Continuous protein precipitation – A robust antibody purification method without the need for steady state conditions during continuous integrated production

Antibody precipitation as capture step is an alternative technology to chromatography, because it is a fully continuous method. Bioreactor effluent from a perfusion reactor can be directly processed without surge tanks and the method can be combined with flocculation. We hypothesized that a continuous precipitation and flocculation reactor can be operated under non-steady state conditions. This will allow the design of reactors with extremely short residence time. In batch precipitation steady stats is achieved when the floc size does not change over time. We developed a continuous capture step for antibodies by protein precipitation with PEG 6000. Precipitation and re-solubilization conditions were established by a high throughput approach in batch mode. Continuous precipitation was realized by a tubular reactor design containing static mixers. The particle size distribution of the product precipitate was on-line monitored by Focused Beam Reflectance Measurement (FBRM) in a flow cell. Various hold times (2 to 20 minutes) of the cell culture harvest and different product concentrations were used to simulate changes during continuous fermentation. Precipitate was collected and separation was performed by centrifugation. High molecular weight impurities were reduced by a factor of 5 to 10, yields of 90% and purity increase by a factor of 2.5 were achieved according to size exclusion chromatography. Using FBRM we were able to demonstrate that different residence times during precipitation do not significantly change the particle size distribution. Stable performance concerning product quality (yield, purity and high molecular weight impurities) for different residence times were demonstrated for up to 90 minutes runtime. The influence of product concentration on the precipitate was quantified by FBRM. We demonstrated that protein precipitation is a robust and feasible capture step for mAb purification that does not require steady state conditions and that such continuous reactors are not compulsory operated at steady state conditions. Optimization potential was identified and can be realized during upscale, and higher yields and better performance can be reasonably expected for larger scales

Continuous protein precipitation – A robust antibody purification method without the need for steady state conditions during continuous integrated production.

Antibody precipitation as capture step is an alternative technology to chromatography, because it is a fully continuous method. Bioreactor effluent from a perfusion reactor can be directly processed without surge tanks and the method can be combined with flocculation. We hypothesized that a continuous precipitation and flocculation reactor can be operated under non-steady state conditions. This will allow the design of reactors with extremely short residence time. In batch precipitation steady stats is achieved when the floc size does not change over time. We developed a continuous capture step for antibodies by protein precipitation with PEG 6000. Precipitation and re-solubilization conditions were established by a high throughput approach in batch mode. Continuous precipitation was realized by a tubular reactor design containing static mixers. The particle size distribution of the product precipitate was on-line monitored by Focused Beam Reflectance Measurement (FBRM) in a flow cell. Various hold times (2 to 20 minutes) of the cell culture harvest and different product concentrations were used to simulate changes during continuous fermentation. Precipitate was collected and separation was performed by centrifugation. High molecular weight impurities were reduced by a factor of 5 to 10, yields of 90% and purity increase by a factor of 2.5 were achieved according to size exclusion chromatography. Using FBRM we were able to demonstrate that different residence times during precipitation do not significantly change the particle size distribution. Stable performance concerning product quality (yield, purity and high molecular weight impurities) for different residence times were demonstrated for up to 90 minutes runtime. The influence of product concentration on the precipitate was quantified by FBRM. We demonstrated that protein precipitation is a robust and feasible capture step for mAb purification that does not require steady state conditions and that such continuous reactors are not compulsory operated at steady state conditions. Optimization potential was identified and can be realized during upscale, and higher yields and better performance can be reasonably expected for larger scales

Three-Dimensional Quantitative Co-Mapping of Pulmonary Morphology and Nanoparticle Distribution with Cellular Resolution in Nondissected Murine Lungs

Deciphering biodistribution, biokinetics, and biological effects of nanoparticles (NPs) in entire organs with cellular resolution remains largely elusive due to the lack of effective imaging tools. Here, light sheet fluorescence microscopy in combination with optical tissue clearing was validated for concomitant three-dimensional mapping of lung morphology and NP biodistribution with cellular resolution in nondissected ex vivo murine lungs. Tissue autofluorescence allowed for label-free, quantitative morphometry of the entire bronchial tree, acinar structure, and blood vessels. Co-registration of fluorescent NPs with lung morphology revealed significant differences in pulmonary NP distribution depending on the means of application (intratracheal instillation and ventilator-assisted aerosol inhalation under anesthetized conditions). Inhalation exhibited a more homogeneous NP distribution in conducting airways and acini indicated by a central-to-peripheral (C/P) NP deposition ratio of unity (0.98 ± 0.13) as compared to a 2-fold enhanced central deposition (C/P = 1.98 ± 0.37) for instillation. After inhalation most NPs were observed in the proximal part of the acini as predicted by computational fluid dynamics simulations. At cellular resolution patchy NP deposition was visualized in bronchioles and acini, but more pronounced for instillation. Excellent linearity of the fluorescence intensity-dose response curve allowed for accurate NP dosimetry and revealed ca. 5% of the inhaled aerosol was deposited in the lungs. This single-modality imaging technique allows for quantitative co-registration of tissue architecture and NP biodistribution, which could accelerate elucidation of NP biokinetics and bioactivity within intact tissues, facilitating both nanotoxicology studies and the development of nanomedicines

Three-Dimensional Quantitative Co-Mapping of Pulmonary Morphology and Nanoparticle Distribution with Cellular Resolution in Non-Dissected Murine Lungs.

Deciphering biodistribution, biokinetics and biological effects of nanoparticles (NPs) in entire organs with cellular resolution remains largely elusive due to the lack of effective imaging tools. Here, light sheet fluorescence microscopy in combination with optical tissue clearing was validated for concomitant three-dimensional mapping of lung morphology and NP biodistribution with cellular resolution in non-dissected ex vivo murine lungs. Tissue autofluorescence allowed for label-free, quantitative morphometry of the entire bronchial tree, acinar structure and blood vessels. Co-registration of fluorescent NPs with lung morphology revealed significant differences in pulmonary NP distribution depending on the means of application (intratracheal instillation and ventilator-assisted aerosol inhalation under anesthetized conditions). Inhalation exhibited a more homogeneous NP distribution in conducting airways and acini indicated by a central-to-peripheral (C/P) NP deposition ratio of unity (0.98 ± 0.13) as compared to a 2-fold enhanced central deposition (C/P = 1.98 ± 0.37) for instillation. After inhalation most of NPs were observed in proximal part of the acini as predicted by Computational Fluid Dynamics simulations. At cellular resolution patchy NP deposition was visualized in bronchioles and acini, but more pronounced for instillation. Excellent linearity of the fluorescence intensity-dose response curve allowed for accurate NP dosimetry and revealed ca. 5% of the inhaled aerosol was deposited in the lungs. This single-modality imaging technique allows for quantitative co-registration of tissue architecture and NP biodistribution, which could accelerate elucidation of NP biokinetics and bioactivity within intact tissues facilitating both nanotoxicology studies and the development of nanomedicines