Flow Imaging Microscopy Research Articles

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.

IgG-loaded hyaluronan-based dissolving microneedles for intradermal protein delivery

Dissolving microneedles are an attractive approach for non-invasive delivery of drugs via the skin, particularly when the doses are in the microgram or low-milligram range. The aim of the study was to develop hyaluronan-based, monoclonal IgG-loaded microneedles for intradermal delivery enabling efficient penetration and rapid dissolution in the skin while preserving protein stability. Microscopic analysis showed successful preparation of sharp microneedles with the tip length of ~280μm and with up to 10% (w/w) of IgG content. The water content of the microneedles was ~12% and was not affected by the protein content. The protein distribution was uniform within microneedle tips and individual arrays but some array-to-array variation of IgG level within a single preparation batch was detected. After dissolution of microneedle arrays in PBS, >80% of protein was recovered and no conformational changes were detected by fluorescence spectroscopy. At submicron level, only weak and reversible interaction between HA and IgG was found by asymmetric flow field flow fractionation analysis after the dissolution of prepared microneedles. Although, the formation of insoluble micron-size particles was detected by flow imaging microscopy the IgG amount incorporated into these particles was negligible (<5%). Finally, microneedles were able to penetrate into the epidermis of ex vivo human skin followed by the rapid dissolution of the microneedle tips in the skin. After 10min of application, the majority of the original tip length was dissolved and IgG and hyaluronan were co-deposited until a depth of 150–200μm in the skin. In conclusion, developed hyaluronan-based dissolving microneedles allow rapid non-invasive intradermal protein delivery.

Measuring Subvisible Particles in Protein Formulations Using a Modified Light Obscuration Sensor with Improved Detection Capabilities.

Although light obscuration is the "gold standard" for subvisible particle measurements in biopharmaceutical products, the current technology has limitations with respect to the detection of translucent proteinaceous particles and particles of sizes smaller and around 2 μm. Here, we describe the evaluation of a modified light obscuration sensor utilizing a novel measuring mode. Whereas standard light obscuration methodology monitors the height (amplitude) of the signal, the new approach monitors its length (width). Experimental evaluation demonstrated that this new detection mode leads to improved detection of subvisible particles of sizes smaller than 2 μm, reduction of artifacts during measurements especially of low concentrations of translucent protein particles, and higher counting accuracy as compared to flow imaging microscopy and standard light obscuration measurements.

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, 0.01mg/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 2min after mixing, while a very high nanoparticle (size up to 500nm) concentration (ca. 9×108/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 1.5×106/ml until it started to slightly decrease after 10h. The average size of the microparticles remained in the low-μm range (1–2μm) with a slight increase and broadening of the size distribution in time. The experimental data could be fitted with Smoluchowski’s 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.

The Effect of Protein PEGylation on Physical Stability in Liquid Formulation

The presence of micron aggregates in protein formulations has recently attracted increased interest from regulatory authorities, industry, and academia because of the potential undesired side effects of their presence. In this study, we characterized the micron aggregate formation of hen egg-white lysozyme (Lyz) and its diPEGylated (5 kDa) analog as a result of typical handling stress conditions. Both proteins were subjected to mechanical stress in the absence and presence of silicone oil (SO), elevated temperatures, and freeze-thaw cycles. Flow imaging microscopy showed that PEGylated Lyz formed approximately half as many particles as Lyz, despite its lower apparent thermodynamic stability and more loose protein fold. Further characterization showed that the PEGylation led to a change from attractive to repulsive protein-protein interactions, which may partly explain the reduced particle formation. Surprisingly, the PEGylated Lyz adsorbed an order of magnitude faster onto SO, despite being much larger in size, as determined by small-angle X-ray scattering and dynamic light scattering measurements. Thus, PEGylation may significantly reduce, but not prevent, micron aggregate formation of a protein during typical handling stresses.

Scalable organic solvent free supercritical fluid spray drying process for producing dry protein formulations

In this study, we evaluated the influence of supercritical carbon dioxide (scCO2) spray drying conditions, in the absence of organic solvent, on the ability to produce dry protein/trehalose formulations at 1:10 and 1:4 (w/w) ratios. When using a 4L drying vessel, we found that decreasing the solution flow rate and solution volume, or increasing the scCO2 flow rate resulted in a significant reduction in the residual water content in dried products (Karl Fischer titration). The best conditions were then used to evaluate the ability to scale the scCO2 spray drying process from 4L to 10L chamber. The ratio of scCO2 and solution flow rate was kept constant. The products on both scales exhibited similar residual moisture contents, particle morphologies (SEM), and glass transition temperatures (DSC). After reconstitution, the lysozyme activity (enzymatic assay) and structure (circular dichroism, HP-SEC) were fully preserved, but the sub-visible particle content was slightly increased (flow imaging microscopy, nanoparticle tracking analysis). Furthermore, the drying condition was applicable to other proteins resulting in products of similar quality as the lysozyme formulations. In conclusion, we established scCO2 spray drying processing conditions for protein formulations without an organic solvent that holds promise for the industrial production of dry protein formulations.

Measurement of subvisible particulates in lyophilised Erwinia chrysanthemi L-asparaginase and relationship with clinical experience.

In order to generate further characterisation data for the lyophilised product Erwinia chrysanthemi l-asparaginase, reconstituted drug product (DP; marketed as Erwinase or Erwinaze) was analysed for subvisible (2–10 μm) particulate content using both the light obscuration (LO) method and the newer flow-imaging microscopy (FIM) technique. No correlation of subvisible particulate counts exists between FIM and LO nor do the counts correlate with activity at both release and on stability. The subvisible particulate content of lyophilised Erwinia l-asparaginase appears to be consistent and stable over time and in line with other parenteral biopharmaceutical products. The majority (ca. 75%) of subvisible particulates in l-asparaginase DP were at the low end of the measurement range by FIM (2–4 μm). In this size range, FIM was unable to definitively classify the particulates as either protein or non-protein. More sensitive measurement techniques would be needed to classify the particulates in lyophilised l-asparaginase into type (protein and non-protein), so the LO technique has been chosen for on-going DP analyses. E. chrysanthemi l-asparaginase has a lower rate of hypersensitivity compared with native Escherichia coli preparations, but a subset of patients develop hypersensitivity to the Erwinia enzyme. A DP lot that had subvisible particulate counts on the upper end of the measurement range by both LO and FIM had the same incidence of allergic hypersensitivity in clinical experience as lots at all levels of observed subvisible particulate content, suggesting that the presence of l-asparaginase subvisible particulates is not important with respect to allergic response.

Flow Imaging Microscopy for Protein Particle Analysis—A Comparative Evaluation of Four Different Analytical Instruments

Flow imaging microscopy was introduced as a technique for protein particle analysis a few years ago and has strongly gained in importance ever since. The aim of the present study was a comparative evaluation of four of the most relevant flow imaging microscopy systems for biopharmaceuticals on the market: Micro-Flow Imaging (MFI)4100, MFI5200, Flow Cytometer And Microscope (FlowCAM) VS1, and FlowCAM PV. Polystyrene standards, particles generated from therapeutic monoclonal antibodies, and silicone oil droplets were analyzed by all systems. The performance was critically assessed regarding quantification, characterization, image quality, differentiation of protein particles and silicone oil droplets, and handling of the systems. The FlowCAM systems, especially the FlowCAM VS1, showed high-resolution images. The FlowCAM PV system provided the most precise quantification of particles of therapeutic monoclonal antibodies, also under impaired optical conditions by an increased refractive index of the formulation. Furthermore, the most accurate differentiation of protein particles and silicone oil droplets could be achieved with this instrument. The MFI systems provided excellent size and count accuracy (evaluated with polystyrene standards) especially the MFI5200 system. This instrument also showed very good performance for protein particles, also in case of an increased refractive index of the formulation. Both MFI systems were easier to use and appeared more standardized regarding measurement and data analysis as compared to the FlowCAM systems. Our study shows that the selection of the appropriate flow imaging microscopy system depends strongly on the main output parameters of interest and it is recommended to decide based on the intended application.

Guidance for Industry: Immunogenicity Assessment for Therapeutic Protein Products [excerpts

Biotechnology Law ReportVol. 32, No. 3 DocumentsGuidance for Industry: Immunogenicity Assessment for Therapeutic Protein Products [excerpts]Published Online:13 Jun 2013https://doi.org/10.1089/blr.2013.9927AboutSectionsPDF/EPUB ToolsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail FiguresReferencesRelatedDetailsCited ByDiscrete, Chiral Polymer–Insulin Conjugates20 September 2022 | Journal of the American Chemical Society, Vol. 100Utility of Three Flow Imaging Microscopy Instruments for Image Analysis in Evaluating four Types of Subvisible Particle in BiopharmaceuticalsJournal of Pharmaceutical Sciences, Vol. 98Protein’s Protein Corona: Nanoscale Size Evolution of Human Immunoglobulin G Aggregates Induced by Serum Albumin13 July 2022 | ACS Applied Materials & Interfaces, Vol. 14, No. 29Phase 1b study of anti-NaPi2b antibody-drug conjugate lifastuzumab vedotin (DNIB0600A) in patients with platinum-sensitive recurrent ovarian cancerGynecologic Oncology, Vol. 158, No. 3Literature review in support of adjuvanticity/immunogenicity assessment of proteinsEFSA Supporting Publications, Vol. 16, No. 1Current possibilities of liquid chromatography for the characterization of antibody-drug conjugatesJournal of Pharmaceutical and Biomedical Analysis, Vol. 147Pharmacological vs . classical approaches in the design of first in man clinical drug trials6 October 2017 | British Journal of Clinical Pharmacology, Vol. 83, No. 12Antibody-drug conjugate characterization by chromatographic and electrophoretic techniquesJournal of Chromatography B, Vol. 1032Immunogenicity/Hypersensitivity of Biologics14 November 2013 | Toxicologic Pathology, Vol. 42, No. 1 Volume 32Issue 3Jun 2013 InformationCopyright 2013, Mary Ann Liebert, Inc.To cite this article:Guidance for Industry: Immunogenicity Assessment for Therapeutic Protein Products [excerpts].Biotechnology Law Report.Jun 2013.172-185.http://doi.org/10.1089/blr.2013.9927Published in Volume: 32 Issue 3: June 13, 2013PDF download