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

Immunogenicity of recombinant interferon beta aggregates : mechanistic studies in transgenic immune tolerant mice

Therapeutic proteins have become an important class of drugs. One of those proteins currently on the market is recombinant human interferon beta (rhIFN?), used to treat multiple sclerosis. After prolonged treatment, a substantial proportion of patients form antibodies against the protein. The immunological response generally starts with the appearance of binding antibodies possibly affecting the clearance of the drug, followed by neutralizing antibodies that block the biological activity of the protein. Such immune response is not expected, since human proteins are in principle tolerated by the human immune system. Several factors contribute to immunogenicity but protein aggregates are believed to play a major role. To study the relation between protein structure and immunogenicity, transgenic animal models immune tolerant for the protein were developed. This thesis aimed at providing mechanistic insight into the relation between rhIFN? aggregation and its immunogenicity. Transgenic C57Bl/6 mice immune tolerant for hIFN? have been applied as tools to study the breaking of immune tolerance by rhIFN?. RhIFN?-1a products formulated with HSA failed to induce antibodies in wildtype C57Bl/6 mice, which led to the conclusion that the transgenic C57Bl/6 mice cannot be used to study the immunogenicity of these products. For this reason, a second mouse model was developed by crossing the original transgenic C57Bl/6 mice with wildtype FVB/N mice. Both wildtype and transgenic C57Bl/6 x FVB/N hybrid offspring were used to evaluate the immunogenicity of three marketed products, Rebif (rhIFN?-1a), Avonex (rhIFN?-1a) and Betaferon (rhIFN?-1b). All products were immunogenic in wildtype hybrid mice, while the immunogenicity in transgenic hybrid mice was product dependent and in line with clinical observations. In conclusion, the hybrid mouse model offered unique possibilities to analyze the immunogenicity of a wide range of rhIFN? preparations, study factors that are of influence and look into the mechanism that is responsible for overcoming immune tolerance. The research described in this thesis led to greater insight in the mechanism by which rhIFN? aggregates overcome immune tolerance for the human protein. The transgenic immune tolerant mouse model that was developed acted as useful instrument for investigating the immunogenicity of a wide range of aggregated products. The transgenic mice enabled us to look into the features of the immune response induced by rhIFN? aggregates, the results of which indicate that CD4+ T cells as well as MZ B cells are involved but NABs and immunological memory are lacking within the time frame of the experiments. Not all rhIFN? aggregates were equally immunogenic, but removing aggregates eliminated the immune response against rhIFN? in transgenic mice. Aggregated rhIFN? preparations that were immunogenic in transgenic mice appeared to contain native-like protein. Moreover, it was shown that different pathways of oxidizing rhIFN? may lead to the formation of aggregates with greatly enhanced immunogenicity. Finally, it was demonstrated that rhIFN? adsorbs readily to common surface materials such as glass, metal and polystyrene. In particular complexes of rhIFN? with metal microparticles induced high levels of anti-rhIFN? IgG in transgenic mice, highlighting the importance of measuring and characterizing (sub)visible particulates in protein formulation

Immunogenicity of recombinant human interferon beta interacting with particles of glass, metal, and polystyrene

Aggregates play a major role in the immunogenicity of recombinant human interferon beta (rhIFNβ), a protein used to treat multiple sclerosis. A possible cause of aggregation is interaction between therapeutic protein and surfaces encountered during processing, storage, and administration. Moreover, proteins may adsorb to particles shed from these surfaces. In this work, we studied the immunogenicity of recombinant human interferon beta-1a (rhIFNβ-1a) interacting with glass microparticles, stainless steel microparticles, and polystyrene nanoparticles. At physiological pH, rhIFNβ-1a readily adsorbed to the particles, while the degree of adsorption was influenced by the ionic strength of the phosphate buffer. Front-face fluorescence showed that the tertiary structure of rhIFNβ-1a slightly changed upon adsorption to glass. The interaction with stainless steel microparticles resulted in increased levels of aggregates in the free protein fraction. Furthermore, protein adsorbed to stainless steel microparticles was more difficult to desorb than protein adsorbed to glass. Incubation with stainless steel considerably enhanced the immunogenicity of rhIFNβ-1a in transgenic mice immune tolerant for human interferon beta. The protein fraction adsorbed on stainless steel particles was responsible for this. In conclusion, rhIFNβ-1a adsorbs to common hydrophilic surface materials, possibly increasing the immunogenicity of the protein

Allergen Ara h 1 occurs in peanuts as a large oligomer rather than as a trimer

Ara h 1, a major peanut allergen, is known as a stable trimeric protein. Nevertheless, upon purification of native Ara h 1 from peanuts using only size exclusion chromatography, the allergen appeared to exist in an oligomeric structure, rather than as a trimeric structure. The oligomeric structure was independent of the salt concentration applied. Subjecting the allergen to anion exchange chromatography induced the allergen to dissociate into trimers. Ammonium sulfate precipitation did not bring about any structural changes, whereas exposing the allergen to hydrophobic interaction chromatography caused it to partly dissociate into trimers, with increasing amounts of trimers at higher ionic strengths. The (partial) dissociation into trimers led to a change in the tertiary structure of the monomeric subunits of the allergen, with the monomers in Ara h 1 oligomers having a more compact tertiary structure compared with the monomers in Ara h 1 trimers. As structural characteristics are important for a protein's allergenicity, this finding may imply a different allergenicity for Ara h 1 than previously described. © 2006 American Chemical Society

Allergen Ara h 1 Occurs in Peanuts as a Large Oligomer Rather Than as a Trimer

Ara h 1, a major peanut allergen, is known as a stable trimeric protein. Nevertheless, upon purification of native Ara h 1 from peanuts using only size exclusion chromatography, the allergen appeared to exist in an oligomeric structure, rather than as a trimeric structure. The oligomeric structure was independent of the salt concentration applied. Subjecting the allergen to anion exchange chromatography induced the allergen to dissociate into trimers. Ammonium sulfate precipitation did not bring about any structural changes, whereas exposing the allergen to hydrophobic interaction chromatography caused it to partly dissociate into trimers, with increasing amounts of trimers at higher ionic strengths. The (partial) dissociation into trimers led to a change in the tertiary structure of the monomeric subunits of the allergen, with the monomers in Ara h 1 oligomers having a more compact tertiary structure compared with the monomers in Ara h 1 trimers. As structural characteristics are important for a protein's allergenicity, this finding may imply a different allergenicity for Ara h 1 than previously described

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

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