Characterization of hydrodynamics and volumetric power input in microtiter plates for the scale-up of downstream operations.

Parameter estimation for scale-up of downstream operations from microtiter plates (MTPs) is mostly done empirically because engineering correlations between microplates and stirred tank reactors (STRs) are not yet available. It is challenging to change the operation mode from shaken MTPs to large-scale STRs. For the scale-up of STRs, volumetric power input is well-established although it is unclear whether this parameter can be used to transfer the operations from MTPs. We determine the volumetric power input in MTPs via the temperature increase caused by the motion of the liquid. The hydrodynamics in MTPs are studied with computational fluid dynamics (CFD). Mixing is investigated in 96-, 24-, and 6-well MTPs to cover different geometries, filling volumes, shaking diameters, and shaking frequencies. All CFD simulations are validated by experimental results, which now allows prediction of the volumetric power input and hydrodynamics at various conditions in MTPs without the need for further experiments. We provide a map of the power input achievable in MTPs. Based on this map, from knowing about large-scale conditions, adequate microscale conditions can be adjusted for process development. This enables the direct scale-up of downstream unit operations from MTPs to STRs.

Media on-demand: Continuous reconstitution of a chemically defined media directly from solids.

Chemically defined media are reconstituted batchwise and stored in hold tanks until use. To avoid large hold tanks and batchwise production of media, we developed continuous on-demand reconstitutions directly from solids consisting of a hopper and a screw conveyor capable of feeding dry powdered media with the required precision ±5% at low dosing rates of 0.171 g min-1 . A commercially available dry powdered cell culture medium was continuously fed over a duration of 12 h into a mixer which was connected to a UV-cell for monitoring and the media were compared to a batchwise production. A comparable amino acid, carbohydrate, and osmolality profile to a batchwise reconstitution could be obtained. Cell cultivation showed comparable performance of batch and continuous reconstitution for two CHO cell lines producing the antibodies adalimumab and trastuzumab on a small and benchtop scale. In-depth analysis of the produced antibodies showed the same glycosylation pattern, other posttranslational profiles such as methionine oxidation and deamidation compared to batchwise reconstitution. Therefore, we conclude a continuous reconstitution of the medium results in the same quality of the product. A continuous on-demand media reconstitution will impact the supply chain and significantly reduce the floor space necessary for preparation and storage.

Continuous integrated antibody precipitation with two-stage tangential flow microfiltration enables constant mass flow.

Continuous precipitation is a new unit operation for the continuous capture of antibodies. The capture step is based on continuous precipitation with PEG6000 and Zn++ in a tubular reactor integrated with a two-stage continuous tangential flow filtration unit. The precipitate cannot be separated with centrifugation, because a highly compressed sediment results in poor resolubilization. We developed a new two-stage tangential flow microfiltration method, where part of the concentrated retentate of the first stage was directly fed to the second stage, together with the wash buffer. Thus, the precipitate was concentrated and washed in a continuous process. We obtained 97% antibody purity, a 95% process yield during continuous operation, and a fivefold reduction in pre-existing high-molecular-weight impurities. For other unit operations, surge tanks are often required, due to interruptions in the product mass flow out of the unit operation (e.g., the bind/elute mode in periodic counter-current chromatography). Our setup required no surge tanks; thus, it provided a truly continuous antibody capture operation with uninterrupted product mass flow. Continuous virus inactivation and other flow-through unit operations can be readily integrated downstream of the capture step to create truly continuous, integrated, downstream antibody processing without the need for hold tanks.

High-capacity protein A affinity chromatography for the fast quantification of antibodies: Two-wavelength detection expands linear range.

The high-throughput analysis of antibodies from processes can be enhanced when the linear range is expanded and sample preparation is kept to a minimum. We developed a fast chromatography method based on a hexameric variant of staphylococcal protein A immobilized on Toyopearl matrix, TSK 5 PW using two wavelengths. A protocol with 5 min runtime and a single-wavelength detection at 280 nm yielded an upper limit of quantification of 2.10 mg/mL and a lower limit of quantification of 0.06 mg/mL. The optimized method with a runtime of 2 min and two-wavelength detection at 280 and 300 nm allowed us to span a valid concentration range of 0.01-5.20 mg/mL using two calibration curves. Sample selectivity was tested using mock supernatant mixed with antibody concentrations of 0.1-2.1 mg/mL, sample stability in the autosampler was shown for at least 24 h. We also tested the capabilities of the method to determine purity of an antibody sample by calculating the ratio of peak area of elution to peak area of flow-through, which correlated well with the expected purity. The method will be very useful for process development and in-process control, spanning concentrations from seed fermentation to harvest and purification.

Monolith affinity chromatography for the rapid quantification of a single-chain variable fragment immunotoxin.

We developed a novel analytical method for concentration determination of tandem single-chain antibody diphtheria toxin (immunotoxin). The method is based on polymethacrylate monoliths with Protein L ligands as the binding moiety. Different buffers were tested for elution of the Protein L-bound immunotoxin and 4.5 M guanidinium hydrochloride performed best. We optimized the elution conditions and the method sequence resulting in a fast and robust method with a runtime <10 min. Fast determination of immunotoxin is critical if any process decisions rely on this data. We determined method performance and a lower limit of detection of 27 µg/mL and a lower limit of quantification of 90 µg/mL was achieved. The validity of the method in terms of residual analysis, precision, and repeatability was proven in a range from 100 to 375 µg/mL. The short runtime and ease of use of a high-performance liquid chromatography method is especially useful for a process analytical tool approach. Bioprocesses related to immunotoxin where fermentation or other process parameters can be adjusted in accordance to the immunotoxin levels will be benefited from this method to achieve the highest possible purity and productivity.

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. RESULTS: Screening of multiple flocculation agents, depth filters, and conditions demonstrated that the best performance was obtained with 0.0375% polydiallyldimethylammonium chloride (pDADMAC; a polycationic flocculation agent) in combination with Clarisolve® depth filters. Using this set-up, a 4-fold decrease of filtration area was achieved relative to standard filtration without flocculation, with yields of ≥97% and DNA depletion of up to 99%. Continuous operation was accomplished using a simple tubular reactor design with parallelization of the filtration. The reactor length was selected to allow a 13.2-min residence time, which was sufficient to complete flocculation in batch experiments. Continuous flocculation performance was monitored on-line using focused beam reflectance measurement. Filter switch cycles based on upstream pressure were controlled by in-line pressure sensors, and were stable from one filter to the next. CONCLUSION: It was demonstrated that stable and efficient continuous flocculation associated with depth filtration can be easily accomplished using tubular reactors and parallelization. Continuous cell separation is essential for the development of fully continuous integrated process trains. This cost-efficient disposable design run in continuous mode significantly reduces facility foot print, process costs and enables great flexbility. © 2017 The Authors. Journal of Chemical Technology & Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

3D printed autoclavable biocompatible biodegradable bioreactor vessels with integrated sparger made from poly-lactic acid.

3D printing has become widespread for the manufacture of parts in various industries and enabled radically new designs. This trend has not spread to bioprocess development yet, due to a lack of material suitable for the current workflow, including sterilization by autoclaving. This work demonstrates that commercially available heat temperature stable poly-lactic acid (PLA) can be used to easily manufacture novel bioreactor vessels with included features like harvest tubes and 3D printed spargers. Temperature responsiveness was tested for PLA, temperature stable PLA (PLA-HP) and glass for temperatures relevant for insect and mammalian cell culture, including temperature shifts within the process. Stability at 27 °C and 37 °C as well as temperature shifts to 22 °C and 32 °C showed acceptable performance with slightly higher temperature overshoot for 3D printed vessels. A stable temperature is reached after 2 h for PLA, 3 h for PLA-HP and 1 h for glass reactors. Temperature can be maintained with a fluctuation of 0.1 °C for all materials. A 3D printed sparger design directly integrated into the vessel wall and bottom was tested under three different conditions (0.3 SLPH and 27 °C, 3 SLPH and 37 °C and 13 SLPH and 37 °C). The 3D printed sparger showed a better kLa than the L-Sparger with more pronounced differences for higher flowrates. An insect cell culture run in the novel vessel exhibited the same growth behavior as that in standard glass vessels, reaching the same maximum cell concentration. Being 3D printed from biodegradable materials, these bioreactors offer design flexibility for novel bioreactor formats. Additionally, their autoclavability allows seamless integration into standard workflows.

An inert tracer: The binding site of a fluorescent dye on the antibody and its effects on Protein A chromatography.

Fluorescently labeled antibodies are widely used to visualize the adsorption process in protein chromatography using confocal laser scanning microscopy (CLSM), but also as a tracer for determination of residence time distribution (RTD) in continuous chromatography. It is assumed that the labeled protein is inert and representative of the unlabeled antibody, ignoring the fact that labeling with a fluorescent dye can change the characteristics of the original molecule. It became evident that the fluorescently labeled antibody has a higher affinity toward protein A resins such as MabSelect Sure. This can be due to slight differences in hydrophobicity and net charge, which are caused by the addition of the fluorescent dye. However, this difference is eliminated when using high salt concentrations in the adsorption studies. In this work, the site occupancy of two labeled antibodies, MAb1 (IgG1 subclass) and MAb2 (IgG2 subclass) conjugated with the fluorescent dye Alexa Fluor™ 488 was elucidated by intact mass spectrometry (MS) and peptide mapping LC-MS/MS, employing a sequential cleavage with Endoproteinase Lys-C and trypsin and in parallel with chymotrypsin alone. It was shown that the main binding site for the dye was a specific lysine in the heavy chains of the MAb1 and MAb2 molecules, in positions 188 and 189 respectively. Other lysine residues distributed throughout the protein sequence were labeled to a lot lesser extent. The labeled antibody had a slightly different affinity to MabSelect Sure although its primary binding site (to Protein A) was not affected by labeling, despite the secondary region responsible for binding to the protein A was partly labeled. Overall, the fluorescent-labeled antibodies are a good compromise as an inert tracer in residence time distribution and chromatography studies because they are much cheaper than isotope-labeled antibodies; However, the differences between the labeled and unlabeled antibodies should be considered.

Robust and resource-efficient production process suitable for large-scale production of baculovirus through high cell density seed train and optimized infection strategy.

The aim of this study was the development of a scalable production process for high titer (108 pfu/mL and above) recombinant baculovirus stocks with low cell line-derived impurities for the production of virus-like particles (VLP). To achieve this, we developed a high cell density (HCD) culture for low footprint cell proliferation, compared different infection strategies at multiplicity of infection (MOI) 0.05 and 0.005, different infection strategies and validated generally applicable harvest criteria of cell viability ≤ 80%. We also investigated online measurable parameters to observe the baculovirus production. The infection strategy employing a very low virus inoculum of MOI 0.005 and a 1:2 dilution with fresh medium one day after infection proved to be the most resource efficient. There, we achieved higher cell-specific titers and lower host cell protein concentrations at harvest than other tested infection strategies with the same MOI, while saving half of the virus stock for infecting the culture compared to other tested infection strategies. HCD culture by daily medium exchange was confirmed as suitable for seed train propagation, infection, and baculovirus production, equally efficient as the conventionally propagated seed train. Online measurable parameters for cell concentration and average cell diameter were found to be effective in monitoring the production process. The study concluded that a more efficient VLP production process in large scale can be achieved using this virus stock production strategy, which could also be extended to produce other proteins or extracellular vesicles with the baculovirus expression system.

3D printing: Economical and supply chain independent single-use plasticware for cell culture.

3D printing represents a democratization of manufacturing processes, and inexpensive 3D printed parts for cell culture have been tested as replacements for single-use plastics currently unavailable due to worldwide supply chain issues. In addition, such distributed manufacturing of cell culture laboratory materials helps remote areas and developing countries with limited resources. HEK293 cells were used to test printed shake flasks for cell culture applications and their ease of manufacture. Recorded growth curves showed that renewable biodegradable poly(lactic acid) (PLA) thermoplastic is an excellent and economical replacement for single-use plastic shake flasks, which have shipment lead times during pandemic situations or other supply chain disruptions of over 6 months. With a price of 0.60 € in materials, and printing machines with prices lower than one box of single-use pre-sterilized plastic shake flasks (<350€), the use of PLA is very affordable. Low-cost photopolymerization resins were also tested, but the inherent cytotoxicity of these materials prevented cell growth. This was also true for plant-based resins marketed as having low volatile organic compounds (VOC). Treatment of parts to reduce VOC content was partially successful, but not sufficient to sustain prolonged cell growth. A high-cost medical device IIa-class material showed no improved cell growth. Nevertheless, with PLA a low-cost printing material was identified and the use as cell culture compatible material was demonstrated, providing low-cost supply chain independence. In the future, the printing of pilot-scale bioreactors with PLA as a green sustainable material at the point of its use will be possible.

Residence time distribution in counter-current protein A affinity chromatography using an inert tracer.

The trend in the biopharmaceutical industry is changing from batch process to continuous process. For continuous biomanufacturing, traceability of the material is required by regulatory authorities. The recent ICH draft guideline Q13 on continuous manufacturing of drug substances and drug products requests an "understanding of process dynamics as a function of input material attributes (e.g., potency, material flow properties), process conditions (e.g., mass flow rates) … One common approach is characterization of residence time distribution (RTD) for the individual unit operations and integrated system." Thus, it is necessary to trace material through individual continuous unit operations and the integrated process. The RTD of a process is obtained experimentally by injecting a pulse of an inert tracer into the inlet and measuring the broadening of the injected pulse in the outlet. We investigated the RTD of three-column periodic counter-current chromatography (PCC) using staphylococcal protein A affinity chromatography, with a focus on how the material distributes over subsequent cycles. A fluorescent-labeled antibody was used as the inert tracer under high salt concentration. The tracer was injected once in each run but at different points of the loading phase. We then analyzed the outlet of the column. In the elution phase, regardless of the point of injection, we observed an even distribution of the tracer. In the loading phase, a constant exchange between the antibody in the solid phase and the liquid phase was observed, meaning that sending the outlet of one chromatography column into another column to improve resin utilization causes higher residence time in the system for some portion of the material.

A comprehensive antigen production and characterisation study for easy-to-implement, specific and quantitative SARS-CoV-2 serotests.

BACKGROUND: Antibody tests are essential tools to investigate humoral immunity following SARS-CoV-2 infection or vaccination. While first-generation antibody tests have primarily provided qualitative results, accurate seroprevalence studies and tracking of antibody levels over time require highly specific, sensitive and quantitative test setups. METHODS: We have developed two quantitative, easy-to-implement SARS-CoV-2 antibody tests, based on the spike receptor binding domain and the nucleocapsid protein. Comprehensive evaluation of antigens from several biotechnological platforms enabled the identification of superior antigen designs for reliable serodiagnostic. Cut-off modelling based on unprecedented large and heterogeneous multicentric validation cohorts allowed us to define optimal thresholds for the tests' broad applications in different aspects of clinical use, such as seroprevalence studies and convalescent plasma donor qualification. FINDINGS: Both developed serotests individually performed similarly-well as fully-automated CE-marked test systems. Our described sensitivity-improved orthogonal test approach assures highest specificity (99.8%); thereby enabling robust serodiagnosis in low-prevalence settings with simple test formats. The inclusion of a calibrator permits accurate quantitative monitoring of antibody concentrations in samples collected at different time points during the acute and convalescent phase of COVID-19 and disclosed antibody level thresholds that correlate well with robust neutralization of authentic SARS-CoV-2 virus. INTERPRETATION: We demonstrate that antigen source and purity strongly impact serotest performance. Comprehensive biotechnology-assisted selection of antigens and in-depth characterisation of the assays allowed us to overcome limitations of simple ELISA-based antibody test formats based on chromometric reporters, to yield comparable assay performance as fully-automated platforms. FUNDING: WWTF, Project No. COV20-016; BOKU, LBI/LBG.

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 0.1-0.2 µm and used this method to characterize particle populations generated by batch as well as continuous precipitation in different shear stress environments. The resulting precipitate structures show a broad distribution in terms of fractal dimension. While the average fractal dimension is significantly different for batch and continuous precipitation, the distribution is broad and samples overlap significantly. The precipitate flocs were monofractal from micro- to nanoscale showing a random but consistent nature of precipitate formation. We showed that the fractal dimension and 3D reconstruction is a valuable tool for characterization of protein precipitate processes. The current switch from batch to continuous manufacturing has to take the 3D structure and population of different protein precipitates into account in their design, engineering, and scale up.

Continuous capture of recombinant antibodies by ZnCl 2 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 ZnCl2. 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. By the use of the crosslinking nature of divalent cations without the use of PEG, we kept viscosity from the supernatant and resolubilization dilution factors very low. This is especially beneficial for the solid-liquid separation for the harvest and wash of the precipitate in continuous mode. For this harvest and wash, we used tangential flow filtration that benefits a lot from low viscosity solutions, which minimizes the membrane fouling. With this precipitation based on ZnCl2, we were able to implement a very lean and efficient process. We demonstrated precipitation studies with three different antibodies, Adalimumab, Trastuzumab, and Denosumab, and a continuous capture case study using tangential flow filtration for precipitate recovery. In this study, we achieved yields of 70%.

Mid-manufacturing storage: Antibody stability after chromatography and precipitation based capture steps.

Antibodies of the IgG2 subclass were captured from the clarified cell culture fluid either by protein A chromatography or by polyethylene glycol precipitation. The captured intermediates were stored as neutralized eluates (protein A chromatography) or in solid form as polyethylene glycol precipitates over a period of 13 months at three temperatures, -20°C, 5°C, and room temperature to compare the capture technologies in regard of the resulting product storability. Monomer content, high molecular mass impurities product loss and changes in the composition of the charge variants were determined at six time points during the storage. At the beginning and end of the study, samples were additionally tested by differential scanning calorimetry, differential scanning fluorimetry, and circular dichroism to determine structural alterations occurring during storage. Protein A purified material was highly stable at all tested temperatures in regard of monomer content and product losses. A transient, acidic isoform was formed during the chromatography step which re-converted to the main charged variant upon storage within a matter of days. Precipitated antibodies could be stored at -20 or 5°C for 3 months without product losses but afterwards recovery yields dropped to 65%. At room temperature, the precipitated antibody was not stable and degraded within 3 months.

Capture and purification of Human Immunodeficiency Virus-1 virus-like particles: Convective media vs porous beads.

Downstream processing (DSP) of large bionanoparticles is still a challenge. The present study aims to systematically compare some of the most commonly used DSP strategies for capture and purification of enveloped viruses and virus-like particles (eVLPs) by using the same staring material and analytical tools. As a model, Human Immunodeficiency Virus-1 (HIV-1) gag VLPs produced in CHO cells were used. Four different DSP strategies were tested. An anion-exchange monolith and a membrane adsorber, for direct capture and purification of eVLPs, and a polymer-grafted anion-exchange resin and a heparin-affinity resin for eVLP purification after a first flow-through step to remove small impurities. All tested strategies were suitable for capture and purification of eVLPs. The performance of the different strategies was evaluated regarding its binding capacity, ability to separate different particle populations and product purity. The highest binding capacity regarding total particles was obtained using the anion exchange membrane adsorber (5.3 × 1012 part/mL membrane), however this method did not allow the separation of different particle populations. Despite having a lower binding capacity (1.5 × 1011 part/mL column) and requiring a pre-processing step with flow-through chromatography, Heparin-affinity chromatography showed the best performance regarding separation of different particle populations, allowing not only the separation of HIV-1 gag VLPs from host cell derived bionanoparticles but also from chromatin. This work additionally shows the importance of thorough sample characterization combining several biochemical and biophysical methods in eVLP DSP.

Temperature dependence of antibody adsorption in protein A affinity chromatography.

Staphylococcal protein A affinity chromatography is a well-established platform for purification of clinical-grade antibodies. The wild type ligand has been mutated to improve caustic stability, elution behavior, and/or to increase binding capacity. Several modified protein A ligands are nowadays commercially available, one of them being the thermosensitive chromatography medium Byzen Pro from Nomadic Bioscience Co., Ltd. According to the manufacturer, Byzen Pro has the ability to release IgG upon a change in temperature. It is based on a thermosensitive mutant of protein A which should allow elution at neutral pH by changing the temperature from binding at 5 °C to elution conditions at 40 °C. We determined equilibrium binding capacities of the thermosensitive protein A medium (Byzen Pro), MabSelect SuRe (GE Healthcare), and TOYOPEARL AF-rProtein A HC-650F (Tosoh Bioscience LLC) for antibodies of the subclass IgG1 and IgG2 at five different temperatures from 4 °C to 40 °C to elucidate the temperature effect. We also observed a temperature dependence of the dynamic binding capacities which were determined for the subclass IgG2 at three temperatures from 4 °C to 40 °C. However, for Byzen Pro, the temperature dependence was only present at a low flow rate and vanished at high flow rates indicating that pore diffusion is the rate-limiting step. Binding of the antibody to MabSelect SuRe and TOYOPEARL AF-rProtein A HC-650F stabilized the conformations as shown by an increase in melting temperature in differential scanning calorimetry measurements. The antibody conformation was slightly destabilized upon binding to the thermosensitive ligand. The conformation change upon binding was fully reversible as shown by circular dichroism, differential scanning calorimetry and size exclusion chromatography. Isothermal titration calorimetry was used to measure the raw heat of adsorption for the IgG2 molecule. The thermosensitive ligand can also be used for antibodies with low stability, because elution can also be effected by salt.

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. Out of eight initially investigated model proteins, two (BSA and myoglobin) showed conformational changes, and in both cases this conformational change was dependent on the size of the nanoparticle. Nanoparticle sizes ranged from 30 to 1000 nm and, in contrast to previous studies, we attempted to use a continuous progression of sizes in the range found in live viruses, which is an interesting size of nanoparticles for the potential use as drug delivery vehicles. Conformational changes were only visible for particles of 200 nm and bigger. Using an optimized circular dichroism protocol allowed us to follow this conformational change with regard to the nanoparticle size and, thanks to the excellent temporal resolution also in time. We uncovered significant differences between the unfolding kinetics of myoglobin and BSA. In this study, we also evaluated the plausibility of commonly used explanations for the phenomenon of nanoparticle size-dependent conformational change. Currently proposed mechanisms are mostly based on studies done with relatively small particles, and fall short in explaining the behavior seen in our studies.

Continuous separation of protein loaded nanoparticles by simulated moving bed chromatography.

For scale up and efficient production of protein loaded nanoparticles continuous separation by size exclusion chromatography in simulated moving bed (SMB) mode helps do reduce unbound protein concentration and increase yields for perfectly covered particles. Silica nanoparticles were loaded with an excess of beta casein or bovine serum albumin (BSA) and the loaded particles purified by size exclusion chromatography using Sephacryl300 as stationary phase in a four zone SMB. We determined our working points for the SMB from batch separations and the triangle theory described by Mazzotti et al. with an SMB setup of one Sephacryl300 26/70mm column per zone with switch times of 5min for BSA and 7min for beta casein. In the case of BSA the Raffinate contained loaded nanoparticles of 63% purity with 98% recovery and the extract was essentially particle free (95% purity). We showed that the low purity of the Raffinate was only due to BSA multimers present in the used protein solution. In the case of beta casein where no multimers are present we achieved 89% purity and 90% recovery of loaded nanoparticles in the Raffinate and an extract free of particles (92% purity). Using a tangential flow filtration unit with 5kDa cutoff membrane we proved that the extract can be concentrated for recycling of protein and buffer. The calculated space-time-yield for loaded nanoparticles was 0.25g of loaded nanoparticles per hour and liter of used resin. This proves that the presented process is suitable for large scale production for industrial purposes.

Ethanol precipitation for purification of recombinant antibodies.

Currently, the golden standard for the purification of recombinant humanized antibodies (rhAbs) from CHO cell culture is protein A chromatography. However, due to increasing rhAbs titers alternative methods have come into focus. A new strategy for purification of recombinant human antibodies from CHO cell culture supernatant based on cold ethanol precipitation (CEP) and CaCl2 precipitation has been developed. This method is based on the cold ethanol precipitation, the process used for purification of antibodies and other components from blood plasma. We proof the applicability of the developed process for four different antibodies resulting in similar yield and purity as a protein A chromatography based process. This process can be further improved using an anion-exchange chromatography in flowthrough mode e.g. a monolith as last step so that residual host cell protein is reduced to a minimum. Beside the ethanol based process, our data also suggest that ethanol could be replaced with methanol or isopropanol. The process is suited for continuous operation.