Rapid purification of mAb using protein a membranes yielding high HCP clearance.

Protein A chromatography remains the crucial step in mAb purification because of the high binding specificity and impurity clearance. In recent years, highly productive membrane adsorbers emerged as an alternative to traditional resins allowing for rapid purification of biomolecules. In this study, we tested three commercially available protein A membranes (Sartobind® Rapid A, HiTrap Fibro™ PrismA and GORE™ Protein Capture Device) regarding flow distribution, permeability and binding performance.

Integration of a perfusion reactor and continuous precipitation in an entirely membrane-based process for antibody capture.

Continuous precipitation coupled with continuous tangential flow filtration is a cost-effective alternative for the capture of recombinant antibodies from crude cell culture supernatant. The removal of surge tanks between unit operations, by the adoption of tubular reactors, maintains a continuous harvest and mass flow of product with the advantage of a narrow residence time distribution (RTD). We developed a continuous process implementing two orthogonal precipitation methods, CaCl precipitation for removal of host-cell DNA and polyethylene glycol (PEG) for capturing the recombinant antibody, with no influence on the glycosylation profile.

Fractal dimension of antibody-PEG precipitate: Light microscopy for the reconstruction of 3D precipitate structures.

Protein and in particular antibody precipitation by PEG is a cost-effective alternative for the first capture step. The 3D structure of precipitates has a large impact on the process parameters for the recovery and dissolution, but current technologies for determination of precipitate structures are either very time consuming (cryo-TEM) or only generate an average fractal dimension (light scattering). We developed a light microscopy based reconstruction of 3D structures of individual particles with a resolution of 0.

Continuous capture of recombinant antibodies by ZnCl precipitation without polyethylene glycol.

The capture of recombinant antibodies from cell culture broth is the first critical step of downstream processing. We were able to develop a precipitation-based method for the capture and purification of monoclonal antibodies based on divalent cations, namely ZnCl. Traditional precipitation processes have to deal with high dilution factors especially for resolubilization and higher viscosity due to the use of PEG as precipitation or co-precipitation agent.

Soft sensor based on 2D-fluorescence and process data enabling real-time estimation of biomass in cultivations.

In bioprocesses, specific process responses such as the biomass cannot typically be measured directly on-line, since analytical sampling is associated with unavoidable time delays. Accessing those responses in real-time is essential for Quality by Design and process analytical technology concepts. Soft sensors overcome these limitations by indirectly measuring the variables of interest using a previously derived model and actual process data in real time.

Continuous cell flocculation for recombinant antibody harvesting.

Background: Integrated continuous production technology is of great interest in biopharmaceutical industry. Efficient, flexible and cost effective methods for continuous cell removal have to be developed, before a fully continuous and integrated product train can be realized. The paper describes the development and testing of such an integrated continuous and disposable set-up for cell separation by flocculation combined with depth filtration.

Protein adsorption onto nanoparticles induces conformational changes: Particle size dependency, kinetics, and mechanisms.

The use of nanomaterials in bioapplications demands a detailed understanding of protein-nanoparticle interactions. Proteins can undergo conformational changes while adsorbing onto nanoparticles, but studies on the impact of particle size on conformational changes are scarce. We have shown that conformational changes happening upon adsorption of myoglobin and BSA are dependent on the size of the nanoparticle they are adsorbing to.