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The effects of 2 squalene-based emulsion adjuvant systems (MedImmune emulsion 0 [ME.0] and Stable Emulsion [SE]) on the structure and stability of the recombinant protein antigen alpha-toxin (AT), a potential vaccine candidate for Staphylococcus aureus infection, were investigated using Fourier-transform infrared spectroscopy and both steady-state and time-resolved intrinsic fluorescence spectroscopy as well as differential scanning calorimetry (DSC). A component study, performed to identify the effects of the individual emulsion's components, showed negligible interactions between AT and ME.0. DSC analysis showed the ME.0 emulsion thermally destabilized AT, probably because of changes in the buffer composition of AT upon mixing. The SE emulsion caused increased alpha-helix and decreased beta-sheet content in AT, and a significant blue shift in the fluorescence spectra relative to that of AT in solution. DSC analysis showed SE exerted a dramatic thermal stabilization effect on AT, probably attributable to an interaction between AT and SE. Size exclusion chromatography showed a complete loss in the recovery of AT when mixed with SE, but not ME.0, indicating a high degree of interaction with SE. This work successfully characterized the biophysical properties of AT in the presence of 2 emulsion adjuvants including a component study to rationalize how emulsion components affect protein antigen stability., (Copyright © 2018 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Cross-reacting material 197 (CRM 197 ), a single amino acid mutant of diphtheria toxoid, is a commonly used carrier protein in commercial polysaccharide protein conjugate vaccines. In this study, CRM 197 proteins from 3 different expression systems and 5 different manufacturers were obtained for an analytical comparability assessment using a wide variety of physicochemical and in vitro antigenic binding assays. A comprehensive analysis of the 5 CRM 197 molecules demonstrate that recombinant CRM 197 's expressed in heterologous systems (Escherichia coli and Pseudomonas fluorescens) are overall highly similar (if not better in some cases) to those expressed in the traditional system (Corynebacterium diphtheriae) in terms of primary sequence/post-translational modifications, higher order structural integrity, apparent solubility, physical stability profile (vs. pH and temperature), and in vitro antigenicity. These results are an encouraging step to demonstrate that recombinant CRM 197 expressed in alternative sources have the potential to replace CRM 197 expressed in C diphtheriae as a source of immunogenic carrier protein for lower cost polysaccharide conjugate vaccines. The physicochemical assays established in this work to monitor the key structural attributes of CRM 197 should also prove useful as complementary characterization methods (to routine quality control assays) to support future process and formulation development of lower cost CRM 197 carrier proteins for use in various conjugate vaccines., (Copyright © 2018 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Objective: In this study, PEGylated rosin derivatives (PRDs) namely D1 and D2 were synthesized and evaluated for their application to produce sustained-release antibacterial films containing sparfloxacin for periodontitis., Significance: PRDs are biodegradable and biocompatible, and therefore sustained-release dental implant of PRD-sparfloxacin can provide an effectual treatment for periodontitis., Methods: Films were produced by solvent casting technique and characterized for morphology, swelling-index, in vitro degradation and drug release kinetics. The impact of type of PRD, concentration of PRDs, and addition of plasticizer (dibutyl phthalate) on various film properties was evaluated. The films were also subjected to stability study at 30 °C and 40 °C for 90 days., Results: Both D1 and D2 produced smooth and non-porous films with sparfloxacin. The D1 films, due to lower amount of polyethylene glycol 400 in D1, exhibited lower swelling-index, slower degradation, and slower drug release compared to D2 films. An increase in PRDs concentration decreased swelling-index, prolonged degradation time, and decreased drug release rate of films; addition of plasticizer showed the similar effect. At pH 7.6, D1 and D2 films showed complete degradation at the end of 58 and 51 days, respectively. At the end of 21 days, D1 and D2 films released 41.85% and 61.53% sparfloxacin, respectively. The drug release from D1 films followed Higuchi square-root kinetics, while D2 films released drug by the zero order kinetics. The stability conditions did not significantly alter PRDs-film properties., Conclusion: Results revealed that PRDs can be used successfully to produce sustained-release antibacterial films containing sparfloxacin for the treatment of periodontitis.

The worldwide switch to inactivated polio vaccines (IPVs) is a key component of the overall strategy to achieve and maintain global polio eradication. To this end, new IPV vaccine delivery systems may enhance patient convenience and compliance. In this work, we examine Nanopatch™ (a solid, polymer microprojection array) which offers potential advantages over standard needle/syringe administration including intradermal delivery and reduced antigen doses. Using trivalent IPV (tIPV) and a purpose-built evaporative dry-down system, candidate tIPV formulations were developed to stabilize tIPV during the drying process and on storage. Identifying conditions to minimize tIPV potency losses during rehydration and potency testing was a critical first step. Various classes and types of pharmaceutical excipients (∼50 total) were then evaluated to mitigate potency losses (measured through D-antigen ELISAs for IPV1, IPV2, and IPV3) during drying and storage. Various concentrations and combinations of stabilizing additives were optimized in terms of tIPV potency retention, and 2 candidate tIPV formulations containing cyclodextrin and a reducing agent (e.g., glutathione), maintained ≥80% D-antigen potency during drying and subsequent storage for 4 weeks at 4°C, and ≥60% potency for 3 weeks at room temperature with the majority of losses occurring within the first day of storage., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

A combination experimental approach, utilizing semi-empirical excipient screening followed by statistical modeling using design of experiments (DOE), was undertaken to identify stabilizing candidate formulations for a lyophilized live attenuated Flavivirus vaccine candidate. Various potential pharmaceutical compounds used in either marketed or investigative live attenuated viral vaccine formulations were first identified. The ability of additives from different categories of excipients, either alone or in combination, were then evaluated for their ability to stabilize virus against freeze-thaw, freeze-drying, and accelerated storage (25°C) stresses by measuring infectious virus titer. An exploratory data analysis and predictive DOE modeling approach was subsequently undertaken to gain a better understanding of the interplay between the key excipients and stability of virus as well as to determine which combinations were interacting to improve virus stability. The lead excipient combinations were identified and tested for stabilizing effects using a tetravalent mixture of viruses in accelerated and real time (2-8°C) stability studies. This work demonstrates the utility of combining semi-empirical excipient screening and DOE experimental design strategies in the formulation development of lyophilized live attenuated viral vaccine candidates., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

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 H 2 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., (Copyright © 2018 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

An automated method using biotinylated GroEL-streptavidin biosensors with biolayer interferometry (GroEL-BLI) was evaluated to detect the formation of transiently formed, preaggregate species in various pharmaceutically relevant monoclonal antibody (mAb) samples. The relative aggregation propensity of various IgG1 and IgG4 mAbs was rank ordered using the GroEL-BLI biosensor method, and the least stable IgG4 mAb was subjected to different stresses including elevated temperatures, acidic pH, and addition of guanidine HCl. The GroEL-BLI biosensor detects mAb preaggregate formation mostly before, or sometimes concomitantly with, observing soluble aggregates and subvisible particles using size-exclusion chromatography and microflow imaging, respectively. A relatively unstable bispecific antibody (Bis-3) was shown to bind the GroEL biosensor even at low temperatures (25 ° C). During thermal stress (50°C, 1 h), increased Bis-3 binding to GroEL-biosensors was observed prior to aggregation by size-exclusion chromatography or microflow imaging. Transmission electron microscopy analysis of Bis-3 preaggregate GroEL complexes revealed, in some cases, potential hydrophobic interaction sites between the Fc domain of the Bis-3 and GroEL protein. The automated BLI method not only enables detection of transiently formed preaggregate species that initiate protein aggregation pathways but also permits rapid mAb formulation stability assessments at low volumes and low protein concentrations., (Copyright © 2018 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

RiVax is a promising recombinant ricin toxin A subunit (RTA) vaccine antigen that has been shown to be safe and immunogenic in humans and effective at protecting rhesus macaques against lethal-dose aerosolized toxin exposure. We previously used a panel of RTA-specific monoclonal antibodies (MAbs) to demonstrate, by competition enzyme-linked immunosorbent assay (ELISA), that RiVax elicits similar serum antibody profiles in humans and macaques. However, the MAb binding sites on RiVax have yet to be defined. In this study, we employed hydrogen exchange-mass spectrometry (HX-MS) to localize the epitopes on RiVax recognized by nine toxin-neutralizing MAbs and one nonneutralizing MAb. Based on strong protection from hydrogen exchange, the nine MAbs grouped into four spatially distinct epitope clusters (namely, clusters I to IV). Cluster I MAbs protected RiVax's α-helix B (residues 94 to 107), a protruding immunodominant secondary structure element known to be a target of potent toxin-neutralizing antibodies. Cluster II consisted of two subclusters located on the "back side" (relative to the active site pocket) of RiVax. One subcluster involved α-helix A (residues 14 to 24) and α-helices F-G (residues 184 to 207); the other encompassed β-strand d (residues 62 to 69) and parts of α-helices D-E (154 to 164) and the intervening loop. Cluster III involved α-helices C and G on the front side of RiVax, while cluster IV formed a sash from the front to back of RiVax, spanning strands b, c, and d (residues 35 to 59). Having a high-resolution B cell epitope map of RiVax will enable the development and optimization of competitive serum profiling assays to examine vaccine-induced antibody responses across species., (Copyright © 2017 American Society for Microbiology.)

A novel protein adjuvant double-mutant Escherichia coli heat-labile toxin, LT (R192G/L211A) or dmLT, is in preclinical and early clinical development with various vaccine candidates. Structural characterization and formulation development of dmLT will play a key role in its successful process development, scale-up/transfer, and commercial manufacturing. This work describes extensive analytical characterization of structural integrity and physicochemical stability profile of dmLT from a lyophilized clinical formulation. Reconstituted dmLT contained a heterogeneous mixture of intact holotoxin (AB 5 , ∼75%) and free B 5 subunit (∼25%) as assessed by analytical ultracentrifugation and hydrophobic interaction chromatography. Intact mass spectrometry (MS) analysis revealed presence of Lys 84 glycation near the native sugar-binding site in dmLT, and forced degradation studies using liquid chromatography-MS peptide mapping demonstrated specific Asn deamidation and Met oxidation sites. Using multiple biophysical measurements, dmLT was found most stable between pH 6.5 and 7.5 and at temperatures ≤50°C. In addition, soluble aggregates and particle formation were observed upon shaking stress. By identifying the physicochemical degradation pathways of dmLT using newly developed stability-indicating analytical methods from this study, we aim at developing more stable candidate formulations of dmLT that will minimize the formation of degradants and improve storage stability, as both a frozen bulk substance and eventually as a liquid final dosage form., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

High protein concentration formulations are required for low-volume administration of therapeutic antibodies targeted for subcutaneous, self-administration by patients. Ultra-high concentrations (≥150 mg/mL) can lead to dramatically increased solution viscosities, which in turn can lead to stability, manufacturing, and delivery challenges. In this study, various categories and individual types of pharmaceutical excipients and other additives (56 in total) were screened for their viscosity reducing effects on 2 different mAbs. The physicochemical stability profile, as well as viscosity ranges, of several candidate antibody formulations, identified and designed based on the results of the excipient screening, were evaluated over a 6-month time period under accelerated and real-time storage conditions. In addition to reducing the solution viscosities to acceptable levels for parenteral administration (using currently available and acceptable delivery devices), the candidate formulations did not result in notable losses of physicochemical stability of the 2 antibodies on storage for 6 months at 25°C. The experiments described here demonstrate the feasibility of a formulation development and selection approach to identify candidate high-concentration antibody formulations with viscosities within pharmaceutically acceptable ranges that do not adversely affect their physicochemical storage stability., (Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

There is growing interest in generating physicochemical and biological analytical data sets to compare complex mixture drugs, for example, products from different manufacturers. In this work, we compare various crofelemer samples prepared from a single lot by filtration with varying molecular weight cutoffs combined with incubation for different times at different temperatures. The 2 preceding articles describe experimental data sets generated from analytical characterization of fractionated and degraded crofelemer samples. In this work, we use data mining techniques such as principal component analysis and mutual information scores to help visualize the data and determine discriminatory regions within these large data sets. The mutual information score identifies chemical signatures that differentiate crofelemer samples. These signatures, in many cases, would likely be missed by traditional data analysis tools. We also found that supervised learning classifiers robustly discriminate samples with around 99% classification accuracy, indicating that mathematical models of these physicochemical data sets are capable of identifying even subtle differences in crofelemer samples. Data mining and machine learning techniques can thus identify fingerprint-type attributes of complex mixture drugs that may be used for comparative characterization of products., (Copyright © 2017 American Pharmacists Association®. All rights reserved.)

Crofelemer is a botanical polymeric proanthocyanidin that inhibits chloride channel activity and is used clinically for treating HIV-associated secretory diarrhea. Crofelemer lots may exhibit significant physicochemical variation due to the natural source of the raw material. A variety of physical, chemical, and biological assays were used to identify potential critical quality attributes (CQAs) of crofelemer, which may be useful in characterizing differently sourced and processed drug products. Crofelemer drug substance was extracted from tablets of one commercial drug product lot, fractionated, and subjected to accelerated thermal degradation studies to produce derivative lots with variations in chemical and physical composition potentially representative of manufacturing and raw material variation. Liquid chromatography, UV absorbance spectroscopy, mass spectrometry, and nuclear magnetic resonance analysis revealed substantial changes in the composition of derivative lots. A chloride channel inhibition cell-based bioassay suggested that substantial changes in crofelemer composition did not necessarily result in major changes to bioactivity. In 2 companion papers, machine learning and data mining approaches were applied to the analytical and biological data sets presented herein, along with chemical stability data sets derived from forced degradation studies, to develop an integrated mathematical model that can identify CQAs which are most relevant in distinguishing between different populations of crofelemer., (Copyright © 2017 American Pharmacists Association®. All rights reserved.)

As the second of a 3-part series of articles in this issue concerning the development of a mathematical model for comparative characterization of complex mixture drugs using crofelemer (CF) as a model compound, this work focuses on the evaluation of the chemical stability profile of CF. CF is a biopolymer containing a mixture of proanthocyanidin oligomers which are primarily composed of gallocatechin with a small contribution from catechin. CF extracted from drug product was subjected to molecular weight-based fractionation and thiolysis. Temperature stress and metal-catalyzed oxidation were selected for accelerated and forced degradation studies. Stressed CF samples were size fractionated, thiolyzed, and analyzed with a combination of negative-ion electrospray ionization mass spectrometry (ESI-MS) and reversed-phase-HPLC with UV absorption and fluorescence detection. We further analyzed the chemical stability data sets for various CF samples generated from reversed-phase-HPLC-UV and ESI-MS using data-mining and machine learning approaches. In particular, calculations based on mutual information of over 800,000 data points in the ESI-MS analytical data set revealed specific CF cleavage and degradation products that were differentially generated under specific storage/degradation conditions, which were not initially identified using traditional analysis of the ESI-MS results., (Copyright © 2017 American Pharmacists Association®. All rights reserved.)

This work describes the formulation design and development of a novel protein based adjuvant, a double mutant of heat labile toxin (dmLT), based on knowledge of the protein's structural integrity and physicochemical degradation pathways. Various classes of pharmaceutical excipients were screened for their stabilizing effect on dmLT during exposure to thermal and agitation stresses as monitored by high throughput analytical assays for dmLT degradation. Sucrose, phosphate, sodium chloride, methionine and polysorbate-80 were identified as potential stabilizers that protected dmLT against either conformational destabilization, aggregation/particle formation or chemical degradation (e.g., Met oxidation and Lys glycation). Different combinations and concentrations of the selected stabilizers were then evaluated to further optimize dmLT stability while maintaining pharmaceutically acceptable ranges of solution pH and osmolality. The effect of multiple freeze-thaw (FT) cycles on the physical stability of candidate bulk formulations was also examined. Increasing the polysorbate-80 concentration to 0.1% in the lead candidate bulk formulation mitigated the loss of protein mass during FT. This formulation development study enabled the design of a new bulk formulation of the dmLT adjuvant and provides flexibility for future use in combination with a variety of different vaccine dosage forms with different antigens., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

A barrier to the use of hydrogen exchange-mass spectrometry (HX-MS) in many contexts, especially analytical characterization of various protein therapeutic candidates, is that differences in temperature, pH, ionic strength, buffering agent, or other additives can alter chemical exchange rates, making HX data gathered under differing solution conditions difficult to compare. Here, we present data demonstrating that HX chemical exchange rates can be substantially altered not only by the well-established variables of temperature and pH but also by additives including arginine, guanidine, methionine, and thiocyanate. To compensate for these additive effects, we have developed an empirical method to correct the hydrogen-exchange data for these differences. First, differences in chemical exchange rates are measured by use of an unstructured reporter peptide, YPI. An empirical chemical exchange correction factor, determined by use of the HX data from the reporter peptide, is then applied to the HX measurements obtained from a protein of interest under different solution conditions. We demonstrate that the correction is experimentally sound through simulation and in a proof-of-concept experiment using unstructured peptides under slow-exchange conditions (pD 4.5 at ambient temperature). To illustrate its utility, we applied the correction to HX-MS excipient screening data collected for a pharmaceutically relevant IgG4 mAb being characterized to determine the effects of different formulations on backbone dynamics.

Multidose formulations of biotherapeutics, which offer better dosage management and reduced production costs, require the addition of antimicrobial preservatives (APs). APs have been shown, however, to decrease protein stability in solution and cause protein aggregation. In this report, the effect of 4 APs, m-cresol, phenol, phenoxyethanol, and benzyl alcohol on conformational stability, aggregation propensity, and backbone flexibility of an IgG1 mAb, mAb-4, is investigated. Compared with no preservative control, each of the APs decreased the conformational stability of mAb-4 as measured by differential scanning calorimetry and extrinsic fluorescence spectroscopy. The addition of APs resulted in increased monomer loss and aggregate accumulation at 50°C over 28 days, as monitored by size-exclusion chromatography. The extent of conformational destabilization and protein aggregation of mAb-4 induced by APs followed their calculated octanol-water partition coefficients. Increases in backbone flexibility, as measured by hydrogen exchange, of a region located in the C H 2 domain of the mAb (heavy chain 237-254) in the presence of APs also correlated with hydrophobicity. Based on these results, the destabilizing effect of APs on mAb-4 correlates with the increased hydrophobicity of the APs and their ability to enhance the local backbone flexibility of an aggregation hot spot within the C H 2 domain of the mAb., (Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

This study describes the physicochemical characterization, stabilization, and formulation design of SM4-AC, an acrylodan-labeled glucose/galactose-binding protein for use in a continuous glucose monitoring device. The physical stability profile of SM4-AC as a function of pH and temperature was monitored using a combination of biophysical techniques and the resulting physical stability profile was visualized using an empirical phase diagram. Forced degradation chemical stability studies (Asn deamidation, Met oxidation) of SM4-AC were performed using a combination of capillary isoelectric focusing, peptide mapping, and reversed-phase HPLC. Differential scanning fluorimetry was then employed to screen various pharmaceutical excipients for their ability to physically stabilize SM4-AC. An optimized formulation of 20% sucrose and 2.5 mM calcium chloride in 10 mM MES buffer, 150 mM NaCl at pH 6.0 increased the conformational stability of SM4-AC by 15°C. Accelerated and real-time stability studies were setup to compare the SM4-AC protein's physicochemical stability and glucose-binding activity in 2 formulations for up to 12 months. SM4-AC in an optimized formulation (vs the original formulation) showed improved physical stability, and similar chemical stability and glucose binding activity profiles during storage up to 52 weeks at various temperatures., (Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Background: Reconstruction of upper extremity deformities continues to be a challenge to the reconstructive surgeon. Various loco regional, distant and free flaps are available for reconstruction. However, each has its own set of advantages and disadvantages. Of the commonly performed local flaps, radial artery forearm flap, and the posterior interosseus artery flap stand out prominently. Recently, perforator propeller flaps have been used for resurfacing the upper extremity. The anterior interosseus artery perforator flap is an uncommonly used and described flap., Methods: This study was divided into anatomical study and clinical application in a IV level of evidence. In the anatomical study, five upper extremities were studied. Clinically, 12 patients underwent reconstruction using the anterior interosseus artery perforator flap. Flaps were performed by a single surgeon. A retrospective review of these cases from November 2008 to May 2014 is presented., Results: The anterior interosseus artery perforator was identified in four out of five cadaver limbs. The septocutaneous perforator was in the fifth extensor compartment around 4 cm proximal to the wrist joint. Of the twelve flaps, there was complete necrosis in one flap, and partial necrosis in one flap. The patient with complete necrosis underwent skin grafting at a later date. The wound healed secondarily in case of partial flap necrosis., Conclusion: Anterior interosseus artery perforator flap must be considered as an important reconstructive option in the armamentarium of the plastic surgeon, while managing hand and wrist defects., Competing Interests: The authors declare no conflict of interest.

Continuous glucose monitoring (CGM) devices offer diabetes patients a convenient approach to assist in controlling blood glucose levels. A prototype CGM has been developed that uses the emission profile of a polarity-sensitive fluorophore (acrylodan) conjugated to a glucose/galactose-binding protein (SM4-AC) to measure the concentration of glucose in vivo. During development, a decrease in the devices signal intensity was observed in vivo over time, which was postulated to be result of oxidative degradation of SM4-AC. A comprehensive physicochemical analysis of SM4-AC was pursued to identify potential mechanisms of signal intensity loss in this CGM during in vitro forced oxidation studies. An assessment of the structural integrity and conformational stability of SM4-AC indicated a relatively decreased polarity and lower tertiary structure stability compared to unconjugated protein (SM4). The stability and polarity of SM4-AC was also altered in the presence of H 2 O 2 . Furthermore, a time-dependent loss in the fluorescence signal of SM4-AC was observed when incubated with H 2 O 2 . An LC-MS peptide mapping analysis of these protein samples indicated that primarily two Met residues in SM4-AC were susceptible to oxidation. When these two residues were genetically altered to an amino acid not prone to oxidation, the glucose binding ability of the protein was retained and no loss of acrylodan fluorescence was observed in the presence of H 2 O 2 . Genetic alteration of these two residues is proposed as an effective approach to increase the long-term stability of SM4-AC within this prototype CGM in vivo., (© 2016 The Protein Society.)

Concentration-dependent reversible self-association (RSA) of monoclonal antibodies (mAbs) poses a challenge to their pharmaceutical development as viable candidates for subcutaneous delivery. While the role of the antigen-binding fragment (Fab) in initiating RSA is well-established, little evidence supports the involvement of the crystallizable fragment (Fc). In this report, a variety of biophysical tools, including hydrogen exchange mass spectrometry, are used to elucidate the protein interface of such non-covalent protein-protein interactions. Using dynamic and static light scattering combined with viscosity measurements, we find that an IgG1 mAb (mAb-J) undergoes RSA primarily through electrostatic interactions and forms a monomer-dimer-tetramer equilibrium. We provide the first direct experimental mapping of the interface formed between the Fab and Fc domains of an antibody at high protein concentrations. Charge distribution heterogeneity between the positively charged interface spanning complementarity-determining regions CDR3H and CDR2L in the Fab and a negatively charged region in C H 3/Fc domain mediates the RSA of mAb-J. When arginine and NaCl are added, they disrupt RSA of mAb-J and decrease the solution viscosity. Fab-Fc domain interactions between mAb monomers may promote the formation of large transient antibody complexes that ultimately cause increases in solution viscosity. Our findings illustrate how limited specific arrangements of amino-acid residues can cause mAbs to undergo RSA at high protein concentrations and how conserved regions in the Fc portion of the antibody can also play an important role in initiating weak and transient protein-protein interactions.

Liquid chromatographic methods, combined with mass spectrometry, offer exciting and important opportunities to better characterize complex vaccine antigens including recombinant proteins, virus-like particles, inactivated viruses, polysaccharides, and protein-polysaccharide conjugates. The current abilities and limitations of these physicochemical methods to complement traditional in vitro and in vivo vaccine potency assays are explored in this review through the use of illustrative case studies. Various applications of these state-of-the art techniques are illustrated that include the analysis of influenza vaccines (inactivated whole virus and recombinant hemagglutinin), virus-like particle vaccines (human papillomavirus and hepatitis B), and polysaccharide linked to protein carrier vaccines (pneumococcal). Examples of utilizing these analytical methods to characterize vaccine antigens in the presence of adjuvants, which are often included to boost immune responses as part of the final vaccine dosage form, are also presented. Some of the challenges of using chromatographic and LC-MS as physicochemical assays to routinely test complex vaccine antigens are also discussed., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Adequate protein solubility is an important prerequisite for development, manufacture, and administration of biotherapeutic drug candidates, especially for high-concentration protein formulations. A previously established method for determining the relative apparent solubility (thermodynamic activity) of proteins using polyethylene glycol (PEG) precipitation is adapted for screening and comparing monoclonal antibody (mAb) candidates where only limited quantities (≤1 mg) are available. This micro-PEG assay is used to evaluate various broadly neutralizing mAb candidates to HIV-1 viral spike (gp120 and gp41 glycoproteins). Using ∼1 mg of VRC01-WT mAb per assay, the precision of the micro-PEG assay was established. A series of 7 different broadly neutralizing mAbs to the HIV-1 viral spike proteins were compared by curve shape (%PEG vs. protein concentration), %PEGmidpoint determinations, and extrapolated apparent solubility values. Numerous formulation conditions were then evaluated for their relative effects on the VRC01-WT mAb. The PEGmidpt and apparent solubility values of VRC01-WT mAb decreased as the solution pH increased and increased as NaCl and arginine were added. A final optimization of the micro-PEG assay established that amounts as low as 0.1-0.2 mg can be used. Thus, the micro-PEG assay has significant potential as a relative solubility screening tool during candidate selection and early formulation development., (Copyright © 2016 American Pharmacists Association®. All rights reserved.)

Introduction: Root Canal Treatment (RCT) has become a mainstream procedure in dentistry. A successful RCT is presented by absence of clinical signs and symptoms in teeth without any radiographic evidence of periodontal involvement. Completing this procedure in one visit or multiple visits has long been a topic of discussion., Aim: To evaluate the incidence of postoperative pain after root canal therapy performed in single visit and two visits., Material and Methods: An unblinded/ open label randomized controlled trial was carried out in the endodontic department of the Dental Institute, where 78 patients were recruited from the regular pool of patients. A total of 66 maxillary central incisors requiring root canal therapy fulfilled the inclusion and exclusion criteria. Using simple randomization by biased coin randomization method, the selected patients were assigned into two groups: group A (n=33) and group B (n=33). Single visit root canal treatment was performed for group A and two visit root canal treatment for group B. Independent sample t-test was used for statistical analysis., Results: Thirty three patients were allotted to group A where endodontic treatment was completed in single visit while 33 patients were allotted to group B where endodontic treatment was completed in two visits. One patient dropped-out from Group A. Hence in Group A, 32 patients were analysed while in Group B, 33 patients were analysed. After 6 hours, 12 hours and 24 hours of obturation, pain was significantly higher in Group B as compared to Group A. However, there was no significant difference in the pain experienced by the patients 48 hours after treatment in both the groups., Conclusion: Incidence of pain after endodontic treatment being performed in one-visit or two-visits is not significantly different.

RiVax is a candidate ricin toxin subunit vaccine antigen that has proven to be safe in human phase I clinical trials. In this study, we introduced double and triple cavity-filling point mutations into the RiVax antigen with the expectation that stability-enhancing modifications would have a beneficial effect on overall immunogenicity of the recombinant proteins. We demonstrate that 2 RiVax triple mutant derivatives, RB (V81L/C171L/V204I) and RC (V81I/C171L/V204I), when adsorbed to aluminum salts adjuvant and tested in a mouse prime-boost-boost regimen were 5- to 10-fold more effective than RiVax at eliciting toxin-neutralizing serum IgG antibody titers. Increased toxin neutralizing antibody values and seroconversion rates were evident at different antigen dosages and within 7 days after the first booster. Quantitative stability/flexibility relationships analysis revealed that the RB and RC mutations affect rigidification of regions spanning residues 98-103, which constitutes a known immunodominant neutralizing B-cell epitope. A more detailed understanding of the immunogenic nature of RB and RC may provide insight into the fundamental relationship between local protein stability and antibody reactivity., (Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

The metal-catalyzed oxidation by [Fe(II)(EDTA)](2-)/H2O2 of IgG-1 leads to the site-specific hydrolysis of peptide bonds in the Fc region. The major hydrolytic cleavage occurs between Met428 and His429, consistent with a mechanism reported for the site-specific hydrolysis of parathyroid hormone (1-34) between Met8 and His9 (Mozziconacci, O.; et al. Mol. Pharmaceutics 2013, 10 (2), 739-755). In IgG-1, to a lesser extent, we also observe hydrolysis reactions between Met252 and Ile253. After 2 h of oxidation (at pH 5.8, 37 °C) approximately 5% of the protein is cleaved between Met428 and His429. For comparison, after 2 h of oxidation, the amount of tryptic peptides containing a Met sulfoxide residue represents less than 0.1% of the protein. The effect of this site-specific hydrolysis on the conformational stability and aggregation propensity of the antibody was also examined. No noticeable differences in structural integrity and conformational stability were observed between control and oxidized IgG-1 samples as measured by circular dichroism (CD), fluorescence spectroscopy, and static light scattering (SLS). Small amounts of soluble and insoluble aggregates (3-6%) were, however, observed in the oxidized samples by UV-visible absorbance spectroscopy and size exclusion chromatography (SEC). Over the course of metal-catalyzed oxidation, increasing amounts of fragments were also observed by SEC. An increase in the concentration of subvisible particles was detected by microflow imaging (MFI).

For therapeutic protein analytical studies related to evaluating lot-to-lot variability, different processes and/or formulations, or biosimilars, there is growing interest in applying novel data visualization tools for fingerprint analysis to identify statistically significant differences between 2 samples. Comparative Signature Diagrams (CSDs) were previously developed to display such differences as colored contour plots using a variety of biophysical data sets. In this study, several improvements are proposed to enhance readability and quantitative determinations of CSDs using protein stability data from more commonly used analytical methods such as size exclusion chromatography and capillary isoelectric focusing. To demonstrate the effectiveness of improved CSDs for data visualization, an accelerated and real-time stability study was set up for an IgG1 mAb (mAb A) and its corresponding triple mutant (mAb E). The stability profiles of both mAbs were compared for differences in aggregation (size exclusion chromatography) and charge heterogeneity (capillary isoelectric focusing) profiles over time. Both traditional data analysis and the improved CSDs conclude that the triple mutant mAb E is more susceptible to physicochemical degradation than mAb A under accelerated conditions. The current abilities and limitations of CSDs to provide fingerprint analysis of protein stability profiles to facilitate the determination of similarity versus nonsimilarity between samples is discussed., (Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

As part of a series of articles in this special issue describing 4 well-defined IgG1-Fc glycoforms as a model system for biosimilarity analysis (high mannose-Fc, Man5-Fc, GlcNAc-Fc and N297Q-Fc aglycosylated), the focus of this work is comparisons of their physical properties. A trend of decreasing apparent solubility (thermodynamic activity) by polyethylene glycol precipitation (pH 4.5, 6.0) and lower conformational stability by differential scanning calorimetry (pH 4.5) was observed with reducing size of the N297-linked oligosaccharide structures. Using multiple high-throughput biophysical techniques, the physical stability of the Fc glycoproteins was then measured in 2 formulations (NaCl and sucrose) across a wide range of temperatures (10°C-90°C) and pH (4.0-7.5) conditions. The data sets were used to construct 3-index empirical phase diagrams and radar charts to visualize the regions of protein structural stability. Each glycoform showed improved stability in the sucrose (vs. salt) formulation. The HM-Fc and Man5-Fc displayed the highest relative stability, followed by GlcNAc-Fc, with N297Q-Fc being the least stable. Thus, the overall physical stability profiles of the 4 IgG1-Fc glycoforms also show a correlation with oligosaccharide structure. These data sets are used to develop a mathematical model for biosimilarity analysis (as described in a companion article by Kim et al. in this issue)., (Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Biosimilarity assessments are performed to decide whether 2 preparations of complex biomolecules can be considered "highly similar." In this work, a machine learning approach is demonstrated as a mathematical tool for such assessments using a variety of analytical data sets. As proof-of-principle, physical stability data sets from 8 samples, 4 well-defined immunoglobulin G1-Fragment crystallizable glycoforms in 2 different formulations, were examined (see More et al., companion article in this issue). The data sets included triplicate measurements from 3 analytical methods across different pH and temperature conditions (2066 data features). Established machine learning techniques were used to determine whether the data sets contain sufficient discriminative power in this application. The support vector machine classifier identified the 8 distinct samples with high accuracy. For these data sets, there exists a minimum threshold in terms of information quality and volume to grant enough discriminative power. Generally, data from multiple analytical techniques, multiple pH conditions, and at least 200 representative features were required to achieve the highest discriminative accuracy. In addition to classification accuracy tests, various methods such as sample space visualization, similarity analysis based on Euclidean distance, and feature ranking by mutual information scores are demonstrated to display their effectiveness as modeling tools for biosimilarity assessments., (Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Four different well-defined IgG1 Fc glycoforms are proposed as a model system to examine important biological and physicochemical features for protein drug biosimilar analyses. The IgG1 Fc glycoforms were produced by yeast expression combined with in vitro enzymatic synthesis as a series of sequentially truncated high-mannose IgG1 Fc glycoforms with an anticipated range of biological activity and structural stability. Initial characterization with mass spectrometry, SDS-PAGE, size exclusion HPLC, and capillary isoelectric focusing confirmed that the glycoproteins are overall highly similar with the only major difference being glycosylation state. Binding to the activating Fc receptor, FcγRIIIa was used to evaluate the potential biological activity of the IgG1 Fc glycoproteins. Two complementary methods using biolayer interferometry, 1 with protein G-immobilized IgG1 Fc and the other with streptavidin-immobilized FcγRIIIa, were developed to assess FcγRIIIa affinity in kinetic binding studies. The high-mannose IgG1 Fc and Man5-IgG1 Fc glycoforms were highly similar to one another with high affinity for FcγRIIIa, whereas GlcNAc-Fc had weak affinity, and the nonglycosylated N297Q-Fc had no measurable affinity for FcγRIIIa. These 4 IgG1 Fc glycoforms were also evaluated in terms of physical and chemical stability profiles and then used as a model system to mathematically assess overall biosimilarity, as described in a series of companion articles., (Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Vaccine formulation development has traditionally focused on improving antigen storage stability and compatibility with conventional adjuvants. More recently, it has also provided an opportunity to modify the interaction and presentation of an antigen/adjuvant to the immune system to better stimulate the desired immune responses for maximal efficacy. In the last decade, there has been a paradigm shift in vaccine antigen and formulation design involving an improved physical understanding of antigens and a better understanding of the immune system. In addition, the discovery of novel adjuvants and delivery systems promises to further improve the design of new, more effective vaccines. Here we describe some of the fundamental aspects of formulation design applicable to virus-like-particle based vaccine antigens (VLPs). Case studies are presented for commercially approved VLP vaccines as well as some investigational VLP vaccine candidates. An emphasis is placed on the biophysical analysis of vaccines to facilitate formulation and stabilization of these particulate antigens., (Published by Elsevier B.V.)

Background and Aims: Obese patients are more vulnerable to residual neuromuscular block (NMB) and its associated complications in the post-operative period. This study was carried out to compare neostigmine induced reversal of vecuronium in normal weight, overweight and obese female patients, objectively using neuromuscular (NM) monitoring., Methods: Twenty female patients each belonging to normal weight, overweight and obese, based on body mass index, requiring general anaesthesia were recruited for this prospective cross sectional study. NMB was induced with vecuronium (0.1 mg/kg) dose based on patient's real body weight (RBW) and monitored using acceleromyographic train of four (TOF). All patients received neostigmine 40 μg/kg and glycopyrrolate 10 μg/kg at 25% of spontaneous recovery of first twitch height (T1) of TOF (DUR 25%) and were allowed to recover to TOF ratio of 0.9. Statistical analysis was done using analysis of variance test., Results: Recovery of TOF ratio to 0.5 was comparable in all three groups. Recovery of TOF ratio to 0.7 was delayed in obese (9.82 ± 3.21 min) compared with normal weight group (7.50 ± 2.52 min). Recovery of TOF to 0.9 was significantly delayed in both overweight (12.18 ± 4.29 min) and obese patients (13.78 ± 4.30 min). DUR 25% was significantly longer in overweight (mean, standard deviation [range]; 30.10 [19-40 min]) and obese (28.8 [12-45 min]) compared with normal weight patients (22.75 [16-30 min])., Conclusion: In overweight and obese patients, when vecuronium induction dose is based on RBW, neostigmine induced recovery of NMB is delayed in late phases (TOF 0.7-0.9), which may result in vulnerability for associated complications of incomplete recovery. Ensuring safe recovery thus requires objective NM monitoring.

Upon exposure to shaking stress, an IgG1 mAb formulation in both the liquid and lyophilized state formed subvisible particles. Because freeze-drying was expected to minimize protein physical instability under these conditions, the extent and nature of aggregate formation in the lyophilized preparation were examined using a variety of particle characterization techniques. The effects of formulation variables such as residual moisture content, reconstitution rate, and reconstitution medium were also examined. Upon reconstitution of shake-stressed lyophilized mAb, differences in protein particle size and number were observed by microflow digital imaging, with the reconstitution medium having the largest impact. Shake stress had minor effects on the structure of protein within the particles as shown by SDS-PAGE and FTIR analysis. The lyophilized mAb was shake stressed to different extents and stored for 3 months at different temperatures. Both extent of cake collapse and storage temperature affected the physical stability of the shake-stressed lyophilized mAb upon subsequent storage. These findings demonstrate that physical degradation upon shaking of a lyophilized IgG1 mAb formulation includes not only cake breakage, but also results in an increase in subvisible particles and turbidity upon reconstitution. The shake-induced cake breakage of the lyophilized IgG1 mAb formulation also resulted in decreased physical stability upon storage., (© 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.)

Bacterium-like particles (BLPs), derived from Lactococcus lactis, offer a self-adjuvanting delivery vehicle for subunit protein vaccines. Proteins can be specifically loaded onto the BLPs via a peptidoglycan anchoring (PA) domain. In this study, the tip proteins IpaD, SipD, and LcrV belonging to type III secretion systems of Shigella flexneri, Salmonella enterica, and Yersinia enterocolitica, respectively, were fused to the PA and loaded onto the BLPs. Herein, we biophysically characterized these nine samples and condensed the spectroscopic results into three-index empirical phase diagrams (EPDs). The EPDs show distinctions between the IpaD/SipD and LcrV subfamilies of tip proteins, based on their physical stability, even upon addition of the PA. Upon attachment to the BLPs, the BLPs become defining moiety in the spectroscopic measurements, leaving the tip proteins to have a subtle yet modulating effect on the structural integrity of the tip proteins-BLPs binding. In summary, this work provides a comprehensive view of physical stability of the tip proteins and tip protein-BLPs and serves as a baseline for screening of excipients to increase the stability of the tip protein-BLPs for future vaccine formulation., (© 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.)

The structural integrity and conformational stability of a genetically modified live, oncolytic herpes simplex virus (o-HSV) were investigated across a wide pH (5.5-8.0) and temperature (10°C-87.5°C) range. A combination of circular dichroism, intrinsic and extrinsic fluorescence, and static light scattering results was visualized using an empirical phase diagram approach to provide a global assessment of physical stability. Distinct phases were identified including the native state of the virus, an intermediate phase that could represent gradual swelling and/or shedding of the viral envelope, and a highly disrupted, aggregated phase. The nature of these altered forms of the virus was further evaluated by transmission electron microscopy and viral plaque assays. The effect of freeze-thaw (F/T) stress on o-HSV was also examined. After one F/T cycle, a loss of infectious virus titers was observed. In addition, the monomeric virus particle concentration decreased during F/T stress, whereas there was a concurrent increase in larger particles (2-10 μm). The comprehensive biophysical characterization of viral stability conducted in this study identified major degradation events leading to loss of infectivity of o-HSV and represents an important step toward stabilization of the virus against thermal and F/T stresses., (© 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.)

Shigella spp. are causative agents of bacillary dysentery, a human illness with high global morbidity levels, particularly among elderly and infant populations. Shigella infects via the fecal-oral route, and its virulence is dependent upon a type III secretion system (T3SS). Two components of the exposed needle tip complex of the Shigella T3SS, invasion plasmid antigen D (IpaD) and IpaB, have been identified as broadly protective antigens in the mouse lethal pneumonia model. A recombinant fusion protein (DB fusion) was created by joining the coding sequences of IpaD and IpaB. The DB fusion is coexpressed with IpaB's cognate chaperone, IpgC, for proper recombinant expression. The chaperone can then be removed by using the mild detergents octyl oligooxyethelene (OPOE) or N,N-dimethyldodecylamine N-oxide (LDAO). The DB fusion in OPOE or LDAO was used for biophysical characterization and subsequent construction of an empirical phase diagram (EPD). The EPD showed that the DB fusion in OPOE is most stable at neutral pH below 55 °C. In contrast, the DB fusion in LDAO exhibited remarkable thermal plasticity, since this detergent prevents the loss of secondary and tertiary structures after thermal unfolding at 90 °C, as well as preventing thermally induced aggregation. Moreover, the DB fusion in LDAO induced higher interleukin-17 secretion and provided a higher protective efficacy in a mouse challenge model than did the DB fusion in OPOE. These data indicate that LDAO might introduce plasticity to the protein, promoting thermal resilience and enhanced protective efficacy, which may be important in its use as a subunit vaccine., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

The ability of a GroEL-based bio-layer interferometry (BLI) assay to detect structurally altered and/or aggregated species of pharmaceutically relevant proteins is demonstrated. Assay development included optimizing biotinylated-GroEL immobilization to streptavidin biosensors, combined with biophysical and activity measurements showing native and biotinylated GroEL are both stable and active. First, acidic fibroblast growth factor (FGF-1) was incubated under conditions known to promote (40°C) and inhibit (heparin addition) molten globule formation. Heat exposed (40°C) FGF-1 exhibited binding to GroEL-biosensors, which was significantly diminished in the presence of heparin. Second, a polyclonal human IgG solution containing 6-8% non-native dimer showed an increase in higher molecular weight aggregates upon heating by size exclusion chromatography (SEC). The poly IgG solution displayed binding to GroEL-biosensors initially with progressively increased binding upon heating. Enriched preparations of the IgG dimers or monomers showed significant binding to GroEL-biosensors. Finally, a thermally treated IgG1 monoclonal antibody (mAb) solution also demonstrated increased GroEL-biosensor binding, but with different kinetics. The bound complexes could be partially to fully dissociated after ATP addition (i.e., specific GroEL binding) depending on the protein, environmental stress, and the assay's experimental conditions. Transmission electron microscopy (TEM) images of GroEL-mAb complexes, released from the biosensor, also confirmed interaction of bound complexes at the GroEL binding site with heat-stressed mAb. Results indicate that the GroEL-biosensor-BLI method can detect conformationally altered and/or early aggregation states of proteins, and may potentially be useful as a rapid, stability-indicating biosensor assay for monitoring the structural integrity and physical stability of therapeutic protein candidates., (© 2014 The Protein Society.)

Instability of vaccines often emerges as a key challenge during clinical development (lab to clinic) as well as commercial distribution (factory to patient). To yield stable, efficacious vaccine dosage forms for human use, successful formulation strategies must address a combination of interrelated topics including stabilization of antigens, selection of appropriate adjuvants, and development of stability-indicating analytical methods. This review covers key concepts in understanding the causes and mechanisms of vaccine instability including (1) the complex and delicate nature of antigen structures (e.g., viruses, proteins, carbohydrates, protein-carbohydrate conjugates, etc.), (2) use of adjuvants to further enhance immune responses, (3) development of physicochemical and biological assays to assess vaccine integrity and potency, and (4) stabilization strategies to protect vaccine antigens and adjuvants (and their interactions) during storage. Despite these challenges, vaccines can usually be sufficiently stabilized for use as medicines through a combination of formulation approaches combined with maintenance of an efficient cold chain (manufacturing, distribution, storage and administration). Several illustrative case studies are described regarding mechanisms of vaccine instability along with formulation approaches for stabilization within the vaccine cold chain. These include live, attenuated (measles, polio) and inactivated (influenza, polio) viral vaccines as well as recombinant protein (hepatitis B) vaccines., (Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The structural integrity and conformational stability of various IgG1-Fc proteins produced from the yeast Pichia pastoris with different glycosylation site occupancy (di-, mono-, and nonglycosylated) were determined. In addition, the physical stability profiles of three different forms of nonglycosylated Fc molecules (varying amino-acid residues at site 297 in the CH 2 domain due to the point mutations and enzymatic digestion of the Fc glycoforms) were also examined. The physical stability of these IgG1-Fc glycoproteins was examined as a function of pH and temperature by high-throughput biophysical analysis using multiple techniques combined with data visualization tools (three index empirical phase diagrams and radar charts). Across the pH range of 4.0-6.0, the di- and monoglycosylated forms of the IgG1-Fc showed the highest and lowest levels of physical stability, respectively, with the nonglycosylated forms showing intermediate stability depending on solution pH. In the aglycosylated Fc proteins, the introduction of Asp (D) residues at site 297 (QQ vs. DN vs. DD forms) resulted in more subtle changes in structural integrity and physical stability depending on solution pH. The utility of evaluating the conformational stability profile differences between the various IgG1-Fc glycoproteins is discussed in the context of analytical comparability studies., (© 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.)

Introduction: There have been rare case reports of damage to adjacent coronary arteries by screw-in pacemaker and implantable cardioverter-defibrillator (ICD) leads. Our aim was to assess the proximity of pacemaker and ICD leads to the major coronary anatomy using cardiac computed tomography (CT)., Methods: Cardiac CT images were retrospectively analyzed to assess the spatial relationship of device lead tips to the major coronary anatomy., Results: Fifty-two right ventricular (RV) leads (17 apical, 35 nonapical) and 35 right atrial (RA) leads were assessed. Leads on the RV antero-septal junction (20 of 52) were close (median 4.7 mm) to, and orientated toward, the left anterior descending (LAD) coronary artery. RA leads in the anterior (26 of 35) and lateral (seven of 35) walls of the RA appendage were not close to (16.9 ± 7.7 mm and 18.9 ± 12.4 mm, respectively) and directed away from the right coronary artery. However, an RA lead adjacent to the superior border of the tricuspid valve was 4.3 mm from the right coronary artery and an RA lead on the medial wall of the RA appendage was 1.6 mm away from the aorta. An RV pacemaker lead in the lateral wall of the RV inlet was 3.4 mm from the right coronary artery., Conclusions: In our cohort, a majority of RV leads were on the antero-septal junction and close to the overlying LAD coronary artery. RA leads adjacent to the tricuspid valve or on the medial RA appendage were in close proximity to the right coronary artery and aorta, respectively., (©2013 Wiley Periodicals, Inc.)

Introduction: We aimed to assess the utility of cardiac computed tomography (CT) in the evaluation of right atrial (RA) and right ventricular (RV) pacemaker and implantable cardiac defibrillator lead perforation., Methods: Images from a 320-slice electrocardiogram-gated cardiac CT scanner were retrospectively independently analyzed by two reviewers for lead position, pericardial effusion, and perforation.Perforation results were correlated with pacing sensing, impedance, and threshold measurements., Results: A total of 52 patients had RV leads and 35 had RA leads. Five of 17 RV apical, one of 35 RV nonapical, and none of the 35 RA leads perforated through the myocardium on CT imaging criteria. Two "clinically" perforated leads (that had protruded 5 mm and 15 mm from the outer edge of the myocardium)had pericardial effusions and changes in pacing parameters, and required RV lead repositioning. In contrast,there were four apparent "radiologic" perforations (that had protruded only an average 1.5±0.5 mm from the outer edge of the myocardium) that did not require repositioning. These had the radiologic appearance of perforation on cardiac CT; however, they were not associated with pericardial effusions or significant changes in RV pacing lead sensing, impedance, and threshold measurements., Conclusions: Cardiac CT scanning with multiplanar reformatting is useful for documenting lead position and assessing for possible cardiac perforation. The clinical significance and natural history of leads with only the appearance of perforation on cardiac CT is uncertain.

Introduction: It is hypothesized that pacing the right ventricular (RV) septum is associated with less deleterious outcomes than RV apical pacing. Our aim was to validate fluoroscopic and electrocardiography (ECG) criteria for describing pacemaker and implantable cardioverter defibrillator RV "septal" lead position against the proposed gold standard: cardiac computed tomography (CT)., Methods: Using the conventional fluoroscopic criteria, we intended to place RV nonapical leads on the interventricular septum. Lead positions were later retrospectively analyzed with CT and correlated with ECGs and fluoroscopic projections: posterior-anterior, 40° left anterior oblique (LAO), 40° right anterior oblique (RAO), and left lateral., Results: Only 21% (nine of 35) of presumed "septal" RV nonapical leads using the conventional fluoroscopic criteria were on the true septum. A schema developed to define septal position in the RAO fluoroscopic view had high agreement with CT images. ECG criteria had only fair to moderate agreement with CT. The paced QRS duration was significantly longer (P < 0.001) with RV apical pacing (176 ± 10.7 ms), compared to RV nonapical pacing (144.5 ± 14.3 ms)., Conclusion: Using the conventional fluoroscopic criteria, only a minority of RV leads were implanted on the true RV septum. Instead, aiming for the middle of the cardiac silhouette in the RAO fluoroscopic view, confirming rightward orientation in the LAO view, and having a paced QRS duration <140 ms may allow the implanting cardiologist a simple, more accurate method to achieve true RV septal lead positioning., (©2013, The Authors. Journal compilation ©2013 Wiley Periodicals, Inc.)

Background: Knowledge of the variations in the arterial supply of hand has reached a point of practical importance with the advent of microvascular surgery for revascularization, replantation and composite tissue transfers. Arterial supply of hand is derived from two anastomotic arches, formed between two main arteries of forearm i.e. radial, ulnar and their branches, in the palm., Objective: The superficial palmar arch shows variation in formation at the radial side. In the present study we have recorded its data which would help in its clinical and surgical implications., Material and Methods: In the present study we have studied the formation of superficial palmar arches and their variations in 100 cadaveric hands at Dr. D . Y. Patil Medical College, Pune and B.J. Government Medical College, Pune, India., Result and Conclusion: According to Adachi's classification the most predominant pattern obseved was of Ulnar type arch (66%). According to Coleman and Anson classification 82% showed complete (Group I) superficial palmar arches and a very low incidence (18%) of incomplete arches (Group II). This suggests that collateral circulation is present in majority of cases. This would result in least number of complications considering radial artery harvesting for coronary bypass. Sub-classification of arches according to Coleman and Anson 1961 indicates that the predominant type in the present study was of Group I (Type B) which is formed entirely by Ulnar Artery (56%). Median artery and ulnar artery forming an incomplete superficial arch under Group II (Type C) having an incidence of 4% was recorded. Thus in such cases radial artery harvesting for coronary artery bypass may prove to be less fatal. This study is an effort to provide data about the formation of superficial palmar arches which has been a centre of attraction for most of the surgical procedures and injuries of the hand.

IgG1 mAb solutions were prepared with and without sodium chloride and subjected to different environmental stresses. Formation of aggregates and particles of varying size was monitored by a combination of size-exclusion chromatography, Nanoparticle Tracking Analysis, Micro-flow Imaging (MFI), turbidity, and visual assessments. Stirring and heating induced the highest concentration of particles. In general, the presence of NaCl enhanced this effect. The morphology of the particles formed from mAb samples exposed to different stresses was analyzed from transmission electron microscopy and MFI images. Shaking samples without NaCl generated the most fibrillar particles, whereas stirring created largely spherical particles. The composition of the particles was evaluated for covalent cross-linking by SDS-PAGE, overall secondary structure by FTIR microscopy, and surface apolarity by extrinsic fluorescence spectroscopy. Freeze-thaw and shaking led to particles containing protein with native-like secondary structure. Heating and stirring produced IgG1-containing aggregates and particles with some non-native disulfide cross-links, varying levels of intermolecular beta sheet content, and increased surface hydrophobicity. These results highlight the importance of evaluating protein particle morphology and composition, in addition to particle number and size distributions, to better understand the effect of solution conditions and environmental stresses on the formation of protein particles in mAb solutions., (© 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.)

Changes in the measurements of a macromolecular biopharmaceutical's physical form are often used to predict changes in the drug's long-term stability. These can in turn be used as important markers of changes to a drug's efficacy and safety. Such stability estimates traditionally require human judgment and are frequently tentative. We introduce methods for developing mathematical models that predict a drug's long-term storage stability profile from measurements of short-term physical form and behavior. We measured the long-term (2 year) chemical and colloidal stability of Granulocyte Colony Stimulating Factor (GCSF) in 16 different liquid formulations. Shortly after formulations were placed on stability, we also employed various spectroscopic techniques to characterize the short-term thermal unfolding response of GCSF in the 16 formulations. The short-term data were processed using several data reduction methods, including reduction to spectra at low temperature, to melt curves, and to transition temperatures. Least squares fitting was used to predict the long-term stability measurements from the reduced short-term spectroscopic measurements. On the basis of the cross-validation and a permutation test, many of the long-term stability predictions have less than 1% probability of occurring by chance., (© 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.)

The recombinant hemagglutinin (rHA)-based influenza vaccine Flublok® has recently been approved in the United States as an alternative to the traditional egg-derived flu vaccines. Flublok is a purified vaccine with a hemagglutinin content that is threefold higher than standard inactivated influenza vaccines. When rHA derived from an H3N2 influenza virus was expressed, purified, and stored for 1 month, a rapid loss of in vitro potency (∼50%) was observed as measured by the single radial immunodiffusion (SRID) assay. A comprehensive characterization of the rHA protein antigen was pursued to identify the potential causes and mechanisms of this potency loss. In addition, the biophysical and chemical stability of the rHA in different formulations and storage conditions was evaluated over time. Results demonstrate that the potency loss over time did not correlate with trends in changes to the higher order structure or hydrodynamic size of the rHA. The most likely mechanism for the early loss of potency was disulfide-mediated cross-linking of rHA, as the formation of non-native disulfide-linked multimers over time correlated well with the observed potency loss. Furthermore, a loss of free thiol content, particularly in specific cysteine residues in the antigen's C-terminus, was correlated with potency loss measured by SRID., (© 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.)

Thirty-eight mutants of RNase Sa (ribonuclease from Streptomyces aureofaciens) were examined for their structure, thermal sensitivity, and tendency to aggregate. Although a biphasic correlation was seen between the effect of temperature on structure and the free energy of transfer changes in many of the mutants, little correlation was seen between the time at which aggregation is initiated or the rate of aggregation and the thermal sensitivity of the mutants. It is hypothesized that the nature of contacts between protein molecules in the associated (aggregated) phase rather than structural changes dominates the aggregation process for these series of mutants., (© 2013 Wiley Periodicals, Inc. and the American Pharmacists Association.)