Host selection as a downstream strategy: polyelectrolyte precipitation of beta-glucuronidase from plant extracts.

Host selection can be a strategy to simplify downstream processing for protein recovery. Advancing capabilities for using plants as hosts offers new host opportunities that have received only limited attention from a downstream processing perspective. Here, we investigated the potential of using a polycationic precipitating agent (polyethylenimine; PEI) to precipitate an acidic model protein (beta-glucuronidase; GUS) from aqueous plant extracts. To assess the potential of host selection to enhance the ease of recovery, the same procedure was applied to oilseed extracts of canola, corn (germ), and soy. For comparison, PEI precipitation of GUS was also evaluated from a crude bacterial fermentation broth. Two versions of the target protein were investigated--the wild-type enzyme (WTGUS) and a genetically engineered version containing 10 additional aspartates on each of the enzyme's four homologous subunits (GUSD10). It was found that canola was the most compatible expression host for use with this purification technique. GUS was completely precipitated from canola with the lowest dosage of PEI (30 mg PEI/g total protein), and over 80% of the initial WTGUS activity was recovered with 18-fold purification. Precipitation from soy gave yields over 90% for WTGUS but only 1.3-fold enrichment. Corn, although requiring the most PEI relative to total protein to precipitate (210 mg PEI/g total protein for 100% precipitation), gave intermediate results, with 81% recovery of WTGUS activity and a purification factor of 2.6. The addition of aspartate residues to the target protein did not enhance the selectivity of PEI precipitation in any of the systems tested. In fact, the additional charge reduced the ability to recover GUSD10 from the precipitate, resulting in lower yields and enrichment ratios compared to WTGUS. Compared to the bacterial host, plant systems provided lower polymer dosage requirements, higher yields of recoverable activity and greater purification factors.     

Affinity precipitation of monoclonal antibodies by nonstoichiometric polyelectrolyte complexes

The nonstoichiometric polyelectrolyte complex (PEC) formed by poly(methacrylic acid) (degree of polymerization 1830) (PMAA)and poly(N-ethyl-4-vinyl-pyridinium bromide) (degree of polymerization 530) (PEVP) undergoes reversible precipitation from aqueous solution at any desired pH-value in the range 4.5–6.5 depending on the ionic strength and PEVP/PMAA ratio in the complex. The antigen, inactivated glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from rabbit was covalently coupled to PEVP. The resulting GAPDH–PEVP/PMAA complex was used for the purification of antibodies from a 6G7 clone specific towards inactivated GAPDH. The crude extract was incubated with GAPDH-containing PEC and the precipitation of the PEC was carried out at 0.01 M NaCl and pH 4.5, 5.3, 6.0 and 6.5 using PEC with PEVP/PMAA ratios of 0.45, 0.3, 0.2 and 0.15, respectively. Purified antibodies were eluted at pH 4.0 where PECs of all compositions used were insoluble.PEC precipitation is accompanied only by small nonspecific coprecipitation of proteins. Precipitated PEC could be dissolved at pH 7.3 and used repeatedly. This revised version was published online in July 2006 with corrections to the Cover Date.     

Purification of chymotrypsin from bovine pancreas using precipitation with a strong anionic polyelectrolyte

The separation of chymotrypsin from a crude filtrate of bovine pancreas homogenate was carried out using precipitation with a commercially available negatively charged strong polyelectrolyte: polyvinyl sulfonate. The zymogen form of chymotrypsin was activated by addition of trypsin (0.01 mg/g homogenate), then, the enzyme was precipitated by polyelectrolyte addition at pH 2.5 in the pancreas homogenate. A stoichiometric ratio of 670 bound molecules of chymotrypsin per polyelectrolyte molecule was found in the non-soluble form of the enzyme–polyelectrolyte complex. The non-soluble complex was separated by simple centrifugation and re-dissolved by a pH change to 8.0. The recovery of chymotrypsin biological activity was 61% of the initial activity in the homogenate with 4.7-fold increase in its specific activity.     

Recovery of residual soluble protein by two-step precipitation process with concomitant COD reduction from the yeast-cultivated cheese whey

The present study was conducted to recover the residual soluble protein after cultivation of yeast (K. marxianus) in cheese whey. Cheese whey continuous fermentation with cell recycle system was carried out at 40 °C and pH 3.5. The yeast biomass was separated from the fermented broth by centrifugation and residual soluble protein from fermented whey supernatant was precipitated by heat treatment (at 100 °C, pH 4.5 and 10 min incubation). The maximum soluble protein recovery up to 53 % was achieved at pH 4.5 with 54 % residual COD removal. However, gravity sedimentable precipitates were obtained at pH 3.5 with 47 % protein recovery. Therefore, the reactor (scale up) study was conducted at pH 3.5 with agitation, which resulted in 68 % of residual soluble protein recovery and simultaneously residual COD removal of 62 %. Further precipitation/coagulation of soluble protein was also evaluated using carboxymethylcellulose (CMC) and then two precipitation (thermal followed by CMC precipitation) processes were combined to increase the protein precipitation, which finally reached up to 81 % of total soluble protein recovery from the supernatant. This optimized process could be applied to recover the residual protein left after fermentation of cheese whey without centrifugation.     

The removal of nucleic acids from microbial extracts by precipitation with lysozyme

It is usually necessary to remove nucleic acids from microbial extracts in order to avoid their interference with the isolation of enzymes from the extract. This may be particularly important where the purification procedure includes chromatography on an anion-exchange column such as DEAE-Sephadex (1). Methods that have been used have included precipitation with Mn, destruction of the nucleic acids with nucleases, and precipitation with basic substances, usually protamine sulfate or streptomycin sulfate. It is likely that there may be advantages, in some cases at least, in using other basic proteins for this purpose, since the results obtained with the previous methods have not always been satisfactory. The procedure described in this paper utilizes high concentrations of lysozyme to precipitate nucleic acids from a bacterial extract. The separation obtained with lysozyme was efficient, reproducible, and superior to the separation obtained with protamine sulfate.     

Recovery of canola meal proteins by precipitation

Recovery of rapeseed proteins from defatted canola meal by precipitation was investigated. The ability of different precipitating agents, such as sodium hexametaphosphate (HMP), carboxymethylcellulose (CMC), ammonium sulphate, and isoelectric precipitation using HCl, were evaluated based on the yield and mean size of protein aggregates. Almost 94% of dissolved protein was precipitated in the presence of 2.7M ammonium sulphate, while the largest mean protein particle size (32 mum) was obtained in the presence of HMP at pH 3.3.     

Fibronectin and cell attachment to cell and protein resistant polyelectrolyte surfaces

Culture of A7r5 smooth muscle cells on a polyelectrolyte multilayer film (PEMU) can influence various cell properties, including adhesion, motility, and cytoskeletal organization, that are modulated by the extracellular matrix (ECM) in vivo. ECM contribution to cell behavior on PEMUs was investigated by determining the amount of fibronectin (FN) bound to charged and hydrophobic PEMUs by optical waveguide lightmode spectroscopy and immunofluorescence microscopy. FN bound best to poly(allylamine hydrochloride) (PAH)-terminated and Nafion-terminated PEMUs. FN bound poorly to PEMUs terminated with a copolymer of poly(acrylic acid) (PAA) and 3-[2-(acrylamido)-ethyl dimethylammonio] propane sulfonate (PAA-co-AEDAPS). Cells adhered and spread well on the Nafion-terminated PEMU surfaces. In contrast, cells spread less and migrated more on both FN-coated and uncoated PAH-terminated PEMU surfaces. Both cells and FN interacted much better with Nafion than with PAA-co-PAEDAPS in a micropatterned PEMU. These results indicate that A7r5 cell adhesion, spreading, and motility on PEMUs can be independent of FN binding to the surfaces.     

Differential inhibition of protein synthesis in reticulocyte lysates and wheat germ extracts by a protein isolated from wheat germ extracts.

A protein with a molecular mass of 35-37 kDa has been isolated and partially purified from the postribosomal supernatant of wheat germ by ammonium sulfate precipitation (60-90%), Sephadex G-75, and DEAE-cellulose chromatography. It inhibited endogenous protein synthesis in rabbit reticulocyte lysates but had no effect on translation in wheat germ extracts. At low concentrations (0.34-1.36 ng/15 microliter assay), inhibition was limited to initiation of peptide synthesis. At higher concentrations (13.6 ng/15 microliter assay), elongation was also suppressed.     

Recovery of a monoclonal antibody from hybridoma culture supernatant by affinity precipitation with Eudragit S-100

An IgG₁ monoclonal antibody (MAB) was isolated from hybridoma culture supernatant by affinity precipitation with an Eudragit S-100-based heterobifunctional ligand. Affinity binding was performed in a homogeneous aqueous phase at pH 7.5 followed by precipitation of the bound affinity complex by lowering the pH to 4.8. After two washing steps, elution of specifically bound MAB was achieved by incubating the precipitate with 0.1 M glycine.HCl pH 2.5. The influence of elution volume and time on the recovery of active MAB and the overall purification factor were studied. The best conditions enabled the recovery of 50.2% of active MAB with a purification factor of 6.2. A further dialysis against 50 mM Tris.HCl pH 8.0 increased the activity yield and the purification factor to 68.4% and 8.3, respectively. This result showed that part of the antibody activity loss during affinity precipitation was due to a reversible inactivation process, being easily recovered after a refining dialysis step.     

Specific binding of cruciform DNA structures by a protein from human extracts.

A gel electrophoresis binding assay has been used to probe extracts from cultured human lymphoblasts for proteins that bind cruciform structures in duplex DNA. Proteins have been detected that form complexes with synthetic X- and Y-junctions. Several lines of evidence suggest that binding is specific for DNA structure rather than sequence: (1) X- and Y-structures were bound whereas linear duplexes containing identical DNA sequences were not, (2) Binding occurred with equal efficiency to two X-junctions that were constructed from DNA strands of different sequence, (3) One X-junction successfully competed with another for binding whereas linear duplex DNA did not; and (4) protein-DNA complexes were observed at probe:non-specific competitor DNA ratios of 1:10,000.     

Analytical biochemistry of DNA–protein assemblies from crude cell extracts

Purification of specific DNA–protein complexes is a challenging task, as the involved interactions can be both electrostatic/H-bond and hydrophobic. The chromatographic stringency needed to obtain reasonable purifications uses salts and detergents. However, these components elicit the removal of proteins unspecifically bound to the chromatographic support itself, thus contaminating the purification products. In this work, a photocleavable linker connected the target oligonucleotidic sequence to the chromatographic beads so as to allow the irradiation-based release of the purified DNA–protein complexes off the beads. Our bioanalytical conditions were validated by purifying the tetracycline repressor protein onto a specific oligonucleotide. The purification factor was unprecedented, with a single contaminant. The robustness of our method was challenged by applying it to the purification of multiprotein assemblies forming onto DNA damage-mimicking oligonucleotides. The purified components were identified as well-known DNA repair proteins, and were shown to retain their enzymatic activities, as seen by monitoring DNA ligation products. Remarkably, kinase activities, also monitored, were found to be distinct on the beads and on the purified DNA–protein complexes, showing the benefits to uncouple the DNA–protein assemblies from the beads for a proper understanding of biochemical regulatory mechanisms involved in the DNA–protein assemblies.     

Controling the rate of protein release from polyelectrolyte complexes

Extended protein release from readily prepared, water-insoluble complexes with oppositely charged polyions is explored. Using hen egg-white lysozyme as a model, its sustained release from such complexes with a number of polyanions under physiological conditions has been demonstrated and rationalized. The rate of release varies orders of magnitude and is controlled by the nature of the polyanion (decreasing upon increase in its linear charge density, length, and hydrophobicity) and the complex particle size (the larger the particles, the slower the release).     

Downstream protein separation by surfactant precipitation: a review

In a conventional protein downstream processing (DSP) scheme, chromatography is the single most expensive step. Despite being highly effective, it often has a low process throughput due to its semibatch nature, sometimes with nonreproducible results and relatively complex process development. Hence, more work is required to develop alternative purification methods that are more cost-effective, but exhibiting nearly comparable performance. In recent years, surfactant precipitation has been heralded as a promising new method for primary protein recovery that meets these criteria and is a simple and cost-effective method that purifies and concentrates. The method requires the direct addition of a surfactant to a complex solution (e.g. a fermentation broth) containing the protein of interest, where the final surfactant concentration is maintained below its critical micelle concentration (CMC) in order to allow for electrostatic and hydrophobic interactions between the surfactant and the target protein. An insoluble (hydrophobic) protein–surfactant complex is formed and backextraction of the target protein from the precipitate into a new aqueous phase is then carried out using either solvent extraction, or addition of a counter-ionic surfactant. Importantly, as highlighted by past researchers, the recovered proteins maintain their activity and structural integrity, as determined by circular dichroism (CD). In this review, various aspects of surfactant precipitation with respect to its general methodology and process mechanism, system parameters influencing performance, protein recovery, process selectivity and process advantages will be highlighted. Moreover, comparisons will be made to reverse micellar extraction, and the current drawbacks/challenges of surfactant precipitation will also be discussed. Finally, promising directions of future work with this separation technique will be highlighted.     

Recognition of target colloid species by conjugates of a linear polyelectrolyte with a vector protein

Redistribution reaction of quaternized poly-4-vinylpyridine polycations and their conjugates with alpha-chymotrypsin by oppositely charged latex particles is disclosed. The polycations are strongly adsorbed on the latex surface. Nevertheless, they are able to migrate between the latex species via occasional interparticle contacts. In the case of a homogeneous latex such interchange results in uniform distribution of polycations by latex particles. The distribution drastically changes, when alpha-chymotrypsin-polycation conjugates interact with a mixture of two latexes: one chemically modified by bovine serum albumin and the other one by specific protein inhibitor of alpha-chymotrypsin. In this case the interchanging polycations are finally fixed on the latex particles carrying the centres of specific binding of the enzyme vector, i.e. recognize them in the latex mixture. The obtained results are considered to mimic physico-chemical interaction and recognition of target supermolecular bio-objects by large macromolecules carrying relatively small molecular vectors.     

Process characterization strategy for a precipitation step for host cell protein reduction

Process characterization using QbD approaches has rarely been described for precipitation steps used for impurity removal in biopharmaceutical processes. We propose a two-step approach for process characterization in which the first step focuses on product quality and the second focuses on process performance. This approach provides an efficient, streamlined strategy for the characterization of precipitation steps under the Quality by Design paradigm. This strategy is demonstrated by a case study for the characterization of a precipitation using sodium caprylate to reduce host cell proteins (HCP) during a monoclonal antibody purification process. Process parameters were methodically selected through a risk assessment based on prior development data and scientific knowledge described in the literature. The characterization studies used two multivariate blocks to decouple and distinguish the impact of product quality (e.g., measured HCP of the recovered product from the precipitation) and process performance (e.g., step yield). Robustness of the precipitation step was further demonstrated through linkage studies across the overall purification process. HCP levels could be robustly reduced to ≤100 ppm in the drug substance when the precipitation step operated within an operation space of ≤1% (m/v) sodium caprylate, pH 5.0–6.0, and filter flux ≤300 L/m²-hr for a load HCP concentration up to 19,000 ppm. This two-step approach for characterization of precipitation steps has several advantages, including tailoring of the experimental design and scale-down model to the intended purpose for each step, use of a manageable number of experiments without compromising scientific understanding, and limited time and material consumption.