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

To investigate the potential of two flow imaging microscopy (FIM) techniques (Micro-Flow Imaging (MFI) and FlowCAM) to determine total cell concentration and cell viability. B-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. All 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). FIM-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. PURPOSE METHODS RESULTS CONCLUSIONDrug Delivery Technolog

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

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 μm for Risperdal Consta; 16–124 μm for Sandostatin LAR). MFI additionally detected a ‘fines’ population (Drug Delivery Technolog

Towards the development of a supercritical carbon dioxide spray process to coat solid protein particles

The aim of this study was to develop a supercritical carbon dioxide (scCO2) spray process to coat solid protein particles with a hydrophilic polymer. The final purpose is to manufacture drug particles exhibiting controlled release behaviour in patients. Lysozyme microparticles (about 20 μm) were suspended in a vessel into which a dextran sulphate (DS) solution was dispersed by scCO2 via a nozzle. Upon interaction with the droplets, DS was deposited onto or mixed with suspended lysozyme particles. Particles of about 100 μm were obtained. The zeta-potential analysis and elemental analysis indicated that the top layer of the particles consisted of both lysozyme and DS. Some of the produced particulate materials showed retarded lysozyme release when exposed to water or phosphate buffered saline, holding promise for future production of controlled drug delivery systems for therapeutic proteins.Drug Delivery TechnologyBiopharmaceutic

Analysis of sub-visible particles in complex injectable formulations

<span style="font-size:12.0pt;font-family:"Times New Roman","serif"; mso-fareast-font-family:Calibri;mso-fareast-theme-font:minor-latin;mso-ansi-language: NL;mso-fareast-language:NL;mso-bidi-language:AR-SA">Complex injectable formulations, such as protein therapeutics, controlled release systems and cell therapy products, are gaining a paramount position in the therapy of many life-threatening and chronic diseases. Most of these products have in common that sub-visible particles (SVP), i.e., particulate matter in the size range of about 1 – 100 µm, are critical quality attributes. Most protein therapeutics are liquid or freeze-dried formulations in which the presence of SVP is unwanted; many injectable controlled release systems are based on particulate drug delivery systems in the sub-visible size range; and cell concentration and viability are important characteristics of cell therapy products. With the continuous improvement of existing and emerging particle analysis techniques, the potentials of these tools in addressing current characterization challenges in the field of complex injectable formulations have to be investigated. Therefore, the aim of this thesis was to develop methods, based on a set of state-of-the-art particle analysis techniques, for characterization of pharmaceutically relevant sub-visible particles and to study the value of these methods in the characterization of complex injectable formulations. </p

Potential Issues With the Handling of Biologicals in a Hospital

Drug Delivery Technolog

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 onprotein aggregation, in particular, in the nanometer and micrometer size range. An overhead stirring setupwas designed in which the presence and magnitude of the contact between the stir bar and thecontainer could be manipulated. A solution of 0.1 mg/mL of a monoclonal antibody (IgG) in phosphatebuffered saline was stirred at 300 rpm at room temperature. At different time points, samples were takenand analyzed by nanoparticle tracking analysis, flow imaging microscopy, and size-exclusion chromatography.In contrast to nonecontact-stirred and unstirred samples, the contact-stirred sample containedseveral-fold more particles and showed a significant loss of monomer. No increase in oligomer contentwas detected. The number of particles formed was proportional to the contact area and the magnitude ofthe normal pressure between the stir bar and the glass container. Extrinsic 9-(2,2-dicyanovinyl) julolidinefluorescence indicated a conformational change for contact-stirred protein samples. Presence ofpolysorbate 20 inhibited the formation of micron-sized aggregates. We suggest a model in whichabrasion of the potentially destabilized, adsorbed protein leads to aggregation and renewal of the surfacefor adsorption of a fresh protein layer.Drug Delivery Technolog

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

Personalised Therapeutic

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

Drug Delivery Technolog

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

PURPOSE: To investigate whether particle sedimentation velocity tracking using a flow imaging microscope (FlowCAM) can be used to determine microparticle porosity. METHODS: Two 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. RESULTS: Polystyrene 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. CONCLUSIONS: We developed two methods to determine average microparticle density and porosity by sedimentation velocity tracking, using only a few milligrams of powder. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11095-017-2120-8) contains supplementary material, which is available to authorized users

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

Drug Delivery Technolog