Biophysical insights on the interaction of anticoagulant drug dicoumarol with calf thymus-DNA: deciphering the binding mode and binding force with thermodynamics.

The biological activity of drugs is exhibited due to their interactions with bio-receptors. Dicoumarol (DIC) is a natural hydroxycoumarin and a well-known anticoagulant. DNA is the genetic material and one of the targets of numerous drugs. The interaction of DIC with calf-thymus DNA (ct-DNA) has been studied using different biophysical techniques and docking studies. The binding constant in the order of 103 to 104 M-1 was observed from spectroscopic studies. Thermodynamic studies at 4 different temperatures revealed the spontaneity of the interaction with the entropy-driven process. Marker displacement studies with competitive markers of intercalators (ethidium bromide) and groove binders (Hoechst 33258) confirmed the groove-binding nature of DIC in DNA. The groove-binding mode of DIC was complemented by different studies like viscosity measurements, DNA melting, and the effect of KI on the binding. A minor perturbation in the DNA viscosity and no significant change in the DNA melting temperature (Tm) after binding with DIC further confirms the groove binding mode. The effect of KI on the DIC and DIC-DNA system suggested the absence of DIC intercalation. The absence of significant electrostatic force was revealed from the ionic-strength effect study. Binding-induced conformational variation in ct-DNA was absent in circular dichroism studies. Molecular docking studies suggested the position of DIC within the minor groove of ct-DNA, covering three base pairs long. The outcome of this report may help in understanding the pharmacodynamics and pharmacokinetics of dicoumarol analogs and related molecules.Communicated by Ramaswamy H. Sarma.

Analysis of Genetic Diversity of cytb gene from Babesia gibsoni Isolates from Naturally Infected Dogs in Karnataka, India.

PURPOSE: The study aimed to investigate genetic diversity in Babesia gibsoni, the causative agent of canine babesiosis, and to assess the presence of atovaquone-resistant isolates in naturally infected dogs. METHODS: A total of 24 blood samples confirmed for B. gibsoni infection was subjected to PCR amplification and sequencing based on cytb gene. Genetic characterization of B. gibsoni as well as attempts to detect the point mutation rendering atovaquone resistance was carried out based on the analysis of nucleotide sequence of cytb gene using bioinformatics software. RESULTS: The findings indicated that the B. gibsoni isolates in the investigation exhibited a high nucleotide identity with the Asian genotype, ranging from 98.41 to 98.69%. Notably, none of the isolates carried cytb gene variants associated with atovaquone resistance. Phylogenetic analysis revealed clustering of most isolates with those from Japan and China, except for one isolate forming a distinct subclade. Haplotype network analysis indicated a high diversity with 22 distinct haplotypes among the B. gibsoni isolates, emphasizing the genetic variability within the studied population. CONCLUSION: In conclusion, the cytb gene exhibited remarkable conservation among the twenty-four B. gibsoni isolates studied and the study represents the first genetic diversity assessment of B. gibsoni using the cytb gene in dogs from India. These findings shed light on the genetic characteristics of B. gibsoni in the region and provide valuable insight for addressing the challenges posed by this life-threatening disease in dogs.

Binding of dicoumarol analog with DNA and its antioxidant studies: A biophysical insight by in-vitro and in-silico approaches.

DNA is the major target for a number of pharmaceutical drugs. The interaction of drug molecules with DNA plays a major role in pharmacokinetics and pharmacodynamics. Bis-coumarin derivatives have diverse biological properties. Here, we have explored the antioxidant activity of 3,3'-Carbonylbis (7-diethylamino coumarin) (CDC) using DPPH, H2O2, and superoxide scavenging studies followed by its binding mode in calf thymus-DNA (CT-DNA) using several biophysical methods including molecular docking. CDC exhibited comparable antioxidant activity to standard ascorbic acid. The UV-Visible and fluorescence spectral variations indicate the CDC-DNA complex formation. The binding constant in the range of 104 M-1 was obtained from spectroscopic studies at room temperature. The fluorescence quenching of CDC by CT-DNA suggested a quenching constant (KSV) of 103 to 104 M-1 order. Thermodynamic studies at 303, 308, and 318 K revealed the observed quenching as a dynamic process besides the spontaneity of the interaction with negative free energy change. Competitive binding studies with site markers like ethidium bromide, methylene blue, and Hoechst 33258 reflect CDC's groove mode of interaction. The result was complemented by DNA melting study, viscosity measurement, and KI quenching studies. The ionic strength effect was studied to interpret the electrostatic interaction and found its insignificant role in the binding. Molecular docking studies suggested the binding location of CDC within the minor groove of CT-DNA, complementing the experimental result.

Chitosan-coated and thymol-loaded polymeric semi-interpenetrating hydrogels: An effective platform for bioactive molecule delivery and bone regeneration in vivo.

Thymol (2-isopropyl-5-methylphenol; Thy) is a monoterpene phenolic phytocompound with medicinal properties; however, its impact on osteogenesis is yet to be thoroughly investigated. Its distribution is often hampered because of its intricate hydrophobic structure, which reduces its bioavailability. In this study, we synthesized a drug delivery vehicle using semi-interpenetrating polymer network (SIPN) hydrogels containing sodium alginate and poly(2-ethyl-2-oxazoline) (SA/Pox) loaded with Thy at varying concentrations (100, 150, and 200 µM). Subsequently, they were coated with chitosan (CS) to increase bioactivity and for sustained and prolonged release of Thy. Thy-loaded CS-coated SIPN hydrogels (SA/Pox/CS-Thy) were developed using ionic gelation and polyelectrolyte-complexation techniques. The addition of CS to hydrogels enhanced their physicochemical and material properties. These hydrogels were cytofriendly toward mouse mesenchymal stem cells (mMSCs). When mMSCs were cultured on hydrogels, Thy stimulated osteoblastic differentiation, as evidenced by calcium deposits at the cellular level. The expression of RUNX2, a key bone transcriptional factor, and other differentiation biomarkers was significantly enhanced in mMSCs cultured on SA/Pox/CS-Thy hydrogels. Notably, Thy in the SA/Pox/CS hydrogels significantly activated the TGF-ß/BMP signaling pathway, which is involved in osteogenesis. A rat tibial bone defect model system revealed that the incorporation of Thy into SA/Pox/CS hydrogels augmented bone regeneration. Thus, sustained and prolonged release of Thy from the SA/Pox/CS hydrogels promoted osteoblast differentiation in vitro and bone formation in vivo. These findings shed light on the effect of Thy bioavailability in fostering osteoblast differentiation and its prospective application in bone rejuvenation.

DNA binding studies of antifungal drug posaconazole using spectroscopic and molecular docking methods.

The binding studies of DNA with small molecules have been an emerging field of research all the time since DNA as the genetic material is a major biological target for various drugs. Interpretation of small molecule-DNA binding helps in understanding their interactions with designing new drugs of greater medicinal activity. Posaconazole is an antifungal drug in the class of triazoles which are known to possess numerous pharmacological properties. In this work, the nature of the binding of posaconazole with calf-thymus DNA has been studied using spectroscopic techniques and molecular docking studies. A binding constant of the order of 103 M-1 was observed from UV-visible and fluorescence studies for the interaction between posaconazole and calf-thymus DNA. The fluorescence property of posaconazole was found to be quenched by calf-thymus DNA with a quenching constant of the order of 103 M-1. Competitive displacement of ethidium bromide and Hoechst 33258 by posaconazole using fluorescence technique suggested minor groove binding of posaconazole in calf-thymus DNA. Confirmation of the binding mode was further complemented by the viscosity measurement and DNA melting studies followed by KI quenching experiments. The studies on the effect of ionic strength on the binding suggested a possible role of electrostatic force in the interaction. Molecular docking studies reflected a crescent shape of the posaconazole within the minor groove of calf-thymus DNA validating the experimental findings showing the residues involved in the interaction.

In-Process Vapor Composition Monitoring in Application to Lyophilization of Ammonium Salt Formulations.

Quality control is of critical importance in manufacturing of lyophilized drug product, which is accomplished by monitoring the process parameters. The residual gas analyzer has emerged as a useful tool in determination of endpoint for primary and secondary drying in lyophilization process as well as leak detection in vacuum systems. This study presents the application of in situ RGA to quantify outgassing rates of species released from aqueous inorganic and organic ammonium salt formulations throughout the freeze-drying process. The determination of ammonia outgassing conditions aids in ensuring product quality where ammonia release is an indication for loss of co-solvent or degradation of active pharmaceutical ingredients (APIs). Data analysis methods are developed to determine ammonia presence under various process conditions. In-situ real time monitoring of vapor dynamics enables RGA to be used as a tool to characterize counter-ion loss throughout the freeze-drying cycle.

Orsellinic acid-loaded chitosan nanoparticles in gelatin/nanohydroxyapatite scaffolds for bone formation in vitro.

AIM: Orsellinic acid (2,4-Dimethoxy-6-methylbenzoic acid) (OA) is a hydrophobic polyphenolic compound with therapeutic potential, but its impact on actuating osteogenesis remains unknown. The bioavailability of OA is hampered by its hydrophobic nature. This study aimed to fabricate nano-drug delivery system-based scaffolds for OA and test its potential for osteogenesis in vitro. MATERIALS AND METHODS: OA was loaded into chitosan nanoparticles (nCS + OA) using the ionic gelation technique at different concentrations. nCS + OA were incorporated onto the scaffolds containing gelatin (Gel) and nanohydroxyapatite (nHAp) by the lyophilization method. Biocomposite scaffolds were examined for their physicochemical and material characteristic properties. The effect of OA in the scaffolds for osteoblast differentiation was determined by alizarin red and von Kossa staining at the cellular level and by reverse transcriptase-qPCR and western blot analysis at the molecular level. KEY FINDINGS: The scaffolds showed excellent physiochemical and material characteristics and remained cyto-friendly to mouse mesenchymal stem cells (mMSCs, C3H10T1/2). The release of OA from Gel/nHAp/nCS scaffolds enhanced the differentiation of mMSCs towards osteoblasts, as observed through cellular and molecular studies. Moreover, the osteogenic potential of OA was mediated by the activation of FAK and ERK signaling pathways through integrins. SIGNIFICANCE: The inclusion of OA into Gel/nHAp/nCS biocomposite scaffolds at 80 µM concentration promoted osteoblast differentiation via cell adhesion mediated signaling, compared with that shown by Gel/nHAp/nCS alone. Overall, this study identified the potential therapeutic OA containing Gel/nHAp/nCS scaffolds, accelerating its potential for clinical application towards bone regeneration.

Chitosan-based 3D-printed scaffolds for bone tissue engineering.

Despite the spontaneous regenerative properties of autologous bone grafts, this technique remains dilatory and restricted to fractures and injuries. Conventional grafting strategies used to treat bone tissue damage have several limitations. This highlights the need for novel approaches to overcome the persisting challenges. Tissue-like constructs that can mimic natural bone structurally and functionally represent a promising strategy. Bone tissue engineering (BTE) is an approach used to develop bioengineered bone with subtle architecture. BTE utilizes biomaterials to accommodate cells and deliver signaling molecules required for bone rejuvenation. Among the various techniques available for scaffold creation, 3D-printing technology is considered to be a superior technique as it enables the design of functional scaffolds with well-defined customizable properties. Among the biomaterials obtained from natural, synthetic, or ceramic origins, naturally derived chitosan (CS) polymers are promising candidates for fabricating reliable tissue constructs. In this review, the physicochemical-biological properties and applications of CS-based 3D-printed scaffolds and their future perspectives in BTE are summarized.

Evaluation of recombinant BgSA3 protein based indirect-ELISA for sero-diagnosis and sero-surveillance of Babesia gibsoni in dogs.

Canine babesiosis, a tick-borne haemoprotozoan disease of dogs, is of significance globally due to its rapid spread. A precise confirmatory diagnosis is required to curtail the rapid spread of infection. Our study described the evaluation of recombinant BgSA3 protein based indirect ELISA for sero-diagnosis and sero-surveillance of Babesia gibsoni infection in dogs. A partial BgSA3 gene segment (1921 bp) of B. gibsoni, encoding for recombinant truncated BgSA3 (75 kDa) protein devoid of predicted signal peptide (23 aa) at N-terminus and transmembrane region (20 aa) at C-terminus, was expressed in E. coli using a pET28a(+) vector. The rBgSA3 protein purified under native conditions using Ni-NTA superflow cartridge was confirmed by SDS-PAGE and Western blotting using sera from dogs infected/uninfected with B. gibsoni, and erythrocyte lysate/ plasma from infected/uninfected dogs. The rBgSA3 protein was specific only to B. gibsoni antibodies but did not react with uninfected sera. Further, rBgSA3 protein was evaluated for sero-diagnosis/sero-surveillance using Indirect-ELISA format. There was no cross reactivity to B. vogeli, E. canis, H. canis and D. repens infected dogs serum samples. The diagnostic sensitivity and specificity of rBgSA3 based I-ELISA was found to be 86.4 and 93.1 % respectively, in comparison with cytb based PCR assay. Additionally, rBgSA3-ELISA evaluated using survey serum samples (n = 287), detected 11.85 % samples as positive. In conclusion, B. gibsoni infection, an emerging disease is prevalent in the present study area and the standardized rBgSA3 protein based indirect-ELISA was found to be a specific and sensitive test for large scale sero-diagnosis and sero-surveillance of B. gibsoni infection in dogs.

An insight into cell-laden 3D-printed constructs for bone tissue engineering.

Escalating bone graft scarcity and donor site morbidity worldwide are alarming reminders that highlight the need for alternatives to gold standard tissue rejuvenation methods. Over the last few decades, many efforts have been made in bone tissue engineering (BTE) to fabricate artificial bone transplants. Conventional BTE techniques do not render pertinent spatial organization of cells, and they fail in mimicking the extracellular matrix of native bone tissue. This setback can be overcome by using the emerging technology of three-dimensional bioprinting (3DBP). 3DBP is a state-of-the-art technology that provides accurate hierarchal biomaterial structures that accommodate live-cell patterning to mimic their native counterparts. Herein, we provide an overview on the recent progress of cell-laden 3DBP technologies and also discuss the various biomaterials utilized (natural polymers such as chitosan, collagen, gelatin, hyaluronic acid, and silk fibroin and synthetic polymers such as PCL, PVP, and ceramics) to engineer scaffolds with requisite structural, mechanical, and biological complexity. We also highlight some of the persisting challenges and the solutions to surmount them, paving the way for progress in the field. Finally, we discuss how the combination of novel modalities with 3DBP can pave the way for new frontiers, like four-dimensional bioprinting (4DBP), to bring customized, stimuli-responsive, and highly effective regenerative scaffolds to bone tissue engineering.

Temperature- and pH-responsive chitosan-based injectable hydrogels for bone tissue engineering.

Spontaneous bone regeneration is heavily restricted because of bone defects, and external mediation is required to enhance repair and regeneration. Bone tissue engineering (BTE) is a multidisciplinary field that offers promising substitutes to traditional methods-namely, autografts, allografts, and xenografts. Amidst the various scaffolds for BTE applications, it has been demonstrated that hydrogels are promising templates for bone regeneration owing to their similarities to the natural extracellular matrix. Regardless of the development of a variety of biomaterials, chitosan (CS) as a natural biopolymer has drawn tremendous attention in recent years for its use as a valuable graft material to form thermo/pH-responsive injectable hydrogels. Formulations of CS-based injectable hydrogels are advantageous in terms of their high-water imbibing capability, minimal invasiveness, porous networks, and ability to mold perfectly into an irregular defect. In this review, the physicochemical properties and applications of thermo/pH-responsive CS-based hydrogels and their future perspectives in BTE are briefly outlined.

An Unusual Cause of Failed Tracheal Decannulation-A Case Report.

Failure of decannulation may occur due to unexpected upper airway problems. However, the presence of a membrane in between the vocal cords is usually rare. We report a case of a 46-year-old female, who presented with focal seizures and progressed to status epilepticus. She was put on a mechanical ventilator because of hypoxic arrest. As she required prolonged ventilatory support, tracheostomy and gradual weaning from ventilator support to T-piece was done. Following stable hemodynamics, decannulation trial was attempted which failed. Subsequently, bronchoscopy was done to assess the upper airway. It revealed a thick membrane in between the vocal cords. Further examination with an indirect laryngoscope under general anesthesia confirmed the findings, and the membrance was excised. Decannulation was successful the very following day and the patient was discharged with stable hemodynamics. HOW TO CITE THIS ARTICLE: Kambhampati S, Lavanya K. An Unusual Cause of Failed Tracheal Decannulation-A Case Report. Indian J Crit Care Med 2019;23(8):378-379.

Pulse Proteolysis: An Orthogonal Tool for Protein Formulation Screening.

Protein formulation stability is difficult to predict a priori and generally involves long-term stability studies. It is of interest to develop an analytical method that can predict stability trends reliably. Here, pulse proteolysis was evaluated as an analytical tool to predict solution-state stability in different formulations. Four proteins formulated in different buffer and excipient compositions were subjected to urea-induced unfolding and brief enzymatic digestion ("pulse" proteolysis), and relative resistance to proteolysis was measured by microfluidics-based capillary electrophoresis-sodium dodecyl sulfate. Biophysical properties of each formulation were measured using orthogonal biophysical techniques such as differential scanning fluorimetry, differential scanning calorimetry, dynamic light scattering, circular dichroism, and fluorescence spectroscopy. Protein stability in all formulations was monitored by size exclusion chromatography on storage at 5°C and 40°C. For all 4 proteins, formulations with greater proteolytic resistance also showed higher monomer content on thermal stability. In contrast, standard biophysical techniques showed reasonable-to-no correlation with size exclusion chromatography data. The data support the use of pulse proteolysis as an orthogonal, quantitative, and predictive tool to measure protein conformational stability and rank-order formulations.

Comparison of imaged capillary isoelectric focusing and cation exchange chromatography for monitoring dextrose-mediated glycation of monoclonal antibodies in infusion solutions.

Intravenous (IV) infusion of therapeutic proteins typically involves dilution of the formulated product into infusion media such as normal saline or dextrose, 5% m/v in water. We report results from a rigorous evaluation of imaged capillary isoelectric focusing (iCIEF) for monitoring dextrose-mediated glycation of proteins in IV infusion solutions. In addition to detecting stable Amadori glycation products, iCIEF was able to detect the labile Schiff base (SB) glycation adducts since the equilibrium with free dextrose is maintained on capillary. Method parameters such as sample dilution factor and ampholyte composition (but not urea) were found to influence the observed level of SB glycation adducts. The impacts of dextrose and urea on the apparent pI values are also reported. iCIEF results were compared with results from cation exchange chromatography, which was found to preferentially detect the more stable Amadori glycation products due to the on-column decomposition of the SB adducts resulting from the separation of the protein from free dextrose which in turn altered the SB adduct- free dextrose equilibrium. These results demonstrate the need for careful consideration when selecting the analytical methodology to investigate protein sensitivity to dextrose and to monitor protein stability in dextrose-containing infusion solutions.

Novel self-assembling conjugates as vectors for agrochemical delivery.

BACKGROUND: Modern agricultural practises rely on surfactant-based spray applications to eliminate weeds in crops. The wide spread and indiscriminate use of surfactants may result in a number of deleterious effects that are not limited to impacts on the crop and surrounding farm eco-system but include effects on human health. To provide a safer alternative to the use of surfactant-based formulations, we have synthesised a novel, self-assembling herbicide conjugate for the delivery of a broad leaf herbicide, picloram. RESULTS: The synthesized self-assembling amphiphile-picloram (SAP) conjugate has three extending arms: a lipophilic lauryl chain, a hydrophilic polyethylene glycol chain and the amphiphobic agrochemical active picloram. We propose that the SAP conjugate maintains its colloidal stability by quickly transitioning between micellar and inverse micellar phases in hydrophilic and lipophilic environments respectively. The SAP conjugate provides the advantage of a phase structure that enables enhanced interaction with the hydrophobic epicuticular wax surface of the leaf. We have investigated the herbicidal efficiency of the SAP conjugate compared against that of commercial picloram formulations using the model plant Arabidopsis thaliana and found that when tested at agriculturally relevant doses between 0.58 and 11.70 mM a dose-dependent herbicidal effect with comparable kill rates was evident. CONCLUSION: Though self-assembling drug carriers are not new to the pharmaceutical industry their use for the delivery of agrochemicals shows great promise but is largely unexplored. We have shown that SAP may be used as an alternative to current surfactant-based agrochemical formulations and has the potential to shift present practises towards a more sustainable approach.

Contracaecum rudolphii Hartwich, 1964 (Nematoda: Anisakidae) in a wild spot-billed pelican (Pelecanus philippensis): a case report.

Post mortem examination of a spot-billed pelican (Pelecanus philippensis) revealed the presence of numerous anisakid nematodes in the proventriculus. The nematodes were processed in lactophenol as temporary mounts and were identified as Contracaecum rudolphii Hartwich, 1964 based on micrometry and morphological description of the anterior end, the sinuous uterus with sub globular thick-shelled eggs in female parasite and the tips of the spicules in male parasite. This is the first record of C. rudolphii in a wild spot-billed pelican from Andhra Pradesh, India.

Influence of Decontaminating Agents and Swipe Materials on Laboratory Simulated Working Surfaces Wet Spilled with Sodium Pertechnetate.

CONTEXT: Decontamination of various working surfaces with sodium pertechnetate minor spillage is essential for maintaining good radiation safety practices as well as for regulatory compliance. AIM: To observe the influences of decontaminating agents and swipe materials on different type of surfaces used in nuclear medicine laboratory work area wet spilled with 99m-technetium (99mTc) sodium pertechnetate. SETTINGS AND DESIGN: Lab-simulated working surface materials. Experimental study design. MATERIALS AND METHODS: Direct decontamination method on dust-free lab simulated new working surfaces [stainless steel, polyvinyl chloride (PVC), Perspex, resin] using four decontaminating agents [tap water, soap water (SW), Radiacwash, and spirit] with four different swipe material [cotton, tissue paper (TP), Whatman paper (WP), adsorbent sheet (AS)] was taken 10 samples (n = 10) for each group. STATISTICAL ANALYSIS: Parametric test two-way analysis of variance is used with significance level of 0.005, was used to evaluate statistical differences between different group of decontaminating agent and swipe material, and the results are expressed in mean ± SD. RESULTS: Decontamination factor is calculated after five cleaning for each group. A total of 160 samples result calculated using four decontaminating agent (tap water, SW, Radiacwash, and spirit), four swipe material (cotton, TP, WP, and AS) for commonly used surface (stainless steel, PVC, Perspex, resin) using direct method by 10 samples (n = 10) for each group. CONCLUSIONS: Tap water is the best decontaminating agent compared with SW, Radiac wash and spirit for the laboratory simulated stainless steel, PVC, and Perspex surface material, whereas in case of resin surface material, SW decontaminating agent is showing better effectiveness. Cotton is the best swipe material compared to WP-1, AS and TP for the stainless steel, PVC, Perspex, and resin laboratory simulated surface materials. Perspex and stainless steel are the most suitable and recommended laboratory surface material compared to PVC and resin in nuclear medicine. Radiacwash may show better result for 99mTc labelled product and other radionuclide contamination on the laboratory working surface area.

Process and Formulation Effects on Protein Structure in Lyophilized Solids Using Mass Spectrometric Methods.

Myoglobin (Mb) was lyophilized in the absence (Mb-A) and presence (Mb-B) of sucrose in a pilot-scale lyophilizer with or without controlled ice nucleation. Cake morphology was characterized using scanning electron microscopy, and changes in protein structure were monitored using solid-state Fourier-transform infrared spectroscopy, solid-state hydrogen-deuterium exchange-mass spectrometry, and solid-state photolytic labeling-mass spectrometry (ssPL-MS). The results showed greater variability in nucleation temperature and irregular cake structure for formulations lyophilized without controlled nucleation. Controlled nucleation resulted in nucleation at ∼(-5°C) and uniform cake structure. Formulations containing sucrose showed better retention of protein structure by all measures than formulations without sucrose. Samples lyophilized with and without controlled nucleation were similar by most measures of protein structure. However, ssPL-MS showed the greatest photoleucine incorporation and more labeled regions for Mb-B lyophilized with controlled nucleation. The data support the use of solid-state hydrogen-deuterium exchange-mass spectrometry and ssPL-MS to study formulation and process-induced conformational changes in lyophilized proteins.

Characterizing Protein Structure, Dynamics and Conformation in Lyophilized Solids.

The long-term stability of protein therapeutics in the solid-state depends on the preservation of native structure during lyophilization and in the lyophilized powder. Proteins can reversibly or irreversibly unfold upon lyophilization, acquiring conformations susceptible to degradation during storage. Therefore, characterizing proteins in the dried state is crucial for the design of safe and efficacious formulations. This review summarizes the basic principles and applications of the analytical techniques that are commonly used to characterize protein structure, dynamics and conformation in lyophilized solids. The review also discusses the applications of recently developed mass spectrometry based methods (solid-state hydrogen deuterium exchange mass spectrometry (ssHDX-MS) and solid-state photolytic labeling mass spectrometry (ssPL-MS)) and their ability to study proteins in the solid-state at high resolution.

Photolytic Cross-Linking to Probe Protein-Protein and Protein-Matrix Interactions in Lyophilized Powders.

Protein structure and local environment in lyophilized formulations were probed using high-resolution solid-state photolytic cross-linking with mass spectrometric analysis (ssPC-MS). In order to characterize structure and microenvironment, protein-protein, protein-excipient, and protein-water interactions in lyophilized powders were identified. Myoglobin (Mb) was derivatized in solution with the heterobifunctional probe succinimidyl 4,4'-azipentanoate (SDA) and the structural integrity of the labeled protein (Mb-SDA) confirmed using CD spectroscopy and liquid chromatography/mass spectrometry (LC-MS). Mb-SDA was then formulated with and without excipients (raffinose, guanidine hydrochloride (Gdn HCl)) and lyophilized. The freeze-dried powder was irradiated with ultraviolet light at 365 nm for 30 min to produce cross-linked adducts that were analyzed at the intact protein level and after trypsin digestion. SDA-labeling produced Mb carrying up to five labels, as detected by LC-MS. Following lyophilization and irradiation, cross-linked peptide-peptide, peptide-water, and peptide-raffinose adducts were detected. The exposure of Mb side chains to the matrix was quantified based on the number of different peptide-peptide, peptide-water, and peptide-excipient adducts detected. In the absence of excipients, peptide-peptide adducts involving the CD, DE, and EF loops and helix H were common. In the raffinose formulation, peptide-peptide adducts were more distributed throughout the molecule. The Gdn HCl formulation showed more protein-protein and protein-water adducts than the other formulations, consistent with protein unfolding and increased matrix interactions. The results demonstrate that ssPC-MS can be used to distinguish excipient effects and characterize the local protein environment in lyophilized formulations with high resolution.