Frank Hilbrig

Protein purification by affinity precipitation

Developing the most efficient strategy for the purification of a (recombinant) protein especially at large scale remains a challenge. A typical problem of the downstream process of mammalian cell products is, for instance, the early capture of the highly diluted product from the complex process stream. Affinity precipitation has been suggested in this context. The technique is known for over 20 years, but has recently received more attention due to the development of new materials for its implementation, but also because it seems ideally suited to specific product capture at large scale. The present review gives a comprehensive overview over this technique. Besides an introduction to the basic principle and a brief summary of the historical development, the main focus is on the current state-of-art of the technique, the available materials, important recent applications, as well as process design strategies and operating procedures. Special consideration is given to affinity precipitation for product recovery at large scale.

Isolation and purification of recombinant proteins, antibodies and plasmid DNA with hydroxyapatite chromatography

Hydroxyapatite and related stationary phases increasingly play a role in the downstream processing of high‐value biological materials, such as recombinant proteins, therapeutic antibodies and pharmaceutical‐grade plasmid DNA. Chromatographic hydroxyapatite is an inorganic, ceramic material identical in composition, if not in structure, to calcium phosphate found in human bones and teeth. The interaction of hydroxyapatite with biomacromolecules is complex and highly dynamic, which can make predicting performance difficult, but also allows the design of very selective isolation processes. This review discusses the currently commercially available chromatographic materials, different retention mechanisms supported by these materials and differential exploitation for the design of highly specific isolation procedures. The state of the art of antibody purification by hydroxy‐ and fluoroapatite is reviewed together with tested routines for method development and implementation. Finally, the isolation of plasmid DNA is discussed, since the purification of DNA therapeutics at a sufficiently large scale is an emerging need in bioprocess development and perhaps the area in bioseparation where apatite chromatography can make its most important contribution to date.

Direct detection of formaldehyde in air by a novel NAD+- and glutathione-independent formaldehyde dehydrogenase-based biosensor.

An amperometric enzyme-based sensor-system for the direct detection of formaldehyde in air is under investigation. The biosensor is based on a native bacterial NAD(+)- and glutathione-independent formaldehyde dehydrogenase as biorecognition element. The enzyme was isolated from Hyphomicrobium zavarzinii strain ZV 580, grown on methylamine hydrochloride in a fed-batch process. The sensor depends on the enzymatic conversion of the analyte to formic acid. Released electrons are detected in an amperometric measurement at 0.2V vs. Ag/AgCl reference electrode by means of a redox-mediator. To optimize the sensing device, Ca(2+) and pyrroloquinoline quinone (PQQ) were added to the buffer solution as reconstitutional substances. At this stage, the sensor shows linear response in the tested ppm-range with a sensitivity of 0.39 microA/ppm. The signal is highly reproducible with respect to sensitivity and base line signal. Reproducibility of sensitivity is more than 90% within the same bacterial batch and even when enzyme of different bacterial batches is used.

Continuous annular chromatography

The principle of continuous annular chromatography (CAC) has been known for several decades. CAC is a continuous chromatographic mode, which lends itself to the separation of multi-component mixtures as well as of bi-component ones. In CAC, the mobile and stationary phases move in a crosscurrent fashion, which allows transformation of the typical one-dimensional batch column separation into a continuous two-dimensional one. With the exception of linear gradient elution, all chromatographic modes have at present been applied in CAC. This review focuses on the capacity of CAC for preparative bioseparation. The historical developments and the predecessors of modern CAC are briefly summarized. The state-of-the-art in the theoretical prediction and simulation of CAC separations is discussed, followed by an overview of current CAC instrumentation and example applications, especially for the isolation of proteins and other bio(macro)molecules. In this context, issues of scale up as well as method development and transfer from batch to continuous CAC columns are discussed using recent bioseparation efforts as pertinent examples.

Integration of Bacteria Capture via Filtration and in Situ Lysis for Recovery of Plasmid DNA under Industry-Compatible Conditions

Combining capture and lysis of the bacteria with partial purification of the plasmid DNA is beneficial for the design of efficient plasmid production processes at larger scale. Such an approach is possible when the bacteria are captured by filtration. Taking industrial requirements into account, however, such a capture requires complex filtration mixtures containing retentive additives such as bentonite and polycations. This makes the straightforward transfer of established lysis protocols to in situ lysis difficult. In this contribution, the different steps of such a protocol are designed for complex filter cakes, including fragilization (by lysozyme), lysis (alkaline pH/acidic pH, 70/37 °C, urea/NaCl/Triton), and specific elution (pH, NaCl, CaCl2, guanidinium hydrochloride). Results are compared in regard to plasmid quality (topoisomeric form) and quantity (compared to the yield obtained by a commercial miniprep of a small aliquot of the bacteria suspension from the bioreactor). Best results in these terms were obtained by the Triton lysis protocol performed at 37 °C (30 min of contact with a lysis buffer composed of 50 mM Tris pH 8, 1% Triton, 1 g/L lysozyme, and 6 M guanidinium hydrochloride) followed by the specific elution of the plasmid DNA in 50 mM Tris buffer pH 8.

Use of azobenzene amino acids as photo-responsive conformational switches to regulate antibody-antigen interaction.

In an attempt to exploit the large geometry changes associated with azobenzene photo-isomerization for the modulation of antibody-antigen interaction, we introduced in the backbone of the FLAG peptide (DYKDDDDK), an azobenzene unit to photo-modulate its conformational states and consequently its interaction with the monoclonal anti-FLAG-tag antibody M1. The FLAG-tag system is an established technique for purifying and detecting the corresponding fusion proteins. In this context, conflicting evidence has been presented regarding the necessity of calcium for stable binding. Using surface plasmon resonance, we showed that not the initial recognition but certainly the stability of the complex improves in the presence of calcium. Subsequently, we substituted two or three of the central aspartate residues for an artificial, azobenzene-based, photo-responsive amino acid. Four structural isomers of the artificial amino acid were considered, in total twelve FLAG-tag analogues were synthesized. Two showed significant differences in their ability to bind to the antibody in their cis versus their trans state. Interestingly, these two peptides are the two shortest of the twelve photo-peptides investigated. Finally, it was shown that for these two FLAG-analogues switching between cis and trans states is possible in the presence of the antibody.

Purification of RT-PCR Competent Poly(A) mRNA from Crude Cell Lysate by Affinity Precipitation

Stimuli‐responsive bioconjugates consisting of avidin covalently linked to poly(N‐isopropylacrylamide) were used for the recovery of poly(A) mRNA hybridized to biotinylated poly(dT)‐tags from crude cell lysates (Jurkat cells) by affinity precipitation. The bioconjugates are soluble in cold water but precipitate readily once a critical solution temperature (33 °C in pure water) is surpassed. The process is fully reversible and shows the expected dependencies on the composition of the aqueous solution and the bioconjugate chemistry. The results of the affinity precipitation were compared to those achieved with an accepted standard purification of poly(A) mRNA using avidin‐activated magnetic beads. Both yield and quality/purity of the affinity precipitated poly(A) mRNA were found to be similar or better (especially removal of rRNA) than for poly(A) mRNA prepared by the magnetic particle‐based protocol, while both mRNA isolates performed equally well in standard reverse transcriptase amplification (RT‐PCR) of a β actin transcript fragment. Poly(A) mRNA purification schemes based on affinity precipitation require no dedicated equipment and should have advantages in terms of scalability, handling, and costs.

Use of the mitochondria toxicity assay for quantifying the viable cell density of microencapsulated jurkat cells

The mitochondria toxicity assay (MTT assay) is an established method for monitoring cell viability based on mitochondrial activity. Here the MTT assay is proposed for the in situ quantification of the living cell density of microencapsulated Jurkat cells. Three systems were used to encapsulate the cells, namely a membrane consisting of an interpenetrating polyelectrolyte network of sodium cellulose sulphate/poly(diallyldimethylammonium chloride) (NaCS/PDADMAC), a calcium alginate hydrogel covered with poly(L‐lysine) (Ca‐alg‐PLL), and a novel calcium alginate‐poly(ethylene glycol) hybrid material (Ca‐alg‐PEG). MTT results were correlated to data obtained by the trypan blue exclusion assay after release of the cells from the NaCS/PDADMAC and Ca‐alg‐PLL capsules, while a resazurin‐based assay was used for comparison in case of the Ca‐alg‐PEG material. Analysis by MTT assay allows quick and reliable determination of viable cell densities of encapsulated cells independent of the capsule material. The assay is highly reproducible with inter‐assay relative standard deviations below 10%.

Capture of Bacteria from Fermentation Broth by Body Feed Filtration: A Solved Problem?

The direct capture of bacteria produced in high cell density fermentation by filtration is not possible once the milliliter‐scale has been surpassed. Filtration in the presence of a filter aid (body feed filtration) constitutes a putative and scalable alternative, but only if conditions proposed by industry for large‐scale filtration processes, namely, flow rates (for aqueous solutions) in the range of 500–1500 L/(m2·h) and a filter aid concentration of ≤20 g/L, can be met. To this end several filter aids (grades of cellulose, diatomaceous earth, mixes thereof) were tested, albeit with no success. Capture and retention of bacteria was low (not possible in the case of cellulose), and the produced filter cakes were unstable and of low permeability. In the case of the diatomaceous earth close to 100% bacterial capture was possible, but only with filter aid concentrations that were 1 order of magnitude above the proposed limit. By using flocculating agents (Fe3+/Al3+, PEI) either alone or in combination with diatomaceous earth and also the use of positively charged cellulose, bacteria capture could be achieved from several liters of cell suspension (OD600 ≤ 15) in filtration experiments adhering to the industrial recommendations. However, the bacteria were only weakly retained in such cakes and easily released by sudden pressure pulses. Satisfactory results were only obtained by filtration of the bacteria suspension at slightly alkaline pH (8.0, 50 mM Tris buffer) after treatment with sodium‐activated bentonite (5 g/L) and PEI (50 mg/L) in the presence of 250 mM NaCl. In such cases 100% of the bacteria could reproducibly be captured in a filtration using 20 g/L of diatomaceous earth as filter aid. The thus produced filter cakes strongly retained the bacteria and showed good filtration performance. The procedure is at present limited to E. coli culture with biomass contents of OD600 ≤ 15. Cultures with higher OD600 have to be diluted prior to filtration.

High cell density cultivation of human leukemia T cells (Jurkat cells) in semipermeable polyelectrolyte microcapsules

The ex vivo expansion of human T cells is of considerable scientific and medical interest. Currently, this requires the addition of massive amounts of stimuli. Here, human leukemia T cells (Jurkat cells) were used as model cells to demonstrate the in vitro expansion of T cells in the absence of added stimuli after encapsulation in semipermeable sodium cellulose sulfate/poly(diallyldimethyl) ammonium chloride polyelectrolyte membrane capsules (molecular weight cutoff <10 kDa, average diameter ca. 800 μm). For comparison, free and encapsulated cells were cultivated in standard T‐flasks and spinner bottles (both 50 mL culture medium) as well as in hanging drops (35 μL, only nonencapsulated cells). Encapsulation led to a significantly higher specific growth rate, a prolonged exponential growth phase together with a reduced tendency for apoptosis, as evidenced by shifts in the cell cycle distribution toward the S and G2/M phases together with a reduced percentage of cells in the sub‐G0/G1 phase. As a consequence, very high cell densities (>140×106 cells/mLcapsule) were obtained in the capsules, particularly for the spinner cultivations. No evidence for nonspecific activation/stimulation, that is IL‐2 and CD25 expression, was found, while specific stimulation by phorbol‐12‐myristate‐13‐acetate/ionomycin was still possible. Since Jurkat cells commonly serve as model cells for primary T lymphocytes, the proposed method may present a strategy for high‐density proliferation of primary human T lymphocytes.