoma Tissue Differentiation.

Rapid tissue differentiation at the molecular level is a prerequisite for precise surgical resection, which is of special value for the treatment of malignant tumors, such as glioblastoma (GBM). Herein, a SERS-active microneedle is prepared by modifying glutathione (GSH)-responsive molecules, 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), on the surface of Au@Ag substrates for the distinction of different GBM tissues. Since the Raman signals on the surface of the DTNB@Au@Ag microneedle can be collected by both portable and benchtop Raman spectrometers, the distribution of GSH in different tissues at centimeter scale can be displayed through Raman spectroscopy and Raman imaging, and the entire analysis process can be accomplished within 12 min. Accordingly, in vivo brain tissues of orthotopic GBM xenograft mice and ex vivo tissues of GBM patients are accurately differentiated with the microneedle, and the results are well consistent with tissue staining and postoperative pathological reports. In addition, the outline of tumor, peritumoral, and normal tissues can be indicated by the DTNB@Au@Ag microneedle for at least 56 days. Considering that the tumor tissues are quickly discriminated at the molecular level without the restriction of depth, the DTNB@Au@Ag microneedle is promising to be a powerful intraoperative diagnostic tool for surgery navigation.

Determination of low molecular thiols and protein sulfhydryl groups using heterocyclic disulfides.

A promising area in the analytical chemistry of thiol-containing compounds is the use of heterocyclic disulfides as analytical agents, but now only a few of them are widely used. In this paper, we evaluate the possibility of using three different heterocyclic disulfides 2,2'-dithiobis[5-phenyl-1,3,4-oxadiazole] (I), 2,2'-dithiobis[benzoxazole] (II) and 8,8'-dithiobis-quinoline (III) as analytical reagents for the low-mass aminothiols cysteine and glutathione determination. The optimal analysis conditions were found. Spectrophotometric, kinetic, CE, and HPLC methods using I, II, III for the determination of cysteine and glutathione were developed. The obtained methods are characterized by accuracy and sensitivity (detection limits in the range of 10-5-10-6 M) sufficient to quantify cysteine and glutathione in their physiological concentrations. Finally, the proposed disulfides were used to determine the SH-content in the bovine serum albumin (BSA). Considering a number of criteria (applicable pH range, absorption properties, susceptibility to hydrolysis) it was concluded that the proposed reagents have advantages over the commonly used ones (such as the Ellman reagent).

Rapid and sensitive detection of rotavirus by surface-enhanced Raman scattering immunochromatography.

A surface-enhanced Raman scattering (SERS) immunochromatographic assay (ICA) has been developed for rapid, ultrasensitive, and quantitative detection of rotavirus in feces using double Raman molecule-labeled Au-core Ag-shell nanoparticles. The Raman signals are generated by 5,5'-dithiobis-(2-nitrobenzoic acid) and the intensity of the characteristic peak at 1334-1 cm was detected as the analytical signal. The Raman signals were enhanced by the SERS-enhanced effect of both Au and Ag, the large amount of Raman molecules, and the hot-spot effect in the narrow gap between the Au core and Ag shell. The SERS ICA can quantitatively detect rotavirus in a concentration range of 8- 40,000 pg/mL, with detection limits of 80 pg/mL and 8 pg/mL based on naked eye observation and SERS signal detection, respectively. No cross-reaction was observed from other common pathogens. The standard deviation of the intra- and inter-batch repetitive tests is less than 10%, and the coincidence between SERS ICA and RT-qPCR as well as commercial colloidal gold ICA is 100%. The results indicated that this SERS ICA is able to quantitatively detect rotavirus in feces in 20 min with high sensitivity, selectivity, reproducibility, and accuracy and might be a promising method for the early detection of rotavirus in clinical analysis.

Au@Ag core-shell nanoparticles for microRNA-21 determination based on duplex-specific nuclease signal amplification and surface-enhanced Raman scattering.

A novel surface-enhanced Raman scattering (SERS) analysis strategy has been designed combining Au@DTNB@Ag core-shell nanoparticles (DTNB attachment on gold nanoparticles, then encapsulated in Ag shell nanoparticles named as ADANPs) and duplex-specific nuclease signal amplification (DSNSA) platform. Firstly, ADANPs and magnetic substrate of Fe3O4 nanoparticles were covalently attached to the 3'- and 5'- end of capture probe (CP) targeting miRNA-21. Upon the addition of target miRNA-21, these heteroduplexes were specifically cleaved by DSN and resulted in ADANPs that were released from the surface of Fe3O4 nanoparticles (Fe3O4 NPs). At the same time, miRNA-21 remained intact and can rehybridize another DNA probe to trigger the signal-amplifying reaction. Based on this principle, the developed SERS method exhibited good linearity in the range 0 to 1 nM for miRNA-21 with a limit of detection (LOD) of 0.084 fM and has an ability to differentiate even a single-base mismatched sequence on the target sequence or other miRNA sequence. The results provide a novel SERS method which can successfully been applied to the miRNA-21 detection in human serum. Graphical abstract a shows the synthesis of Fe3O4 NPs and the conjugation of Au@DTNB@Ag NPs (ADANPs) for the detection of miRNA-21, b shows the operating principle of DSN-assisted signal amplification strategy for miRNA detection based on Fe3O4@CP@ADA NPs.

Duplex Surface Enhanced Raman Scattering-Based Lateral Flow Immunosensor for the Low-Level Detection of Antibiotic Residues in Milk.

A duplex surface enhanced Raman scattering (SERS)-based lateral flow immunosensor was established for the simultaneous detection of two common antibiotic residues including tetracycline and penicillin in milk. The newly synthesized Au@Ag nanoparticles were labeled with different Raman molecules including 5,5-dithiobis-2-nitrobenzoic acid (DTNB) or 4-mercaptobenzoic acid (MBA), followed by the conjugation of anti-tetracycline monoclonal antibody or anti-penicillin receptor, forming two kinds of SERS nanoprobes. The two nanoprobes can recognize tetracycline-BSA and ampicillin-BSA, respectively, which facilitates the simultaneous detection of the two types of antibiotics on a single test line. After optimization, detection limits of tetracycline and penicillin as low as 0.015 ng/mL and 0.010 ng/mL, respectively, were achieved. These values were far below those of most of other documented bio-analytical approaches. Moreover, the spiking test demonstrates an excellent assay accuracy with recoveries of 88.8% to 111.3%, and satisfactory assay precision with relative standard deviation below 16%. Consequently, the results demonstrate that the SERS-based lateral flow immunosensor developed in this study has the advantages of excellent assay sensitivity and remarkable multiplexing capability, thus it will have great application potential in food safety monitoring.

Thiol-disulfide exchange reactions occurring at modified bovine serum albumin detected using ellman's reagent (5, 5'-dithiobis (2-itrobenzoic acid).

Bovine serum albumin (BSA) is usually employed as a model protein because of being homologous with human serum albumin. Cysteine-34 of BSA has been oxidised with Ellman's reagent to produce BSA labelled with an Ellman's moiety (BSA-SE). The BSA-SE was then reacted with glutathione, N-acetylcysteine and D-penicillamine (D-pen). The two were able to release the Ellman's moiety bound at cysteine-34 while D-pen did not. Albumin labeled using Ellman's reagent was used to demonstrate the cleavage of a protein mixed disulphide. The kinetics of thiol disulfide interchange reactions involving formation of a chromophoric thiolate were determined by UV-visible spectroscopy. The reaction of thiolates with excess Ellman's reagent is used for quantitative estimation of thiol by measuring the absorption at λ, 412 nm. The disulfide exchange reactions occurring at Cys-34 of BSA was determined and the reduction of oxidized Cys-34 was studied in order to understand the reverse reaction. Spectroscopic evidence suggested that glutathione and N-acetylcysteine remove the label and produce BSA in a disulfide form. In contrast, D-pen reaction returned BSA to its thiolate form via mediation. It was observed that thio-disulfide exchange occurred at cysteine-34 labelled with Ellman's moiety. The implications to the redox status of plasma are discussed.

Cysteine Disulfide Traps Reveal Distinct Conformational Ensembles in Dengue Virus NS2B-NS3 Protease.

The dengue virus protease (NS2B-NS3pro) plays a critical role in the dengue viral life cycle, making it an attractive drug target for dengue-related pathologies, including dengue hemorrhagic fever. A number of studies indicate that NS2B-NS3pro undergoes a transition between two widely different conformational states: an "open" (inactive) conformation and a "closed" (active) conformation. For the past several years, the equilibrium between these states and the resting conformation of NS2B-NS3pro have been debated, although a strong consensus is emerging. To investigate the importance of such conformational states, we developed versions of NS2B-NS3pro that allow us to trap the enzyme in various distinct conformations. Our data from these variants suggest that the enzymatic activity appears to be dependent on the movement of NS2B and may rely on the flexibility of the protease core. Locking the enzyme into the "closed" conformation dramatically increased activity, strongly suggesting that the "closed" conformation is the active conformation. The observed resting state of the enzyme depends largely on the construct used to express the NS2B-NS3pro complex. In an "unlinked" construct, in which the NS2B and NS3 regions exist as independent, co-expressed polypeptides, the enzyme rests predominantly in a "closed", active conformation. In contrast, in a "linked" construct, in which NS2B and NS3 are attached by a nine-amino acid linker, NS2B-NS3pro adopts a more relaxed, alternative conformation. Nevertheless, even the unlinked construct samples both the "closed" and other alternative conformations. Given our findings, and the more realistic resemblance of NS2B-NS3pro to the native enzyme, these data strongly suggest that studies should focus on the "unlinked" constructs moving forward. Additionally, the results from these studies provide a more detailed understanding of the various poses of the dengue virus NS2B-NS3 protease and should help guide future drug discovery efforts aimed at this enzyme.

SERS-based rapid assay for sensitive detection of Group A Streptococcus by evaluation of the swab sampling technique.

Beta-hemolytic, Group A Streptococcus pyogenes (GAS) is a life-threating pathogen and the reason for prominent disease, pharyngitis. The conventional analysis of GAS, gold standard, takes 48 hours and the related rapid tests lack in accuracy and sensitivity. In this study, firstly, the efficiency of swab sampling, which is a must in the GAS detection, was discussed with the proposed surface-enhanced Raman spectroscopy (SERS)-based batch assay and each step was controlled by the plate-counting method. Secondly, SERS-based lateral flow immunoassay (LFIA) test strips were constructed and the variation in the SERS intensity of 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) was observed. Thus, a linear correlation was found with a R2 value of 0.9926 and the LOD was calculated to be 0.2 CFU mL-1 of GAS which could be counted as one cell. The combination of the gold standard with the LFIA-SERS technique enabled the fast and accurate pathogen detection. In addition, GAS was quantified with paper-based test strips up to 100 CFU ml-1 level of bacteria for the first time without any interference. Besides, this study was featured with the discussion of the whole cell and pretreated cell detection of pathogens with LFIAs. Therefore, this work enlightens the points that have never been discussed on pathogen detection with paper-based platforms.

Effects of aryl methanesulfonate derivatives on acetylcholinesterase and butyrylcholinesterase.

There is a dire need for new treatments for Alzheimer's disease (AD). Principal drugs have reached maturity, and the number of people affected by AD is growing at a rapid rate. After years of research and many clinical trials, only symptomatic treatments are available. An effective disease-modifying drug for AD needs to be discovered. The research presented in this paper aims to facilitate in the discovery of new potential targets that could help in the ongoing AD research. Aryl methanesulfonate derivatives were screened for their acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) inhibitory activities. IC50 values between 0.660 and 3.397 µM against AChE and 0.885 and 2.596 µM against BuChE were obtained.

Identification of human flavin-containing monooxygenase 3 substrates by a colorimetric screening assay.

Human hepatic flavin-containing monooxygenase 3 is a phase I drug-metabolizing enzyme that is responsible for the oxidation of a variety of drugs and xenobiotics. This work reports on a high throughput rapid colorimetric assay for the screening of substrates or inhibitors of this enzyme. The method is based on the competition of two substrates for access to the active site of hFMO3 whereby the enzymatic product of the first drug converts nitro-5-thiobenzoate (TNB, yellow) to 5,5'-dithiobis (2-nitrobenzoate) (DTNB, colourless). Upon addition of a competing substrate, the amount of detected DNTB is decreased. The assay is validated testing three known substrates of hFMO3, namely benzydamine, tozasertib and tamoxifen. The latter drugs resulted in 41%-55% inhibition. In addition, two other drugs also classified as doping drugs, selegiline and clomiphene, were selected based on their chemical structure similarity to known substrates of hFMO3. These drugs showed 21% and 60% inhibition in the colorimetric assay and therefore were proven to be hFMO3 substrates. LC-MS was used to confirm their N-oxide products. Further characterisation of these newly identified hFMO3 substrates was performed determining their Km and kcat values that resulted to be 314 µM and 1.4 min-1 for selegiline and, 18 µM and 0.1 min-1 for clomiphene. This method paves the way for a rapid automated high throughput screening of nitrogen-containing compounds as substrates/inhibitors of hFMO3.

Determination of thiol-to-protein ratio and drug-to-antibody ratio by in-line size exclusion chromatography with post-column reaction.

An in-line size-exclusion (SE) ultra-high-performance liquid chromatography (UHPLC)- 5,5-dithio-bis-(2-nitrobenzoic acid) (DTNB) method to quantify thiols in monoclonal antibodies (mAb) when manufacturing antibody-drug conjugates (ADCs) was developed. The mAbs are separated on an SE-UHPLC column and monitored with a UV detector at a wavelength of 280 nm. Eluents are channeled into a reaction coil and mixed with DTNB to form 5-thio-2-nitrobenzoic acid (TNB). Thiol concentration is calculated using absorption at 412 nm. Using optimized conditions, partially reduced mAbs can be separated from low-molecular weight contaminants and undergo the DTNB reaction. The standard curve of L-cysteine had good linearity between 100 and 1000 µM. The selectivity, linearity, repeatability, and robustness of this method were evaluated. The calculated free-SH:protein ratios of partially reduced mAbs were consistent between in-line SE-UHPLC-DTNB and conventional methods. The SE-UHPLC-DTNB method showed time- and temperature-dependent changes in the free-SH:protein ratio of mAbs during reduction. The changes in drug-antibody ratio (DAR) of ADCs during the conjugation reaction were also evaluated. This method is an inexpensive and versatile alternative to conventional methods of estimating the free-SH:protein ratio of mAbs and the DAR of ADCs. This method also minimizes assay time.

Novel Gold(I) Thiolate Derivatives Synergistic with 5-Fluorouracil as Potential Selective Anticancer Agents in Colon Cancer.

New gold(I) thiolate complexes have been synthesized and characterized, and their physicochemical properties and anticancer activity have been tested. The coordination of PTA derivatives provides optimal hydrophilicity/lipophilicity properties to the complexes, which present high solution stability. Moreover, the complexes show a high anticancer activity against Caco-2 cells, comparable to that of auranofin, and a very low cytotoxic activity against enterocyte-like differentiated cells. Their activity has been shown to produce cell death by apoptosis and arrest of the cell cycle because of interaction with the reductase enzymes and consequent reactive oxygen species production. Some of these new complexes are also able to decrease the necessary dose of 5-fluorouracil, a drug used for the treatment of colon cancer, by a synergistic mechanism.

Phase Transfer and Surface Functionalization of Hydrophobic Nanoparticle using Amphiphilic Poly(amino acid).

Functionalization of nanoparticles with chemical and biochemical is essential for their biomedical and other application. However, most of the high quality nanoparticles are hydrophobic in nature due to surfactant capping and their conversion into water-soluble functional nanoparticle via appropriate coating and conjugation chemistry is extremely critical issue. Here we report amphiphilic poly(amino acid)-based one-pot coating and conjugation approach that can transform hydrophobic nanoparticle into water-soluble nanoparticle functionalized with primary amine, thiol, and biomolecule. We have designed amphiphilic polyaspartimide that can anchor hydrophobic nanoparticle through octadecyl groups, leaving the polar polyethylene glycol and aspartimide groups exposed outwards. The aspartimide group is then reacted with primary amine containing chemical/biomolecule with the formation of water-soluble functional nanoparticle. This approach has been extended to different hydrophobic nanoparticles and biomolecules. The present approach has advantages over existing approaches as coating and functionalization can be performed in one pot and functional nanoparticles have <12 nm hydrodynamic size, high colloidal stability, and biocompartibility. This developed approach can be used to derive biocompatible nanobioconjugates for various biomedical applications.

Quantification of PEG-maleimide ligands and coupling efficiencies on nanoparticles with Ellman's reagent.

The surface modification of nanometer- and micrometer-sized particles and planar substrates with polyethylene glycol (PEG) ligands of varying length is a very common strategy to tune the hydrophilicity and biocompatibility of such materials, minimize unspecific interactions, improve biofunctionalization efficiencies, and enhance blood circulation times. Nevertheless, simple methods for the quantification of PEG ligands are comparatively rare. Here, we present a new concept for the quantification of PEG ligands for maleimide-functionalized PEG molecules and the determination of PEG coupling efficiencies, exploiting the quantitative reaction of maleimide with l-cysteine, and the subsequent determination of the unreacted thiol with the photometric Ellman's test. This is shown for heterobifunctional PEG spacers of varying length and amino-functionalized polystyrene nanoparticles (PS NP) without and with differently charged encoding dyes. The reaction of l-cysteine with the Ellman's reagent was monitored photometrically and with electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS) to derive the reaction mechanism and to obtain the stoichiometry factor for l-cysteine quantification. Mass balances and quantification of l-cysteine via its sulfur concentration using elemental analysis and inductively coupled plasma mass spectrometry (ICP-MS) confirmed the accuracy and reliability of this approach that can be extended to other surface groups and ligands.

Kinetic and thermodynamic studies on the disulfide-bond reducing potential of hydrogen sulfide.

The significance of persulfide species in hydrogen sulfide biology is increasingly recognized. However, the molecular mechanisms of their formation remain largely elusive. The obvious pathway of the reduction of biologically abundant disulfide moieties by sulfide was challenged on both thermodynamic and kinetic grounds. Using DTNB (5,5'-dithiobis-(2-nitrobenzoic acid), also known as Ellman's reagent) as a model disulfide we conducted a comprehensive kinetic study for its reaction with sulfide. The bimolecular reaction is relatively fast with a second-order rate constant of 889 ± 12 M(-1)s(-1) at pH = 7.4. pH dependence of the rate law revealed that the reaction proceeds via the bisulfide anion species with an initial nucleophilic thiol-disulfide exchange reaction to give 5-thio-2-nitrobenzoic acid (TNB) and TNB-persulfide with a pH independent second-order rate constant of 1090 ± 12 M(-1)s(-1). However, kinetic studies and stoichiometric analyses in a wide range of reactant ratios together with kinetic simulations revealed that it is a multistep process that proceeds via kinetically driven, practically irreversible reactions along the disulfide → persulfide → inorganic polysulfides axis. The kinetic model postulated here, which is fully consistent with the experimental data, suggests that the TNB-persulfide is further reduced by sulfide with a second-order rate constant in the range of 5 × 10(3) - 5 × 10(4) M(-1)s(-1) at pH 7.4 and eventually yields inorganic polysulfides and TNB. The reactions of cystine and GSSG with sulfide were found to be significantly slower and to occur via more complicated reaction schemes. (1)H NMR studies suggest that these reactions also generate Cys-persulfide and inorganic polysulfide species, but in contrast with DTNB, in consecutive equilibrium processes that are sensitive to changes in the reactant and product ratios. Collectively, our results demonstrate that the reaction of disulfides with sulfide is a highly system specific process from both thermodynamic and kinetic aspects, which together with the considerable steady-state concentrations of the reactants in biological systems signifies physiological relevance.

A chromogenic assay of substrate depletion by thiol dioxygenases.

A fast and easy method for enzyme activity assays using the chromogenic Ellman reagent, 5,5'-dithiobis(2-nitrobenzoic acid), was developed. The method was used to measure the activity of the nonheme mono-iron enzyme cysteine dioxygenase. Quantifying the depletion of the substrate, cysteine, allowed standard kinetic parameters to be determined for the enzyme from Rattus norvegicus. The assay was also used to quickly test the effects of ionic strength, pH, enzyme storage conditions, and potential inhibitors and activators. This assay facilitates a higher throughput than available HPLC-based assays, as it enjoys the advantages of fewer sample handling steps, implementation in a 96-well format, and speed. In addition, the relative specificity of Ellman's reagent, coupled with its reaction with a wide range of thiols, means that this assay is applicable to many enzymes. Finally, the use of readily available reagents and instrumentation means that this assay can be used by practically any research group to compare results with those of other groups.

Thiolated human serum albumin cross-linked dextran hydrogels as a macroscale delivery system.

Hydrogels play an important role in macroscale delivery systems by enabling the transport of cells and molecules. Here we present a facile and benign method to prepare a dextran-based hydrogel (Dex-sHSA) using human serum albumin (HSA) as a simultaneous drug carrier and covalent cross-linker. Drug binding affinity of the albumin protein was conserved in the thiolation step using 2-iminothiolane and subsequently, in the in situ gelation step. Oscillation rheometry studies confirmed the formation of a three-dimensional viscoelastic network upon reaction of dextran and the HSA protein. The mechanical properties of Dex-sHSA hydrogel can be tuned by the protein concentration, and the degree of thiolation of sHSA. Sustained release of hydrophobic drugs, such as ibuprofen, paclitaxel and dexamethasone, from the Dex-sHSA network was shown over one week. Hence, this albumin-based dextran hydrogel system demonstrates its potential as a macroscale delivery system of hydrophobic therapeutics for a wide range of biomedical applications.

Atypical thioredoxins in poplar: the glutathione-dependent thioredoxin-like 2.1 supports the activity of target enzymes possessing a single redox active cysteine.

Plant thioredoxins (Trxs) constitute a complex family of thiol oxidoreductases generally sharing a WCGPC active site sequence. Some recently identified plant Trxs (Clot, Trx-like1 and -2, Trx-lilium1, -2, and -3) display atypical active site sequences with altered residues between the two conserved cysteines. The transcript expression patterns, subcellular localizations, and biochemical properties of some representative poplar (Populus spp.) isoforms were investigated. Measurements of transcript levels for the 10 members in poplar organs indicate that most genes are constitutively expressed. Using transient expression of green fluorescent protein fusions, Clot and Trx-like1 were found to be mainly cytosolic, whereas Trx-like2.1 was located in plastids. All soluble recombinant proteins, except Clot, exhibited insulin reductase activity, although with variable efficiencies. Whereas Trx-like2.1 and Trx-lilium2.2 were efficiently regenerated both by NADPH-Trx reductase and glutathione, none of the proteins were reduced by the ferredoxin-Trx reductase. Only Trx-like2.1 supports the activity of plastidial thiol peroxidases and methionine sulfoxide reductases employing a single cysteine residue for catalysis and using a glutathione recycling system. The second active site cysteine of Trx-like2.1 is dispensable for this reaction, indicating that the protein possesses a glutaredoxin-like activity. Interestingly, the Trx-like2.1 active site replacement, from WCRKC to WCGPC, suppresses its capacity to use glutathione as a reductant but is sufficient to allow the regeneration of target proteins employing two cysteines for catalysis, indicating that the nature of the residues composing the active site sequence is crucial for substrate selectivity/recognition. This study provides another example of the cross talk existing between the glutathione/glutaredoxin and Trx-dependent pathways.

Preparation of 2-nitro-5-thiobenzoate for the routine determination of reagent hypochlorous acid concentrations.

Solutions of hypochlorous acid (HOCl) decay over time. This decay indicates the necessity for methods and reagents for the routine measurement of this oxidant. 2-Nitro-5-thiobenzoate is commonly used to measure HOCl concentrations. This article describes a method for the preparation of 2-nitro-5-thiobenzoate that is stable for at least 3 months. This method relies on the partial rather than full reduction of 5,5'-dithiobis-(2-nitrobenzoic acid) and the resulting equilibrium between the substrate and the product.

Evaluation of protein disulfide conversion in vitro using a continuous flow dialysis system.

Recombinant therapeutic proteins are heterogeneous due to chemical and physical modifications. Understanding the impact of these modifications on drug safety and efficacy is critical for optimal process development and for setting reasonable specification limits. In this study, we describe the development of an in vitro continuous flow dialysis system to evaluate potential in vivo behavior of thiol adducted species and incorrectly disulfide bonded species of therapeutic proteins. The system is capable of maintaining the low-level cysteine concentrations found in human blood. Liabilities of cysteamine adducted species, incorrectly disulfide bonded species, and the correctly disulfide bonded form of an Fc-fusion protein were studied using this system. Results showed that 90% of the cysteamine adduct converted into the correctly disulfide bonded form and incorrectly disulfide bonded species in approximately 4 h under physiological conditions. Approximately 50% of incorrectly disulfide bonded species converted into the correctly bonded form in 2 days. These results provide valuable information on potential in vivo stability of the cysteamine adduct, incorrectly disulfide bonded species, and the correctly bonded form of the Fc-fusion protein. These are important considerations when evaluating the criticality of product quality attributes.