Chemical Degradation Pathways Research Articles

Modeling of a continuous photocatalytic reactor for isovaleraldehyde oxidation: Effect of different operating parameters and chemical degradation pathway

An investigation of isovaleraldehyde (ISOV) photocatalytic oxidation was conducted at initial concentrations ranging from 25 to 150mg/m3 and different relative humidities (5–90% RH) in order to characterize the process performances close to indoor air conditions. Experiments were carried out in two different reactors: cylinder and flat-plate photoreactor (planar reactor) at different air gap (20–60mm) and gas residence times (0.67–5.0s). A plug flow reactor system was developed in order to perform kinetic studies of (i) isovaleraldehyde removal, (ii) selectivity of CO2, (iii) by-products formation and removal. It appears that ISOV removal efficiencies increased with lower inlet concentrations, lower air gap and higher gas residence times.In small amounts, the presence of water vapor has a promoting effect on the degradation due to the formation of OH radicals.Evaluating different kinetic models by least squares analysis, it was shown that the Langmuir-Hinshelwood (L-H) model could give a good correlation with the experimental results. Thus, the effect of ISOV gas-phase concentration, air gap and light intensity on chemical conversion rates is discussed.A kinetic model based on Langmuir-Hinshelwood (L-H) approach and taking into account the mass transfer step was developed. This allows us to determine L-H constants regardless of the transfer aspect. This last is estimated by semi-empirical correlation. The separation between the mass transfer and the chemical reaction steps is obtained. The effect of UV light intensity is also considered.

Chemical evolution of Macondo crude oil during laboratory degradation as characterized by fluorescence EEMs and hydrocarbon composition

The fluorescence EEM technique, PARAFAC modeling, and hydrocarbon composition were used to characterize oil components and to examine the chemical evolution and degradation pathways of Macondo crude oil under controlled laboratory conditions. Three major fluorescent oil components were identified, with Ex/Em maxima at 226/328, 262/315, and 244/366nm, respectively. An average degradation half-life of ∼20d was determined for the oil components based on fluorescence EEM and hydrocarbon composition measurements, showing a dynamic chemical evolution and transformation of the oil during degradation. Dispersants appeared to change the chemical characteristics of oil, to shift the fluorescence EEM spectra, and to enhance the degradation of low-molecular-weight hydrocarbons. Photochemical degradation played a dominant role in the transformation of oil components, likely an effective degradation pathway of oil in the water column. Results from laboratory experiments should facilitate the interpretation of field-data and provide insights for understanding the fate and transport of oil components in the Gulf of Mexico.

Simultaneous Assessment of Asp Isomerization and Asn Deamidation in Recombinant Antibodies by LC-MS following Incubation at Elevated Temperatures

The degradation of proteins by asparagine deamidation and aspartate isomerization is one of several chemical degradation pathways for recombinant antibodies. In this study, we have identified two solvent accessible degradation sites (light chain aspartate-56 and heavy chain aspartate-99/101) in the complementary-determining regions of a recombinant IgG1 antibody susceptible to isomerization under elevated temperature conditions. For both hot-spots, the degree of isomerization was found to be significantly higher than the deamidation of asparagine-(387, 392, 393) in the conserved CH3 region, which has been identified as being solvent accessible and sensitive to chemical degradation in previous studies. In order to reduce the time for simultaneous identification and functional evaluation of potential asparagine deamidation and aspartate isomerization sites, a test system employing accelerated temperature conditions and proteolytic peptide mapping combined with quantitative UPLC-MS was developed. This method occupies the formulation buffer system histidine/HCl (20 mM; pH 6.0) for denaturation/reduction/digestion and eliminates the alkylation step. The achieved degree of asparagine deamidation and aspartate isomerization was adequate to identify the functional consequence by binding studies. In summary, the here presented approach greatly facilitates the evaluation of fermentation, purification, formulation, and storage conditions on antibody asparagine deamidation and aspartate isomerization by monitoring susceptible marker peptides located in the complementary-determining regions of recombinant antibodies.

Identification of Potential Sites for Tryptophan Oxidation in Recombinant Antibodies Using tert-Butylhydroperoxide and Quantitative LC-MS

Amino acid oxidation is known to affect the structure, activity, and rate of degradation of proteins. Methionine oxidation is one of the several chemical degradation pathways for recombinant antibodies. In this study, we have identified for the first time a solvent accessible tryptophan residue (Trp-32) in the complementary-determining region (CDR) of a recombinant IgG1 antibody susceptible to oxidation under real-time storage and elevated temperature conditions. The degree of light chain Trp-32 oxidation was found to be higher than the oxidation level of the conserved heavy chain Met-429 and the heavy chain Met-107 of the recombinant IgG1 antibody HER2, which have already been identified as being solvent accessible and sensitive to chemical oxidation. In order to reduce the time for simultaneous identification and functional evaluation of potential methionine and tryptophan oxidation sites, a test system employing tert-butylhydroperoxide (TBHP) and quantitative LC-MS was developed. The optimized oxidizing conditions allowed us to specifically oxidize the solvent accessible methionine and tryptophan residues that displayed significant oxidation in the real-time stability and elevated temperature study. The achieved degree of tryptophan oxidation was adequate to identify the functional consequence of the tryptophan oxidation by binding studies. In summary, the here presented approach of employing TBHP as oxidizing reagent combined with quantitative LC-MS and binding studies greatly facilitates the efficient identification and functional evaluation of methionine and tryptophan oxidation sites in the CDR of recombinant antibodies.

Peptide and Protein Delivery at a Glance

Peptide and Protein Delivery at a Glance

Identification of Racemization Sites Using Deuterium Labeling and Tandem Mass Spectrometry

Racemization of amino acids is a common chemical degradation pathway observed in biopharmaceuticals and is particularly prevalent in synthetic peptides. The identification of racemized amino acid residue(s) by mass spectrometry is particularly challenging due to isobaric mass between the isomeric forms. In this paper, we present a novel methodology combining stable deuterium labeling with collisionally induced dissociation-tandem mass spectrometry (CID-MS/MS) to elucidate racemized amino acid residues in immunoglobulin samples. Immunoglobulin G subclasses IgG1, IgG2, and IgG4 samples were first stressed in protonated or deuterated buffer (pH 8 or 9) at 40 or 50 degrees C storage for days or weeks. These forced degraded samples were reduced, S-carbamidomethylated, and digested with trypsin in protonated solution, and the tryptic digests were then analyzed via liquid chromatography/mass spectrometry (LC-MS) or sequenced via liquid chromatography/tandem mass spectrometry (LC-MS/MS) to detect racemized peptides and elucidate the location of racemized amino acid residues. The methodology successfully identified several racemized amino acid residues in the constant region of the heavy chains of the three IgG subclasses. Although the IgG subclasses have very similar primary protein sequences, our results interestingly indicated different racemization rates for specific amino acid residues.

Stability of optical and electroluminescence properties of a semiconducting polymer over a decade

Optical and electrical properties of conjugated polymers are often strongly affected by aging, not only during operation but also during storage, because of the variety of chemical degradation pathways available. Here we report the properties of a phenylated poly(arylene vinylene), after a decade of storage in powder form, in air, at room temperature, and in the dark. We find that the values for both the photoluminescence efficiency (60% or higher) and the electroluminescence are comparable with those reported for a similar but (then) freshly prepared batch of material, measured over 13 years ago. The optical properties of this material relevant for display applications are thus essentially stable over more than a decade for storage in the dark.

Origin and Propagation of an Incorrect Chemical Degradation Pathway in the Literature: cis-1,2-Dichloroethylene as a Daughter Product of 1,1,1-Trichloroethane

The correct understanding of contaminant degradation pathways is essential for well-informed decision processes regarding site assessment, monitored natural attenuation, and environmental liability proceedings. Over a 20-year period, and including at least one recent regulatory publication, it has been erroneously reported that cis-1,2-dichloroethylene (cis-DCE) is the daughter product of 1,1,1-trichloroethane (1,1,1-TCA). To clarify the degradation pathways of 1,1,1-TCA, and to corroborate that cis-DCE is not a degradation product of 1,1,1-TCA as suggested and propagated by some sources, we conducted a detailed review of the available literature and mapped out a citation history of 27 publications and reports which contain both the correct and incorrect degradation pathways. Other than the sources containing the erroneous degradation pathways, the reviewed literature shows that cis-DCE is not a proven transformation product of 1,1,1-TCA.

Stabilization of Human Growth Hormone Against Deamidation by Interaction with Strongly Basic Protamines from Salmon

Deamidation of proteins is probably one of the most common chemical degradation pathways in protein pharmaceuticals. [...]

The effect of sucrose hydrolysis on the stability of protein therapeutics during accelerated formulation studies

Stability studies of protein therapeutics are often accelerated by storing potential formulations at elevated temperatures where the rates of various chemical and physical degradation pathways are increased. An often overlooked caveat of using these studies is the potential degradation of the formulation components themselves. In this report, we show that the monoclonal antibody MAB001 aggregated at a faster rate when formulated with sucrose compared to samples that contained sorbitol or no excipient during accelerated stability studies following an initial lag phase where the rates of aggregate formation were similar in all formulations. The duration of the lag phase was both pH and temperature dependent and a significant increase of protein glycation was noticed during this time. These observations indicate that the enhanced rate of antibody aggregation in sucrose containing formulations is likely due to protein glycation following sucrose hydrolysis under accelerated conditions. This hypothesis was confirmed by demonstrating that antibody directly glycated with glucose aggregated at a faster rate than nonglycated antibody stored in the identical formulation. These findings question the utility of using accelerated stability data for predicting protein stability in sucrose containing formulations stored at 2–8°C, where no glycation or change in aggregation rate was observed. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:4501–4510, 2009

Evaluation of chemical degradation of a trivalent recombinant protein vaccine against botulinum neurotoxin by LysC peptide mapping and MALDI-TOF mass spectrometry

Vaccines utilizing recombinant protein antigens typically require an adjuvant to enhance immune response in the recipients. However, the consequences of antigen binding to adjuvant on both the short- and long-term stability of the protein remain poorly defined. In our companion paper (Vessely et al., in press, J Pharm Sci), we characterized the effects of binding to adjuvant on the conformation and thermodynamic stability of three antigen variants for botulinum vaccines: rBoNTA(Hc), rBoNTB(Hc), and rBoNTE(Hc). In the current study, we evaluated the effect of binding to adjuvant (Alhydrogel™, aluminum hydroxide) on chemical stability of these antigens during long-term storage in aqueous suspension. We developed methods that employ LysC peptide mapping in conjunction with MALDI-TOF mass spectrometry. Peptide maps were developed for the proteins for a vaccine formulation of rBoNTE(Hc) as well as a trivalent rBoNT(Hc) vaccine formulation. This method provided high sequence coverage for the proteins in part due to the implementation of a postdigestion elution fractionation method during sample preparation, and was also successfully utilized to evaluate the chemical integrity of adjuvant-bound rBoNT(Hc) protein antigens. We found that all three of the rBoNT(Hc) proteins were susceptible to degradation via both oxidation and deamidation. In many cases, such reactions occurred earlier with the adjuvant-bound protein formulations when compared to the proteins in control samples that were not bound to adjuvant. Additionally, some chemical modifications were found in the adjuvant-bound protein formulations but were not detected in the unbound solution controls. Our studies indicate that binding to aluminum-based adjuvants can impact the chemical stability and/or the chemical degradation pathways of protein during long-term storage in aqueous suspension. Furthermore, the methods we developed should be widely useful for assessing chemical stability of adjuvant-bound recombinant protein antigens. © 2008 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:2994–3012, 2009

Degradation of HTL layers during device operation in PhOLEDs

Different analytical tools and methodologies are currently employed to determine degradation products of organic blue light emitting devices in order to identify the failure mechanisms which determine the lifetime of these devices. This article provides a deeper understanding of degradation mechanisms of organic light emitting diodes (OLEDs) during device operation. Degradation products of blue emitting devices containing 8% of the phosphorescent emitter iridium(III)bis(4,6-difluorophenyl)-pyridinato-N,C 2′ picolinate (FIrpic) in a matrix containing bis(2-methyl-8-quinolinolato)(4-phenylphenolato)aluminium (BAlq) as electron transport layer (ETL), 4,4′,4″-tri( N-carbazolyl)triphenylamine (TCTA) and N, N′-diphenyl- N, N′-bis(1-naphthyl)-1,1′-biphenyl-4,4″-diamine (α-NPD) were investigated using laser desorption ionization (LDI) coupled with a time of flight mass spectrometry (TOF/MS). Especially chemical degradation pathways of the hole transport materials TCTA and α-NPD were investigated. The comparison of experimental data of unstressed and stressed device revealed that new reaction products are formed during the device operation. The linkage of TCTA fragments to the α-NPD core in an interfacial reaction as well as a dimerization of TCTA itself was observed. Ten new reaction products could be characterized via LDI-TOF-MS. Some of these compounds might possess a negative influence on the drop of efficiency and lifetime of blue light emitting devices based on FIrpic.

The effects of sucrose on stability of human brain natriuretic peptide [hBNP (1–32)] and human parathyroid hormone [hPTH (1–34)

Although the effect of sucrose on the physical stability of proteins has been well documented, its impact on their chemical stability is largely unknown. The aim of this study was to investigate the potential effects of sucrose on the structural conformation of human brain natriuretic peptide [hBNP (1-32)] and the synthetic human parathyroid hormone [hPTH (1-34)], and link these effects to chemical degradation pathways of these peptides. The stability of hBNP (1-32) and hPTH (1-34) was studied at pH 5.5. Aggregation was monitored using size exclusion high-performance liquid chromatography (SE-HPLC), whereas oxidation and deamidation products were measured by reversed phase (RP) HPLC. Fourier transform infrared (FT-IR) spectroscopy was used to study the peptides' conformation. Sucrose retarded aggregation, deamidation, and oxidation of hBNP (1-32) and hPTH (1-34), with a maximum effect at relatively high concentrations (as much as 1 m). FT-IR spectroscopy indicated that sucrose maintained the native conformation of hBNP (1-32) and induced small conformation changes in the hPTH (1-34) structure. Sucrose enhanced the stability of hBNP (1-32) and hPTH (1-34) in liquid formulations. The stabilizing effect of sucrose was due to a large extent to retardation of oxidation and deamidation of hBNP (1-32) and hPTH (1-34).

Prediction of CL-20 chemical degradation pathways, theoretical and experimental evidence for dependence on competing modes of reaction

Highest occupied and lowest unoccupied molecular orbital energies, formation energies, bond lengths and FTIR spectra all suggest competing CL-20 degradation mechanisms. This second of two studies investigates recalcitrant, toxic, aromatic CL-20 intermediates that absorb from 370 to 430 nm. Our earlier study (Struct. Chem., 15, 2004) revealed that these intermediates were formed at high OH− concentrations via the chemically preferred pathway of breaking the C–C bond between the two cyclopentanes, thereby eliminating nitro groups, forming conjugated π bonds, and resulting in a pyrazine three-ring aromatic intermediate. In attempting to find and make dominant a more benign CL-20 transformation pathway, this current research validates hydroxylation results from both studies and examines CL-20 transformations via photo-induced free radical reactions. This article discusses CL-20 competing modes of degradation revealed through: computational calculation; UV/VIS and SF spectroscopy following alkaline hydrolysis; and photochemical irradiation to degrade CL-20 and its byproducts at their respective wavelengths of maximum absorption.

Micro-tensile bond strength of adhesives bonded to class-I cavity-bottom dentin after thermo-cycling

A widely used artificial aging methodology is thermo-cycling. The ISO TR 11450 standard (1994) recommends 500 cycles in water between 5 and 55 degrees C. Recent literature revealed that more cycles are needed to mimic long-term bonding effectiveness. Furthermore, the artificial aging effect induced by thermo-cycling is not clearly established. Two underlying mechanisms can be advanced: (1) hot water may accelerate hydrolysis and elution of interface components and (2) repetitive contraction/expansion stress can be generated. The purpose of this study was to evaluate the relative contribution of both chemical (hydrolysis and elution of interface components) and mechanical (repetitive contraction/expansion stress) degradation pathways on the thermo-cycling-induced artificial aging of dentin-adhesive interfaces at the bottom of class-I cavities. The micro-tensile bond strength (muTBS) of contemporary adhesives (a three-step etch and rinse, a two-step and a one-step self-etch adhesive) bonded to class-I cavity-bottom dentin was determined after 20,000 cycles as well as after 20 days of water storage (control). Restored class-I cavities (repetitive contraction/expansion stress) as well as prepared micro-specimens (diffusion-dependent hydrolysis and elution) were subjected to the thermo-cycling regimen. Thermo-cycling did not enhance chemical or mechanical degradation of the bonds produced by a two-step self-etch and a three-step etch and rinse adhesive to dentin. The one-step self-etch adhesive tested was, however, not able to withstand polymerization shrinkage stress, nor thermo-cycling, when applied in class-I cavities. Thermo-cycling results in combined contraction/expansion stress and accelerated chemical degradation. However, the relative contribution of each is strongly dependent on the specific test set-up and the adhesive used.

Effect of jet nebulization on DNA: identifying the dominant degradation mechanism and mitigation methods

Jet nebulization is a common technique by which gene therapies can be delivered to the lungs, however current research has shown a reduction in activity and effectiveness of these treatments when not complexed with cationic agents. To date, a systematic investigation of chemical and mechanical degradation pathways has yet to be undertaken to identify the proximate cause of DNA instability. The purpose of this paper is to identify the dominant chemical and/or mechanical mechanism(s) by which DNA degrades when subjected to jet nebulization. Investigation of multiple degradation pathways revealed hydrodynamic shear to be primarily responsible for the observed loss in DNA integrity. Complexation of DNA with cationic agents polyethyleneimine or poly- L-lysine to DNA was sufficient to overcome these mechanical forces via a reduction in applied hydrodynamic forces.

Rational design of stable lyophilized protein formulations: theory and practice.

For ease of preparation and cost containment by the manufacturer, and ease of handling by the end user, an aqueous therapeutic protein formulation usually is preferred. However, with many proteins it is not possible—especially considering the time constraints for product development—to develop sufficiently stable aqueous formulations. Unacceptable denaturation and aggregation can be induced readily by the numerous stresses to which a protein in aqueous solution is sensitive; e.g., heating, agitation, freezing, pH changes, and exposure to interfaces or denaturants (Arakawa et al., 1993; Cleland et al., 1993; Brange, 2000; Bummer and Koppenol, 2000). Furthermore, even under conditions that thermodynamically greatly favor the native state of proteins, aggregation can arise during months of storage in aqueous solution (e.g., Gu et al., 1991; Arakawa et al., 1993; Chen et al., 1994; Chen et al., 1994; Chang et al., 1996a). In addition, several chemical degradation pathways (e.g., hydrolysis and deamidation) are mediated by water. In aqueous formulations, the rates of these and other (e.g., oxidation) chemical degradation reactions can be unacceptably rapid on the time scale of storage (e.g., 18–24 months) for pharmaceutical products (Manning et al., 1989; Cleland et al., 1993; Goolcharran et al., 2000; Bummer and Koppenol, 2000).

Effect of moisture on the stability of a lyophilized humanized monoclonal antibody formulation.

To determine the effect of moisture and the role of the glass transition temperature (Tg) on the stability of a high concentration, lyophilized, monoclonal antibody. A humanized monoclonal antibody was lyophilized in a sucrose/histidine/polysorbate 20 formulation. Residual moistures were from 1 to 8%. Tg values were measured by modulated DSC. Vials were stored at temperatures from 5 to 50 degrees C for 6 or 12 months. Aggregation was monitored by size exclusion chromatography and Asp isomerization by hydrophobic interaction chromatography. Changes in secondary structure were monitored by Fourier transform infrared (FTIR). T. values varied from 80 degrees C at 1% moisture to 25 degrees C at 8% moisture, there was no cake collapse and were no differences in the secondary structure by FTIR. All formulations were stable at 5 degrees C. High moisture cakes had higher aggregation rates than drier samples if stored above their Tg values. Intermediate moisture vials were more stable to aggregation than dry vials. High moisture samples had increased rates of Asp isomerization at elevated temperatures both above and below their Tg values. Chemical and physical degradation pathways followed Arrhenius kinetics during storage in the glassy state. Only Asp isomerization followed the Arrhenius model above the Tg value. Both chemical and physical stability at T > or = Tg were fitted to Williams-Landel-Ferry (WLF) kinetics. The WLF constants were dependent on the nature of the degradation system and were not characteristic of the solid system. High moisture levels decreased chemical stability of the formulation regardless of whether the protein was in a glassy or rubbery state. In contrast, physical stability was not compromised, and may even be enhanced, by increasing residual moisture if storage is below the Tg value.

The Excluding Effects of Sucrose on a Protein Chemical Degradation Pathway: Methionine Oxidation in Subtilisin

The conformational stabilization of proteins by sucrose has been previously attributed to a preferential exclusion mechanism. The present study links this mechanism to stability against a chemical degradation pathway for subtilisin. Oxidation of a methionine residue adjacent to the active site to the sulfoxide form compromises subtilisin's enzymatic activity. In the presence of hydrogen peroxide and borate buffer, a borate–hydrogen peroxide complex binds to subtilisin's active site prior to the formation of methionine sulfoxide. Sucrose decreases the oxidation rate by limiting the accessibility of the complex to the methionine at the partially buried active site. The stabilization mechanism of sucrose is based on shifting the equilibrium of transiently expanding native conformations of subtilisin to favor the most compact states. Enzymatic parameter determination (kcat, KM) and hydrogen–deuterium exchange measurements confirm the limited conformational mobility of the enzyme in the presence of sucrose. Further support for limited mobility as the cause of oxidation inhibition by sucrose comes from the findings that neither viscosity nor possible interactions of sucrose with hydrogen peroxide, hydroxyl radicals, or borate can adequately explain the inhibition. The volume exclusion of sucrose from subtilisin is used to estimate the extent by which the native state of subtilisin must expand in solution to allow oxidation. The surface area of the oxidation-competent state is ca. 3.9% greater than that of the native state.

Degradation mechanism and kinetic studies of a novel anticancer agent, AG2034

AG2034 is an anticancer drug designed using protein structure-based techniques. As demonstrated in preformulation studies [ Wang et al., 1997. Preformulation studies of a novel glycinamide ribonucleotide formyl transferase inhibitor, AG2034 (in preparation)], hydrolysis and oxidation were the two major degradation pathways for AG2034. AG2034 was initially formulated as a lyophilized powder for injection for parenteral administration. The hydrolysis was an unusual problem because of the erratic degradation kinetics. Further investigation in this study suggested that the hydrolysis of AG2034 was not through chemical degradation pathway, rather it was enzymatic—caused by the microbial growth in the solution. The enzymatic mechanism was supported by the observations that hydrolysis was dependent on temperature and pH, and could be effectively inhibited by the presence of EDTA, some preservatives, as well as by aseptic filtration. Due to the sensitivity of AG2034 to enzymatic degradation, sterility was crucial in maintaining the solution stability. Oxidation was the other degradation pathway which was found to be more significant at higher pH range. The pH-oxidation profile was studied in the pH range of 6–9, and the results indicated that the optimization of pH alone could not effectively minimize oxidation rate and result in a sufficiently stable liquid formulation. Consequently, a number of antioxidants and chelating agent EDTA were evaluated. It was observed that the effect of EDTA on oxidation was minimal. Sodium thiosulfate could slightly stabilize AG2034 against oxidation, while monothioglycerol, sodium sulfite and cysteine had no positive effects. Oxidation of AG2034 was shown to be sensitive to the presence of oxygen. The replacement of headspace gas with nitrogen significantly reduced the amount of oxidation product formation. Based on the kinetic study, it was estimated that liquid formulation could be stabilized with the headspace nitrogen control and the use of appropriate antioxidant.