Ahmad S. Sediq

No Touching! Abrasion of Adsorbed Protein Is the Root Cause of Subvisible Particle Formation During Stirring.

This study addressed the effect of contact sliding during stirring of a monoclonal antibody solution on protein aggregation, in particular, in the nanometer and micrometer size range. An overhead stirring set-up was designed in which the presence and magnitude of the contact between the stir bar and the container could be manipulated. A solution of 0.1 mg/mL of a monoclonal antibody (IgG) in phosphate buffered saline was stirred at 300 rpm at room temperature. At different time points, samples were taken and analyzed by nanoparticle tracking analysis, flow imaging microscopy, and size-exclusion chromatography. In contrast to non-contact-stirred and unstirred samples, the contact-stirred sample contained several-fold more particles and showed a significant loss of monomer. No increase in oligomer content was detected. The number of particles formed was proportional to the contact area and the magnitude of the normal pressure between the stir bar and the glass container. Extrinsic 9-(2,2-dicyanovinyl) julolidine fluorescence indicated a conformational change for contact-stirred protein samples. Presence of polysorbate 20 inhibited the formation of micron-sized aggregates. We suggest a model in which abrasion of the potentially destabilized, adsorbed protein leads to aggregation and renewal of the surface for adsorption of a fresh protein layer.

Protein–polyelectrolyte interactions: Monitoring particle formation and growth by nanoparticle tracking analysis and flow imaging microscopy

The purpose of this study was to investigate the formation and growth kinetics of complexes of proteins and oppositely charged polyelectrolytes. Equal volumes of IgG and dextran sulfate (DS) solutions, 0.01 mg/ml each in 10mM phosphate, pH 6.2, were mixed. At different time points, samples were taken and analyzed by nanoparticle tracking analysis (NTA), Micro-Flow Imaging (MFI) and size-exclusion chromatography (SEC). SEC showed a huge drop in monomer content (approximately 85%) already 2 min after mixing, while a very high nanoparticle (size up to 500 nm) concentration (ca. 9 × 10(8)/ml) was detected by NTA. The nanoparticle concentration gradually decreased over time, while the average particle size increased. After a lag time of about 1.5h, a steady increase in microparticles was measured by MFI. The microparticle concentration kept increasing up to about 1.5 × 10(6)/ml until it started to slightly decrease after 10h. The average size of the microparticles remained in the low-μm range (1-2 μm) with a slight increase and broadening of the size distribution in time. The experimental data could be fitted with Smoluchowskis perikinetic coagulation model, which was validated by studying particle growth kinetics in IgG:DS mixtures of different concentrations. In conclusion, the combination of NTA and MFI provided novel insight into the kinetics and mechanism of protein-polyelectrolyte complex formation.

Determination of the Porosity of PLGA Microparticles by Tracking Their Sedimentation Velocity Using a Flow Imaging Microscope (FlowCAM)

PurposeTo investigate whether particle sedimentation velocity tracking using a flow imaging microscope (FlowCAM) can be used to determine microparticle porosity.MethodsTwo different methods were explored. In the first method the sedimentation rate of microparticles was tracked in suspending media with different densities. The porosity was calculated from the average apparent density of the particles derived by inter- or extrapolation to the density of a suspending medium in which the sedimentation velocity was zero. In the second method, the microparticle size and sedimentation velocity in one suspending fluid were used to calculate the density and porosity of individual particles by using the Stokes’ law of sedimentation.ResultsPolystyrene beads of different sizes were used for the development, optimization and validation of the methods. For both methods we found porosity values that were in excellent agreement with the expected values. Both methods were applied to determine the porosity of three PLGA microparticle batches with different porosities (between about 4 and 52%). With both methods we obtained microparticle porosity values similar to those obtained by mercury intrusion porosimetry.ConclusionsWe developed two methods to determine average microparticle density and porosity by sedimentation velocity tracking, using only a few milligrams of powder.

Potential Issues With the Handling of Biologicals in a Hospital

A master student, who surveyed the procedures in a hospital pharmacy with regard to the handling of biologicals, identified several issues that might have jeopardized product quality. This case may be a tip of the iceberg and illustrates the urgent need for a better education of end-users about how to handle biologicals.

Micro-Flow Imaging as a quantitative tool to assess size and agglomeration of PLGA microparticles

Graphical abstract Figure. No caption available. ABSTRACT The purpose of this study was to explore the potential of flow imaging microscopy to measure particle size and agglomeration of poly(lactic‐co‐glycolic acid) (PLGA) microparticles. The particle size distribution of pharmaceutical PLGA microparticle products is routinely determined with laser diffraction. In our study, we performed a unique side‐by‐side comparison between MFI 5100 (flow imaging microscopy) and Mastersizer 2000 (laser diffraction) for the particle size analysis of two commercial PLGA microparticle products, i.e., Risperdal Consta and Sandostatin LAR. Both techniques gave similar results regarding the number and volume percentage of the main particle population (28–220 &mgr;m for Risperdal Consta; 16–124 &mgr;m for Sandostatin LAR). MFI additionally detected a ‘fines’ population (<28 &mgr;m for Risperdal Consta; <16 &mgr;m for Sandostatin LAR), which was overlooked by Mastersizer. Moreover, MFI was able to split the main population into ‘monospheres’ and ‘agglomerates’ based on particle morphology, and count the number of particles in each sub‐population. Finally, we presented how MFI can be applied in process development of risperidone PLGA microparticles and to monitor the physical stability of Sandostatin LAR. These case studies showed that MFI provides insight into the effect of different process steps on the number, size and morphology of fines, monospheres and agglomerates as well as the extent of microparticle agglomeration after reconstitution. This can be particularly important for the suspendability, injectability and release kinetics of PLGA microparticles.

Label-Free, Flow-Imaging Methods for Determination of Cell Concentration and Viability

PurposeTo investigate the potential of two flow imaging microscopy (FIM) techniques (Micro-Flow Imaging (MFI) and FlowCAM) to determine total cell concentration and cell viability.MethodsB-lineage acute lymphoblastic leukemia (B-ALL) cells of 2 different donors were exposed to ambient conditions. Samples were taken at different days and measured with MFI, FlowCAM, hemocytometry and automated cell counting. Dead and live cells from a fresh B-ALL cell suspension were fractionated by flow cytometry in order to derive software filters based on morphological parameters of separate cell populations with MFI and FlowCAM. The filter sets were used to assess cell viability in the measured samples.ResultsAll techniques gave fairly similar cell concentration values over the whole incubation period. MFI showed to be superior with respect to precision, whereas FlowCAM provided particle images with a higher resolution. Moreover, both FIM methods were able to provide similar results for cell viability as the conventional methods (hemocytometry and automated cell counting).ConclusionFIM-based methods may be advantageous over conventional cell methods for determining total cell concentration and cell viability, as FIM measures much larger sample volumes, does not require labeling, is less laborious and provides images of individual cells.

Cathodic Corrosion of a Bulk Wire to Nonaggregated Functional Nanocrystals and Nanoalloys

A key enabling step in leveraging the properties of nanoparticles (NPs) is to explore new, simple, controllable, and scalable nanotechnologies for their syntheses. Among “wet” methods, cathodic corrosion has been used to synthesize catalytic aggregates with some control over their size and preferential faceting. Here, we report on a modification of the cathodic corrosion method for producing a range of nonaggregated nanocrystals (Pt, Pd, Au, Ag, Cu, Rh, Ir, and Ni) and nanoalloys (Pt50Au50, Pd50Au50, and AgxAu100–x) with potential for scaling up the production rate. The method employs poly(vinylpyrrolidone) (PVP) as a stabilizer in an electrolyte solution containing nonreducible cations (Na+, Ca2+), and cathodic corrosion of the corresponding wires takes place in the electrolyte under ultrasonication. The ultrasonication not only promotes particle–PVP interactions (enhancing NP dispersion and diluting locally high NP concentration) but also increases the production rate by a factor of ca. 5. Further increase in the production rate can be achieved through parallelization of electrodes to construct comb electrodes. With respect to applications, carbon-supported Pt NPs prepared by the new method exhibit catalytic activity and durability for methanol oxidation comparable or better than the commercial benchmark catalyst. A variety of AgxAu100–x nanoalloys are characterized by ultraviolet–visible absorption spectroscopy and high-resolution transmission electron microscopy. The protocol for NP synthesis by cathodic corrosion should be a step toward its further use in academic research as well as in its practical upscaling.

A Flow Imaging Microscopy–Based Method Using Mass-to-Volume Ratio to Derive the Porosity of PLGA Microparticles

The release of drugs from poly(lactic-co-glycolic acid) (PLGA) microparticles depends to a large extent on the porosity of the particles. Therefore, porosity determination of PLGA microparticles is extremely important during pharmaceutical product development. Currently, mercury intrusion porosimetry (MIP) is widely used despite its disadvantages, such as the need for a large amount of sample (several hundreds of milligrams) and residual toxic waste. Here, we present a method based on the estimation of the volume of a known mass (a few milligrams) of particles using micro-flow imaging (MFI) to determine microparticle batch porosity. Factors that are critical for the accuracy of this method (i.e., density of the suspending fluid, particle concentration, and postsample rinsing) were identified and measures were taken to minimize potential errors. The validity of the optimized method was confirmed by using nonporous polymethylmethacrylate microparticles. Finally, the method was employed for the analysis of 7 different PLGA microparticle batches with various porosities (4.0%-51.9%) and drug loadings (0%-38%). Obtained porosity values were in excellent agreement with the MIP-derived porosities. Altogether, the developed MFI-based method is a valuable tool for deriving the total volume of a known mass of PLGA particles and therewith their porosity.