Recovery of lysozyme from hen egg white by selective magnetic cake filtration

A new concept for the improvement of the downstream processing and purification is the so‐called magnetic separation. By using surface functionalized magnetic substrate particles, which selectively adsorb the target product, it can be directly separated out of a crude bioprocess stream. These methods are already used for analytical purposes, where only small amounts of functionalized particles are necessary. To apply the same concept at a larger scale, effective and economical procedures have to be provided. First, suitable process equipment has to be developed. Second, the magnetic particles have to be manufactured with a stable surface functionalization and long‐term stability for their reuse. Up to now mainly high‐gradient magnetic separation filter devices are applied for selective magnetic separation. They consist of a magnetic matrix in which the magnetic particles are trapped. In this work, a new magnetic filter is introduced that overcomes the capacity limitations of the current high‐gradient magnetic separation technology. The principle is demonstrated by selective recovery of lysozyme from hen egg white. Prior to the separation experiments magnetic beads with a strong acid cation‐exchange surface functionalization are synthesized. The separation procedure is implemented in only one unit operation. With the implementation of the displacement elution sequence lysozyme can be separated out of a hen egg white solution with a purification factor of PF=36 and a purity P=0.83.     

Optimizing a rotor‐stator filter matrix for high‐gradient magnetic separation of functionalized magnetic particles

The processing of wines with enzymes is a process chain in which losses of biocatalyst are unavoidable. A promising technique for the minimization of these losses and for the reduction of processing time is the high‐gradient magnetic separation in combination with enzymes, which are immobilized onto functionalized magnetic particles. When magnetizable particles are used and magnetic separation is applied to separate these particles from nonmagnetizable particles and solutes, the enzymes can be recycled and used for several production batches. The magnetic filter used in this study had a filter matrix with concentrically stacked circular rotor and stator plates which are arranged in an alternating order. Different geometries of the filter plate notches were examined to optimize the reproducibility of particle retention. In computational fluid dynamic studies, the influence of the notch geometries on the shear rate generation was analyzed for the rinsing procedure. Separation experiments with an optimized geometry of the filter plates were carried out in water and white wine suspensions.     

First comprehensive view on a magnetic separation based protein purification processes: From process development to cleaning validation of a GMP‐ready magnetic separator

Magnetic separation processes are known as integrated bioanalytical protein purification method since decades and are well described. However, use of magnetic separation processes in a regulated industrial production environment has been prevented by the lack of suitable process equipment and prejudice against the productivity of the process and its qualification for cleaning‐in‐place operation. With the aim of overcoming this prejudice, a comprehensive process development approach is presented, based on a GMP‐compliant magnetic separator, including an optimization of the batch adsorption process, implementation into a technical‐scale, and the development and validation of cleaning routines for the device. By the implementation of a two‐step counter‐current binding process, it was possible to raise the yields of the magnetic separation process even for very low concentrated targets in a vast surplus of competing proteins, like the hormone equine chorionic gonadotropin in serum, from 74% to over 95%. For the validation of the cleaning process, a direct surface swabbing method combined with a total organic carbon analysis was established for the determination of two model contaminants. The cleanability of the process equipment was proven for both model contaminants by reliably meeting the 10 ppm criteria.     

Development of a 3D‐printed single‐use separation chamber for use in mRNA‐based vaccine production with magnetic microparticles

Laboratory protocols using magnetic beads have gained importance in the purification of mRNA for vaccines. Here, the produced mRNA hybridizes specifically to oligo(dT)‐functionalized magnetic beads after cell lysis. The mRNA‐loaded magnetic beads can be selectively separated using a magnet. Subsequently, impurities are removed by washing steps and the mRNA is eluted. Magnetic separation is utilized in each step, using different buffers such as the lysis/binding buffer. To reduce the time required for purification of larger amounts of mRNA vaccine for clinical trials, high‐gradient magnetic separation (HGMS) is suitable. Thereby, magnetic beads are selectively retained in a flow‐through separation chamber. To meet the requirements of biopharmaceutical production, a disposable HGMS separation chamber with a certified material (United States Pharmacopeia Class VI) was developed which can be manufactured using 3D printing. Due to the special design, the filter matrix itself is not in contact with the product. The separation chamber was tested with suspensions of oligo(dT)‐functionalized Dynabeads MyOne loaded with synthetic mRNA. At a concentration of c_B = 1.6–2.1 g·L^–1 in lysis/binding buffer, these 1 μm magnetic particles are retained to more than 99.39% at volumetric flows of up to 150 mL·min^–1 with the developed SU‐HGMS separation chamber. When using the separation chamber with volumetric flow rates below 50 mL·min^–1, the retained particle mass is even more than 99.99%.     

Dewatering of phosphorus extracted from liquid swine waste

Phosphorus (P) recovery from liquid swine manure is an attractive technology when on-farm application of liquid swine manure is not an option. We developed a technology that enables separation of this P, but its high moisture content makes transportation difficult. In this work, we investigated dewatering procedures to concentrate the P product. Sludge rich in calcium phosphate (> 20% P2O5) was obtained using a field prototype, and it was further dewatered using a combination of polymer treatment and filter bags. Anionic polyacrylamide polymer treatment (> or = 20 mg/L) was effective to flocculate the P-rich sludge, which enhanced filtration and dewatering. Without polymer, filtration was incomplete due to clogging of filters. Non-woven polypropylene and monofilament filter bag fabrics with mesh size < or = 200 microm retained > 99% of suspended solids and total P. Solids content dramatically increased from about 1.5% to > 90%. These dewatered solids can be transported more economically off the farm for use as a valuable fertilizer material.     

Strategy for selecting disposable bags for cell culture media applications based on a root‐cause investigation

The use of disposable bags for cell culture media storage has grown significantly in the past decade. Some of the key advantages of using disposable bags relative to non‐disposable containers include increased product throughput, decreased cleaning validation costs, reduced risk of cross contamination and lower facility costs. As the scope of use of disposable bags for cell culture applications increases, problematic bags and scenarios should be identified and addressed to continue improving disposables technologies and meet the biotech industry's needs. In this article, we examine a cell culture application wherein media stored in disposable bags is warmed at 37°C before use for cell culture operations. A problematic bag film was identified through a prospective and retrospective cell culture investigation. The investigation provided information on the scope and variation of the issue with respect to different Chinese hamster ovary (CHO) cell lines, cell culture media, and application‐specific parameters. It also led to the development of application‐specific test methods and enabled a strategy for disposable bag film testing. The strategy was implemented for qualifying an alternative bag film for use in our processes. In this test strategy, multiple lots of 13 bag film types, encompassing eight vendors were evaluated using a three round, cell culture‐based test strategy. The test strategy resulted in the determination of four viable bag film options based on the technical data. The results of this evaluation were used to conclude that a volatile or air‐quenched compound, likely generated by gamma irradiation of the problematic bag film, negatively impacted cell culture performance. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1535–1549, 2013     

Cost Comparison of Protein Capture from Cultivation Broths by Expanded and Packed Bed Adsorption

In the downstream processing of recombinant protein production, the reduction of unit operations required for product capture and purification, is of the utmost priority due to its cost diminishing effect. In this regard, target protein capture from cell suspensions in a fluidized bed of affinity particles with different sizes (expanded bed adsorption (EBA) with classified particles), presents an efficient tool since EBA may substitute cell disintegration, separation by centrifugation or filtration, and packed bed adsorption. However, as illustrated by experiments with the BSA/yeast cells system, the entire broth processing used in EBA also has detrimental influences due to the cell (or cell debris) binding on the affinity carrier. In particular, external mass transfer may become more dominant, and the lifetime of the affinity particles may reduce as a result of other cleaning procedures. Using simulations performed with a commercial software package, the cost superiority of alternate process routes (EBA or packed bed adsorption with preceding steps) can be evaluated. This elucidates the favorable application range for each route.     

A closed system for the filtration of Mycobacterium bovis liquid cultures using disposable capsule filters

A filtration system was designed to sterilize large volumes of Mycobacterium bovis BCG Tokyo culture safely, needed to purify protein antigens for immunodiagnosis of bovine tuberculosis. A closed system consists of culture bottles connected to three disposable filter capsules of decreasing pore size in series : a depth prefilter over a 1·2 μm filter ; a 0·8 μm prefilter over a 0·45 μm filter ; and a 0·2 μm sterile filter. Low air pressure (3 psi) forces liquid from below the bacillary pellicle. The system features a stainless steel clamp to hold rubber stoppers on the culture bottles, pleated filters to exclude bacillary clumps, a quick disconnector to minimize aerosols, and a closed system with plastic disposable filters that can be autoclaved as a unit without dismantling.     

Thiophilic paramagnetic particles as a batch separation medium for the purification of antibodies from various source materials

A preparation of thiophilic agarose-based paramagnetic particles (T-Gel) has been developed with physical characteristics (particle size and particle density) that facilitate its use as a batch separation medium suitable for the large-scale purification and isolation of immunoglobulins. The medium was used to extract immunoglobulins from a wide range of starting materials, including sera, ascites fluid, tissue culture medium, and whole blood. None of these starting materials required pretreatment such as clarification by centrifugation or filtration prior to antibody extraction. The antibody purity obtained using T-Gel compared well with that obtained using protein A agarose column chromatography. Yields were approximately 30 mg of immunoglobulins per milliliter of T-Gel, and little was required in the way of specialist equipment. The method is uncomplicated and involves a roll mix extraction overnight, followed by magnetic separation to facilitate supernatant removal and subsequent washing of the particles. Elution of bound antibodies was carried out at neutral pH to yield a concentration of immunoglobulins that was approximately 7 mg/ml. The method was found to be applicable to antibody purification from the blood serum of seven different mammalian species and for all immunoglobulin classes.     

Modification of a commercial cell sorter to support efficient and reliable preparation of ALDH-bright cells for clinical use

BACKGROUND: Cell populations manufactured by conventional commercial cell sorters have been safely infused into patients, but reliably sterilizing these instruments remains challenging. We are developing clinical protocols involving use of ALDH bright cells manufactured by cell sorting in patients. However, we encountered problems when we attempted to reliably sterilize the FACSAria cell sorter using standard methods. RESULTS: We have identified and modified potential sources of microbial contamination in several FACSAria systems. We added new filter systems to the sheath and sample air lines, to the wet cart fluid supply, and to the sample line. Sheath was provided from an external sterile, disposable bag through sterile disposable tubing sets. The plenum reservoirs were modified in several ways to allow efficient decontamination of internal surfaces. A new bubble filter assembly was added and one valve was eliminated from the sample pathway to improve flow cell sterilization. A new cleaning and sterilization protocol was developed and validated. All cell products manufactured using the modified instrument and validated cleaning protocol have met lot release criteria for prevention of microbial contamination and safe clinical use. DISCUSSION: The instrument modification and cleaning protocol described enable reliable manufacture of ALDH bright cell populations that are suitable for clinical trials. We have manufactured nineteen consecutive samples that meet all clinical release criteria in an on-going Phase 1 human trial.     

Recent advances in bioprocessing application of membrane chromatography

Compared to traditional chromatography using resins in packed-bed columns, membrane chromatography is a relatively new and immature bioseparation technology based on the integration of membrane filtration and liquid chromatography into a single-stage operation. Over the past decades, advances in membrane chemistry have yielded novel membrane devices with high binding capacities and improved mass transfer properties, significantly increasing the bioprocessing efficiency for purification of biomolecules. Due to the disposable nature, low buffer consumption, and reduced equipment costs, membrane chromatography can significantly reduce downstream bioprocessing costs. In this review, we discuss technological merits and disadvantages associated with membrane chromatography as well as recent bioseparation applications with a particular attention on purification of large biomolecules.     

Protein purification using magnetic adsorbent particles

The application of functionalised magnetic adsorbent particles in combination with magnetic separation techniques has received considerable attention in recent years. The magnetically responsive nature of such adsorbent particles permits their selective manipulation and separation in the presence of other suspended solids. Thus, it becomes possible to magnetically separate selected target species directly out of crude biological process liquors (e.g. fermentation broths, cell disruptates, plasma, milk, whey and plant extracts) simply by binding them on magnetic adsorbents before application of a magnetic field. By using magnetic separation in this way, the several stages of sample pretreatment (especially centrifugation, filtration and membrane separation) that are normally necessary to condition an extract before its application on packed bed chromatography columns, may be eliminated. Magnetic separations are fast, gentle, scaleable, easily automated, can achieve separations that would be impossible or impractical to achieve by other techniques, and have demonstrated credibility in a wide range of disciplines, including minerals processing, wastewater treatment, molecular biology, cell sorting and clinical diagnostics. However, despite the highly attractive qualities of magnetic methods on a process scale, with the exception of wastewater treatment, few attempts to scale up magnetic operations in biotechnology have been reported thus far. The purpose of this review is to summarise the current state of development of protein separation using magnetic adsorbent particles and identify the obstacles that must be overcome if protein purification with magnetic adsorbent particles is to find its way into industrial practice.     

A feasibility study of a filtration type autotransfusion device

This paper describes a feasibility study of a disposable autotransfusion device for blood salvage during surgery. The goal was to concentrate hemolyzed blood at 20% hematocrit to 50% while reducing the plasma free hemoglobin concentration from 10 to 1.5 g/l. The device should have a total membrane area of less than 0.6 m² and should be able to process ten 500 ml blood bags. The processing time for each blood bag should not exceed 5 min. The basic idea was to use several polypropylene hollow fibre plasma filters of 0.1 m² in series with saline addition between them. Since the mean pore size is 0.5μ m, anticoagulant and plasma hemoglobin can pass freely across the membrane and their concentration is reduced by dilution. The process was first modelled using mass balance equations for red blood cells and plasma hemoglobin in order to find the best device configuration (number of filters and dilutions). It was found that a three filter system could theoretically meet the requirements, if the last filter had a larger surface area (0.3 m²). Some experiments permitted us to prove the validity of this model and to define fully the third filtration stage. Finally, it was shown that the treatment of a 500 ml blood bag required three filtration stages (whose surface areas were respectively 0.1, 0.1 and 0.3 m²) and the use of 750 ml of saline solution added between the filters. This configuration also offers the possibility of using a vacuum driving force instead of pumps, so that the device becomes completely disposable.     

Membrane‐Based Approach for the Downstream Processing of Influenza Virus‐Like Particles

Currently, marketed influenza vaccines are only efficient against homologous viruses, thus requiring a seasonal update based on circulating subtypes. This constant reformulation adds several challenges to manufacturing, particularly in purification due to the variation of the physicochemical properties of the vaccine product. A universal platform approach capable of handling such variation is therefore of utmost importance. In this work, a filtration‐based approach is explored to purify influenza virus‐like particles. Switching from adsorptive separation to size‐based purification allows overcoming the differences in retention observed for different influenza strains. The proposed process employs a cascade of ultrafiltration and diafiltration steps, followed by a sterile filtration step. Different process parameters are assessed in terms of product recovery and impurities’ removal. Membrane chemistry, pore size, operation modes, critical flux, transmembrane pressure, and permeate control strategies are evaluated. After membrane selection and parameter optimization, concentration factors and diafiltration volumes are also defined. By optimizing the filtration mode of operation, it is possible to achieve product recoveries of approximately 80%. Overall, the process time is decreased by 30%, its scalability is improved, and the costs are reduced due to the removal of chromatography and associated buffer consumptions, cleaning, and its validation steps.     

Downscaling cake filtration—Correlations between compressibility of lysozyme crystal filter cakes and filter area

Crystallization is a commonly used method for the purification and formulation of proteins. In order to harmonize crystallization with the mechanical separation process, it is necessary to estimate early the separation conditions of protein crystals. To fulfill this requirement, the feed material for filtration is minimized by reducing the filter area. The filtration behavior of lysozyme crystals in pressure filters with two different filter areas was compared. Crystal slurries with different mean crystal sizes and shapes were produced and the influence of the size and shape on the scalability of filtration data was examined. It was found that for different aggregated crystal structures and isometric crystals, the larger the compressibility of the cake was, the larger were the deviations for the two considered filter areas. For needle‐shaped crystals, the compressibility was not subject to deviations, but the absolute filter cake resistance was. Furthermore, the deviations of the cake resistances increased with higher pressures for all product systems. It was indicated that upscaling from the small filter area is possible for low compressible product systems and at low pressures. With high compressible products and for needle‐like crystals, the filtration time is underestimated with the small filter area.     

Continuous capture of recombinant antibodies by ZnCl2 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 ZnCl_2, 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%.     

Membrane filtration affinity purification (MFAP) of dehydrogenases using cibacron blue

The method for purification of biomolecules by a combination of affinity interactions and membrane filtration for separation of unwanted material has been found to be of interest for large-scale work. This study examines the suitability of silica nanoparticles as carriers in the process. Alcohol dehydrogenase and lactate dehydrogenases were chosen as target molecules to be purified. The binding capacity was found to be comparative to what is obtained for high-performance liquid chromatography (HPLC) packing material. Both binding and desorption of the enzymes were found to be effective. The limiting factor of the process was the filtration flow rate.     

Magnetic particles for the separation and purification of nucleic acids

Nucleic acid separation is an increasingly important tool for molecular biology. Before modern technologies could be used, nucleic acid separation had been a time- and work-consuming process based on several extraction and centrifugation steps, often limited by small yields and low purities of the separation products, and not suited for automation and up-scaling. During the last few years, specifically functionalised magnetic particles were developed. Together with an appropriate buffer system, they allow for the quick and efficient purification directly after their extraction from crude cell extracts. Centrifugation steps were avoided. In addition, the new approach provided for an easy automation of the entire process and the isolation of nucleic acids from larger sample volumes. This review describes traditional methods and methods based on magnetic particles for nucleic acid purification. The synthesis of a variety of magnetic particles is presented in more detail. Various suppliers of magnetic particles for nucleic acid separation as well as suppliers offering particle-based kits for a variety of different sample materials are listed. Furthermore, commercially available manual magnetic separators and automated systems for magnetic particle handling and liquid handling are mentioned.     

Magnetic seeding to aid recovery of biological precipitates

A new approach to solid/liquid separation for biological precipitates is presented. The precipitate is seeded with small dense and/or magnetic particles to increase the density difference between precipitate and liquid or to make the precipitate amenable to magnetic separation. Experiments on seeding of ammonium sulphate precipitates of casein and separation of the seeded precipitate by gravity settling and batch centrifugation have shown that the approach holds promise. Seeded precipitates exhibit hindered settling under gravity with typical settling velocities of 0.6 cm/min for iron oxide seed and 2.4 cm/min for nickel seed. Calculations indicate that seeding of these precipitates causes a 23 fold increase in density difference between precipitate particle and supernatant liquid. The design of a magnetic seeding step for use in an enzyme isolation process is discussed.     

High gradient magnetic separation of yeast

High gradient magnetic separation (HGMS) is used to separate nonmagnetic microorganisms from solution by a technique known as seeding. Fine magnetic particles are adhered to the cells' surfaces, making them magnetic and amenable to magnetic separation. Attachment of the sub-micron, acicular gamma-Fe(2)O(3) seed to the yeast surface occurs irrespective of the solution pH and surface charge and is essentially irreversible. A model is developed to predict the separation of yeast in a high gradient magnetic separator. The effective capture radius is assumed to be proportional to the derived magnetic parameter gamma for the case where the dominant competing force to magnetic attraction is the magnetic floc's inertia. Using this parameter, yeast separation in an HGMS unit is predicted. The measured separation of Saccharomyces cerevisiae at differing magnetic seed concentrations and two flow rates supports the above model.