Comparison of Ensemble and Single Molecule Methods for Particle Characterization and Binding Analysis of a PEGylated Single-Domain Antibody.

Domain antibodies (dAbs) are single immunoglobulin domains that form the smallest functional unit of an antibody. This study investigates the behavior of these small proteins when covalently attached to the polyethylene glycol (PEG) moiety that is necessary for extending the half-life of a dAb. The effect of the 40 kDa PEG on hydrodynamic properties, particle behavior, and receptor binding of the dAb has been compared by both ensemble solution and surface methods [light scattering, isothermal titration calorimetry (ITC), surface Plasmon resonance (SPR)] and single-molecule atomic force microscopy (AFM) methods (topography, recognition imaging, and force microscopy). The large PEG dominates the properties of the dAb-PEG conjugate such as a hydrodynamic radius that corresponds to a globular protein over four times its size and a much reduced association rate. We have used AFM single-molecule studies to determine the mechanism of PEG-dependent reductions in the effectiveness of the dAb observed by SPR kinetic studies. Recognition imaging showed that all of the PEGylated dAb molecules are active, suggesting that some may transiently become inactive if PEG sterically blocks binding. This helps explain the disconnect between the SPR, determined kinetically, and the force microscopy and ITC results that demonstrated that PEG does not change the binding energy.

Investigation of monoclonal antibody fragmentation artifacts in non-reducing SDS-PAGE.

Fragmentation of monoclonal antibodies has been routinely observed in non-reducing SDS-PAGE, mainly due to disulfide-bond scrambling catalyzed by free sulfhydryl groups, resulting in a method induced artifact. To minimize this artifact, alkylating agents like iodoacetamide (IAM) and N-ethylmaleimide (NEM) were commonly included in SDS sample buffer to block free sulfhydryls. However, the selection of agents and the applied concentrations differ from study to study. In addition, there is no direct comparison of these agents thus far, resulting in difficulties in selecting the suitable agent. To address these questions, we have tested the activities of IAM and NEM in inhibiting the fragment-band artifact of IgG4 monoclonal antibodies. Our data suggest that the inhibition activity of both agents is concentration dependent. Interestingly, 5mM NEM can achieve the same inhibition effect as 40 mM IAM. In addition, NEM still retained strong activity after prolonged sample heating, whereas IAM lost most of its activity. Overall, NEM appears to have a better inhibition effect than IAM on all tested IgG4 proteins, either with SDS-PAGE or CE-SDS methods. These observations demonstrate that NEM has stronger fragmentation inhibition activity than IAM, and thus is a more suitable alkylating agent for both SDS-PAGE and CE-SDS method to reduce this fragmentation artifact.

Application of the Gyrolab™ platform to ligand-binding assays: a user's perspective.

In many areas of drug discovery and development, scientists are in a constant search for methods and platforms to reduce assay time and cost. The Gyrolab™ microfluidics platform that we describe here promises to deliver faster ligand-binding assays with lower reagent and sample consumption, while maintaining good accuracy and precision. Due to its limited track record, we evaluated its performance on assays currently used to support pharmacokinetic and immunogenicity studies, and detection of host cell protein impurities in samples from biotechnology processes. This article summarizes our preliminary conclusions about the utility of the Gyrolab microfluidics platform from Gyros AB.

Imaging glycosylation.

The glycosylation state of individual antibodies was imaged using an atomic force microscope with a probe modified with lectins and an image acquisition system that permits simultaneous acquisition of sample topography data along with a map of lectin binding sites.

Controlled inactivation of recombinant viruses with vitamin B2.

Inactivated viruses are important tools for vaccine development and gene transfer. 8-Methoxypsoralen (8-MOP) and long-wavelength ultraviolet irradiation (LWUVI) inactivates many viruses. Toxicity limits its use in animals and humans. Toxicological and photosensitizing properties of riboflavin make it suitable for virus inactivation in preparations for biological use. Viruses expressing beta-galactosidase were mixed with either 8-MOP (1.5mM) or riboflavin (50 microM) and exposed to LWUVI (365 nm) for 2 h. Virus activity was determined by limiting dilution. The half-life of the adenovirus preparation treated with 8-MOP was 8.28 ns(-1) and 36.5 ns(-1) after treatment with riboflavin. Despite the difference in half-life, both preparations were completely inactivated within 45 min. In contrast, the half-lives for adeno-associated virus (AAV) preparations were similar (63 ns(-1) 8-MOP vs. 67 ns(-1) riboflavin). Each AAV preparation was fully inactivated within 90 min. The half-life of lentivirus was 193.4 ns(-1) after treatment with 8-MOP and 208 ns(-1) after exposure to riboflavin. Virus treated with riboflavin was inactivated within 20 min. Virus exposed to 8-MOP was inactivated in 90 min. DNA and RNA viruses can be inactivated by riboflavin and LWUVI and used in physiological systems sensitive to other photochemicals.

Particle size coarsening induced by valve silicone in a metered dose inhaler.

The objective of this study was to evaluate the effect of valve silicone on the delivered particle size distribution of a suspension metered dose inhaler (MDI). Valves were manufactured with distinct levels of silicone, which could be differentiated with Fourier transform infrared spectroscopy (FT-IR). The amount of silicone in the valve was proportional to the amount of silicone that entered the formulation and the subsequent decrease in fine particle fraction (FPF) of the active pharmaceutical ingredient (API) measured by Andersen cascade impaction. The effect of silicone content was not linear as even small amounts of silicone made a significant contribution to particle size coarsening. This coarsening was also a function of storage time and temperature. Accelerated stability conditions greatly increased coarsening kinetics as 1 month at 40 degrees C and 75% RH induced significantly more coarsening than 12 months at room temperature. Field emission scanning electron micrograph images suggest that the primary mechanism of particle size change may be aggregation as particle clusters were seen. This study indicates that silicone can be a critical process parameter for particle size distribution of a suspension MDI product. Thus, the amount of silicone in the valves needs to be minimized and controlled.

Comparative thermal stabilities of recombinant adenoviruses and hexon protein.

Differential scanning calorimetry was used to identify the thermal stability profile of the replication deficient and protein IX deleted recombinant adenovirus type 5 that contains the p53 transgene (rAd/p53) in phosphate buffered saline (vPBS) or 10% glycerol (TRIS/phosphate buffer). The wildtype adenovirus (Ad/WT) and purified hexon protein (major capsid protein) were also evaluated in 10% glycerol (TRIS/phosphate buffer) as controls. The thermal profile of rAd/p53 revealed three endothermic transitions (T1, T2 and T3) occurring between 25 degrees C and 90 degrees C. T1, which occurred at 46.7 degrees C in vPBS and 49.4 degrees C in TRIS/PO4 10% glycerol buffer, was irreversible following repeated scanning and attributed to the degradation of the intact vector. The latter two endothermic transitions, T2 and T3, occurring at 69 degrees C and 78 degrees C, respectively, corresponded with the two transitions of purified hexon in temperature and amount of heat absorbed. The thermal profile of Ad/WT revealed four endothermic transitions at 51.5 degrees C (T1), 70.5 degrees C (T2A), 73.6 degrees C (T2B), and 77.4 degrees C (T3). The higher temperature of degradation as well as additional transition was attributed to the presence of protein IX associated with the hexon. The positions and excess molar heat capacities of the intact rAds were found to be affected by pH, glycerol, vector concentration and the presence or absence of protein IX in the capsid. Irreversibility of T1 implied that the degradation of the intact virus may follow first-order kinetics. The thermal scan rate dependence of T1 further confirmed that degradation of the intact virus may be first-order. The apparent activation energies for the degradation of the intact vectors were determined from the scan rate dependence of T1 and shown to be affected by protein IX in the capsid and solution conditions. Analysis of rAd samples incubated at 45 degrees C by Field Emission Electron Microscopy (FESEM) confirmed that loss of single particles was first-order. Although aggregates were observed in the samples, degradation appeared to be the dominant reaction leading to disappearance of single virions from the aqueous matrix. Based on thermal and FESEM analysis, an empirical model was proposed that accounted for the disappearance of single rAd particles. At or near T1, degradation of rAd particles followed a unidirectional, pseudo-first order reaction. However, at lower temperatures, disappearance of single virions resulted from competing irreversible degradation and aggregation reactions.

Use of an automated image processing program to quantify recombinant adenovirus particles.

Electron microscopy has a pivotal role as an analytical tool in pharmaceutical research. However, digital image data have proven to be too large for efficient quantitative analysis. We describe here the development and application of an automated image processing (AIP) program that rapidly quantifies shape measurements of recombinant adenovirus (rAd) obtained from digitized field emission scanning electron microscope (FESEM) images. The program was written using the macro-recording features within Image-Pro Plus software. The macro program, which is linked to a Microsoft Excel spreadsheet, consists of a series of subroutines designed to automatically measure rAd vector objects from the FESEM images. The application and utility of this macro program has enabled us to rapidly and efficiently analyze very large data sets of rAd samples while minimizing operator time.

Influence of the size of micronized active pharmaceutical ingredient on the aerodynamic particle size and stability of a metered dose inhaler.

Pharmaceutical inhalers are often used to treat pulmonary diseases. Only active pharmaceutical ingredient (API) particles from these inhalers that are less than approximately 5 microm are likely to reach the lung and be efficacious. This study was designed to investigate the impact of micronized API particle size on the aerodynamic particle size distribution (PSD) profile and the particle size stability of a suspension metered dose inhaler (MDI) containing propellant HFA-227 (1,1,1,2,3,3,3 heptafluoropropane) and a corticosteroid. The median API particle size ranged from 1.1 microm to 1.8 microm (97% to 70% of particles <3 microm, respectively). This study showed that increasing the particle size of the API used to manufacture a suspension MDI product increased the aerodynamic PSD of the MDI product. Furthermore, upon storage of the MDI product under temperature cycling conditions, samples containing larger-size API particles were less stable with respect to their aerodynamic PSD than those with smaller-size API particles. It was found that size-dependent particle growth and/or aggregation of the suspended API may be occurring as a result of temperature cycling. In conclusion, this study has shown that the particle size of the raw API impacts the properties and stability of the emitted aerosol spray. Based on the findings from this study, it is recommended that the API particle size be carefully controlled in order to meet specifications set for the finished MDI product.

Development of a biosensor-based method for detection and isotyping of antibody responses to adenoviral-based gene therapy vectors.

A biosensor-based assay, using a surface plasmon resonance detection system, was developed to detect and isotype anti-adenoviral antibodies in patients dosed with an adenoviral-based gene therapy vector. In the assay, whole, intact virus was immobilized onto the sensor chip surface. Electron microscopy and monoclonal antibody studies provide evidence that the virus remains intact after immobilization. The patients tested had preexisting serum levels of anti-adenoviral antibodies. A classic anamnestic response was observed in patients dosed with the gene-therapy agent. Isotyping experiments indicated that IgG antibodies predominated in serum even at the predose time point. Analysis of ascites fluid samples from some patients indicated detectable levels of IgA in addition to IgG. Results obtained using the biosensor-based assay corresponded to an existing enzyme-linked immunosorbent assay. The assay was easy to perform and the automated instrument reduced the required "hands on" time. In addition to studying the development of anti-adenoviral antibodies, the techniques described may be applied to virus:receptor interaction studies or antiviral drug:virus interaction studies.

The use of field emission scanning electron microscopy to assess recombinant adenovirus stability.

A field emission scanning electron microscopy (FESEM) method was developed to assess the stability of a recombinant adenovirus (rAd). This method was designed to simultaneously sort, count, and size the total number of rAd viral species observed within an image field. To test the method, a preparation of p53 transgene-expressing recombinant adenovirus (rAd/p53) was incubated at 37 degrees C and the viral particles were evaluated by number, structure, and degree of aggregation as a function of time. Transmission electron microscopy (TEM) was also used to obtain ultrastructural detail. In addition, the infectious activity of the incubated rAd/p53 samples was determined using flow cytometry. FESEM image-analysis revealed that incubation at 37 degrees C resulted in a time-dependent decrease in the total number of detectable single rAd/p53 virus particles and an increase in apparent aggregates composed of more than three adenovirus particles. There was also an observed decrease in both the diameter and perimeter of the single rAd/p53 viral particles. TEM further revealed the accumulation of damaged single particles with time at 37 degrees C. The results of this study demonstrate that FESEM, coupled with sophisticated image analysis, may be an important tool in quantifying the distribution of aggregated species and assessing the overall stability of rAd samples.