Monoclonal antibody purification using cationic polyelectrolytes: an alternative to column chromatography

The potential of cationic polyelectrolytes to precipitate host cell and process related impurities was investigated, to replace one or more chromatography steps in monoclonal antibody purification. The impact of antibody isoelectric point, solution properties (pH and ionic strength), and polyelectrolyte properties (structure, molecular weight and pK(a)) on the degree of precipitation was studied. At neutral pH, increasing solution ionic strength impeded the ionic interaction between the polyelectrolyte and impurities, reducing impurity precipitation. Increasing polyelectrolyte molecular weight and pK(a) enabled precipitation of impurities at higher ionic strength. PoIy(arginine) was selected as the preferred polyelectrolyte in unconditioned cell culture fluid. PoIy(arginine) precipitation achieved consistent host cell protein clearance and antibody recovery for multiple antibodies across a wider range of polyelectrolyte concentrations. Poly(arginine) precipitation was evaluated as a flocculant and as a functional replacement for anion exchange chromatography in an antibody purification process. Upstream treatment of cell culture fluid with poly(arginine) resulted in flocculation of solids (cells and cell debris), and antibody recovery and impurity clearance (host cell proteins, DNA and insulin) comparable to the downstream anion exchange chromatography step.     

Development of an alternative approach to protein crystallization

We are developing an alternate strategy for the crystallization of macromolecules that does not, like current methods, depend on the optimization of traditional variables such as pH and precipitant concentration, but is based on the hypothesis that many conventional small molecules might establish stabilizing, intermolecular, non covalent crosslinks in crystals, and thereby promote lattice formation. To test the hypothesis, we carried out preliminary experiments encompassing 18,240 crystallization trials using 81 different proteins, and 200 chemical compounds. Statistical analysis of the results demonstrated the validity of the idea. In addition, we conducted X-ray diffraction analyses of some of the crystals grown in the experiments. These clearly showed incorporation of conventional molecules into the protein crystal lattices, and further validated the underlying hypothesis. We are currently extending the investigations to include a broader and more diverse set of proteins, an expanded search of conventional and biologically active small molecules, and a wider range of precipitants. The strategy proposed here is essentially orthogonal to current approaches and has an objective of doubling the success rate of today.     

A simple strategy towards membrane protein purification and crystallization

A simple and cost-efficient detergent screening strategy has been developed, by which a number of detergents were screened for their efficiency to extract and purify the recombinant ammonium/ammonia channel, AmtB, from Escherichia coli, hence selecting the most efficient detergents prior to large-scale protein production and crystallization. The method requires 1 ml cell culture and is a combination of immobilized metal ion affinity chromatography and filtration steps in 96-well plates. Large-scale protein purification and subsequent crystallization screening resulted in AmtB crystals diffracting to low resolution with three detergents. This strategy allows exclusion of detergents with the lowest probability in yielding protein crystals and selecting those with higher probability, hence, reducing the number of detergents to be screened prior to large-scale membrane protein purification and perhaps also crystallization.     

Chromosomal His-tagging: an alternative approach to membrane protein purification

Membrane proteins are of keen interest to structural biologists, as they are known to act as receptors, adhesins, sensors, transporters, and signal-transducers of living cells. During the past few decades, the efforts made to study the bacterial membrane proteins have been impaired by the problems encountered during the production and purification of native proteins. Herein we demonstrate that the Campylobacter jejuni CadF protein, which was isolated using a novel purification strategy, exhibits biological activity as evidenced by channel activity in lipid bilayers. CadF, an E. coli OmpA-like protein, facilitates the binding of C. jejuni to the extracellular matrix component, fibronectin.     

Enzyme purification by hydrophobic chromatography: an alternative approach illustrated in the purification of aspartate transcarbamoylase from wheat germ (Short Communication)

Two adsorbents containing similar numbers of hydrocarbon (C(10)) chains but different numbers of carboxyl groups were made by chemical modification of Sepharose. The use of these adsorbents to purify proteins, under conditions where hydrophobic adsorption is partly resisted by electrostatic repulsion, is illustrated in the purification of aspartate transcarbamoylase (EC 2.1.3.2) from wheat germ.     

Toluene dioxygenase: purification of an iron-sulfur protein by affinity chromatography

Toluene dioxygenase, from $\text{Pseudomonas}\text{putida}$, oxidizes toluene to (+)-$\text{cis}$-1(S),2(R)-dihydroxy-3-methylcyclohexa-3,5-diene. The oxygenase-component of this multienzyme system was purified to homogeneity by a two-step procedure that utilized affinity and ion exchange chromatography. The purified enzyme would oxidize toluene only in the presence of NADH, ferrous iron and partially purified preparations of NADH cytochrome c reductase and an iron-sulfur protein (ferredoxin_TOL). Spinach NADPH cytochrome c reductase and NADPH could substitute for the $\text{Pseudomonas}$ reductase and NADH. The molecular weight of the oxygenase-component was determined to be 151,000 and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicated that the enzyme is composed of two subunits with molecular weights of 52,500 and 20,800. The absorption spectrum showed maxima at 550 (Shoulder), 450, 326 and 278 nm and preliminary experiments have indicated the presence of 2 gram atoms of iron and 2 gram atoms of acid-labile sulfur per mole of protein. The results indicate that the oxygenase-component of the toluene dioxygenase enzyme system is an iron-sulfur protein that has been designated ISP_TOL.     

The D-channel of cytochrome oxidase: an alternative view

The D-pathway in A-type cytochrome c oxidases conducts protons from a conserved aspartate on the negatively charged N-side of the membrane to a conserved glutamic acid at about the middle of the membrane dielectric. Extensive work in the past has indicated that all four protons pumped across the membrane on reduction of O(2) to water are transferred via the D-pathway, and that it is also responsible for transfer of two out of the four "chemical protons" from the N-side to the binuclear oxygen reduction site to form product water. The function of the D-pathway has been discussed in terms of an apparent pK(a) of the glutamic acid. After reacting fully reduced enzyme with O(2), the rate of formation of the F state of the binuclear heme-copper active site was found to be independent of pH up to pH~9, but to drop off at higher pH with an apparent pK(a) of 9.4, which was attributed to the glutamic acid. Here, we present an alternative view, according to which the pH-dependence is controlled by proton transfer into the aspartate residue at the N-side orifice of the D-pathway. We summarise experimental evidence that favours a proton pump mechanism in which the proton to be pumped is transferred from the glutamic acid to a proton-loading site prior to proton transfer for completion of oxygen reduction chemistry. The mechanism is discussed by which the proton-pumping activity is decoupled from electron transfer by structural alterations of the D-pathway. This article is part of a Special Issue entitled: Allosteric cooperativity in respiratory proteins.     

Tetanus toxoid purification: chromatographic procedures as an alternative to ammonium-sulphate precipitation

Given an existing demand to establish a process of tetanus vaccine production in a way that allows its complete validation and standardization, this paper focuses on tetanus toxoid purification step. More precisely, we were looking at a possibility to replace the widely used ammonium-sulphate precipitation by a chromatographic method. Based on the tetanus toxin's biochemical characteristics, we have decided to examine the possibility of tetanus toxoid purification by hydrophobic chromatography, and by chromatographic techniques based on interaction with immobilized metal ions, i.e. chelating chromatography and immobilized metal affinity chromatography. We used samples obtained from differently fragmented crude tetanus toxins by formaldehyde treatment (assigned as TTd-A and TTd-B) as starting material for tetanus toxoid purification. Obtained results imply that purification of tetanus toxoid by hydrophobic chromatography represents a good alternative to ammonium-sulphate precipitation. Tetanus toxoid preparations obtained by hydrophobic chromatography were similar to those obtained by ammonium-sulphate precipitation in respect to yield, purity and immunogenicity. In addition, their immunogenicity was similar to standard tetanus toxoid preparation (NIBSC, Potters Bar, UK). Furthermore, the characteristics of crude tetanus toxin preparations had the lowest impact on the final purification product when hydrophobic chromatography was the applied method of tetanus toxoid purification. On the other hand, purifications of tetanus toxoid by chelating chromatography or immobilized metal affinity chromatography generally resulted in a very low yield due to not satisfactory tetanus toxoid binding to the column, and immunogenicity of the obtained tetanus toxoid-containing preparations was poor.     

Method of purification of glucose oxidase by means of affinity chromatography on immunoabsorbent]

The possibility to purify glucose oxidase from Penicillium vitale on immunosorbent containing specific antibodies to the enzyme covalently bound with Sepharose 4B is studied. The method of affinity chromatography was applied, beside routine methods of fractionating blood serum proteins, to isolate specific antibodies from antiserum of rabbits immunized with glucose oxidase. Immobilized on Sepharose glucose oxidase was used as biospecific sorbent. Specific antibodies to the enzyme were isolated using chromatograpy of gamma-globulins mixture followed by protein desorption from the column with 1 M NaC1 and 3% glucose. Antibodies were immobilized by their covalent binding to activated Sepharose. The immunosorbent obtained was used to purify low active preparation of glucose oxidase by means of affinity chromatography under conditions worked out for the antibodies isolation. The enzyme was eluted from the column with 1 M NaC1 (pH 3.0) containing 3% glucose. 5-Fold purified enzyme preparation was isolated.     

Trypanosome alternative oxidase: from molecule to function

Trypanosome alternative oxidase (TAO) is the cytochrome-independent terminal oxidase of the mitochondrial electron transport chain. TAO is a diiron protein that transfers electrons from ubiquinol to oxygen, reducing the oxygen to water. The mammalian bloodstream forms of Trypanosoma brucei depend solely on TAO for respiration. The inhibition of TAO by salicylhydroxamic acid (SHAM) or ascofuranone is trypanocidal. TAO is present at a reduced level in the procyclic form of T. brucei, where it is engaged in respiration and is also needed for developmental processes. Alternative oxidases similar to TAO have been found in a wide variety of organisms but not in mammals, thus rendering TAO an important chemotherapeutic target for African trypanosomiasis.     

Using precipitation by polyamines as an alternative to chromatographic separation in antibody purification processes

Polyamine precipitation conditions for removing host cell protein impurities from the cell culture fluid containing monoclonal antibody were studied. We examined the impact of polyamine concentration, size, structure, cell culture fluid pH and ionic strength. A 96-well microtiter plate based high throughput screening method was developed and used for evaluating different polyamines. Polyallylamine, polyvinylamine, branched polyethyleneimine and poly(dimethylamine-co-epichlorohydrin-ethylenediamine) were identified as efficient precipitants in removing host cell protein impurities. Leveraging from the screening results, we incorporated a polyamine precipitation step into a monoclonal antibody purification process to replace the Protein A chromatography step. The optimization of the overall purification process was performed by taking the mechanisms of both precipitation and chromatographic separation into account. The precipitation-containing process removed a similar amount of process-related impurities, including host cell proteins, DNA, insulin and gentamicin and maintained similar product quality in respect of size and charge variants to chromatography based purification. Overall recovery yield was comparable to the typical Protein A affinity chromatography based antibody purification process.     

Rapid purification of synthetic oligonucleotides: a convenient alternative to high-performance liquid chromatography and polyacrylamide gel electrophoresis

A new method has been developed to purify and detritylate milligram amounts of synthetic oligonucleotides. Dimethoxytrityl oligonucleotides from 15 to 100 nucleotides in length are applied in triethylammonium acetate or concentrated ammonium hydroxide to a disposable chromatographic cartridge, the NENSORB PREP Nucleic Acid Purification Cartridge. Salts, failure sequences and synthetic by-products are washed away while the desired, full-length, dimethoxytrityl oligonucleotide remains bound to the cartridge. The trityl group is hydrolyzed from the 5'-end of the oligonucleotide with an acid wash and then the purified oligonucleotide is eluted with 35% methanol. Oligonucleotides are recovered salt-free with purities greater than 95%. NENSORB PREP-purified primers provide superior sequence data compared to similar primers used without purification and equivalent data to primers purified by polyacrylamide gel electrophoresis when used in manual radiometric Sanger sequencing.     

Caprylic acid precipitation method for impurity reduction: An alternative to conventional chromatography for monoclonal antibody purification

We report the use of caprylic acid based impurity precipitation as (1) an alternative method to polishing chromatography techniques commonly used for monoclonal antibody purification and (2) an impurity reduction step prior to harvesting the bioreactor. This impurity reduction method was tested with protein A purified antibodies and with cell culture fluid. First, the operational parameters influencing precipitation of host cell proteins and high molecular weight aggregate in protein A pools were investigated. When used as a polishing step, the primary factor affecting purification and yield was determined to be pH. Caprylic acid precipitation was comparable to polishing IEX chromatography in reducing host cell protein and aggregate levels. A virus reduction study showed complete clearance of a model retrovirus during caprylic acid precipitation of protein A purified antibody. Caprylic acid mediated impurity precipitation in cell culture showed that the impurity clearance was generally insensitive to pH and caprylic acid concentration whereas yield was a function of caprylic acid concentration. Protein A purification of caprylic acid precipitated cell culture fluid generated less turbid product pool with reduced levels of host cell proteins and high molecular weight aggregate. The results of this study show caprylic acid precipitation to be an effective purification method that can be incorporated into a production facility with minimal cost as it utilizes existing tanks and process flow. Eliminating flow through chromatography polishing step can provide process intensification by avoiding the process tank volume constraints for high titer processes. Biotechnol. Bioeng. 2012; 109: 2589–2598. © 2012 Wiley Periodicals, Inc.     

Rapid purification by affinity chromatography of rat brain pyridoxal kinase and pyridoxamine-5-phosphate oxidase

Rat brain pyridoxal kinase and pyridoxamine 5′ phosphate oxidase have been purified to electrophoretic homogeneity using pyridoxyl Sepharose and phosphopyridoxyl Sepharose columns as the first stages in the purification procedures. These affinity supports were synthesized by two simple steps consisting of reacting pyridoxal and pyridoxal 5′ phosphate with commercially available ω-aminohexyl Sepharose and subsequent reduction of the resultant Schiff's bases with sodium borohydride. This method allows total purification of both enzymes from the same tissue source in 4–5 days.     

Protein purification by bulk crystallization: the recovery of ovalbumin

Crystallization is used industrially for the recovery and purification of many inorganic and organic materials. However, very little is reported on the application of bulk crystallization for proteins. In this work, ovalbumin was selected as a model protein to investigate the feasibility of using bulk crystallization for the recovery and purification of proteins. A stirred 1-L seeded batch crystallizer was used to obtain the crystal growth kinetics of ovalbumin in ammonium sulfate solutions at 30 degrees C. The width of the metastable region, in which crystal growth can occur without any nucleation, is equivalent to a relative supersaturation of about 20. The bulk crystallizations were undertaken within this range (using initial relative supersaturations less than 10) and nucleation was not observed. The ovalbumin concentration in solution was measured by UV absorbance and checked by crystal content measurement. Crystal size distributions were measured both by using a Malvern Mastersizer and by counting crystals through a microscope. The crystal growth rate was found to have a second-order dependence upon the ovalbumin supersaturation. While there is no discernible effect of ammonium sulfate concentration at pH 4.90, there is a slight effect at higher pH values. Overall the effect of ammonium sulfate concentration is small compared to the effect of pH, for which there is a 10-fold increase in the growth rate constant, k(Gsigma) over the range pH 4.6-5.4. To demonstrate the degree of purification which can be achieved by bulk crystallization, ovalbumin was crystallized from a solution containing conalbumin (80,000 Da) and lysozyme (14, 600 Da). After one crystallization and a crystal wash, ovalbumin crystals were produced with a protein purity greater than 99%. No contamination by the other proteins was observed when using overloaded sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) stained with Coomassie blue stain and only trace amounts of lysozyme were observed using a silver stain. The presence of these other proteins in solution did not effect the crystal growth rate constant, k(Gsigma). The study demonstrates the feasibility of using bulk crystallization for the recovery and purification of ovalbumin. It should be readily applicable to other protein systems. (c) 1995 John Wiley & Sons, Inc.     

Cynomolgus monkey liver aldehyde oxidase: extremely high oxidase activity and an attempt at purification

Aldehyde oxidase (EC 1.2.3.1) in monkey (Macaca fascicularis) liver was characterized. Liver cytosol exhibited extremely high benzaldehyde and phthalazine oxidase activities based on aldehyde oxidase, compared with those of rabbits, rats, mice and guinea pigs. Monkey liver aldehyde oxidase showed broad substrate specificity distinct from that of the enzyme from other mammals. Purified aldehyde oxidase from monkey liver cytosol showed two major bands and two minor bands in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). These bands were also observed in Western blotting analysis using anti-rat aldehyde oxidase. The molecular mass of the enzyme was estimated to be 130-151 kDa by SDS-PAGE, and to be about 285 kDa by HPLC gel filtration. The results suggest that isoforms of aldehyde oxidase exist in monkey livers.     

Gradient thin-layer chromatography of oligonucleotides on DEAE-cellulose: an alternative to homochromatography.

Homochromatography fingerprints are widely used for sequence studies of labeled RNA: however, the more general use of this method is restricted in several aspects by the large excess of RNA introduced by the homomix. Gradient thin-layer chromatography on DEAE-cellulose plates using ammonium formate gradients in 9 m urea provides an efficient procedure for preparing fingerprints of labeled as well as unlabeled RNA, and allows the isolation of oligonucleotides free of salt, urea, and carrier RNA. This method produces fingerprints similar to those obtained by homochromatography under appropriate conditions. Consequently, gradient thin-layer chromatography is a convenient alternative to homochromatography without some of its limitations.     

Human lysosomal beta-glucosidase: purification by affinity chromatography

Two Sepharose-bound substrate analogs, 6'-aminohexanoyl-(2-N-sphingosyl-O-beta-D-glucoside) and 6'-aminohexyl-dodecanedioyl-1-(2-N-sphingosyl-1-O-beta-D-glu coside), were synthesized and used sequentially for the affinity purification of lysosomal beta-glucosidase (N-acyl-sphingosyl-1-O-beta-D-glucoside:glucohydrolase, EC 3.2.1.45). The capacities of these nondegradable affinity supports were 0.1 and 0.15 mg enzyme/ml settled gel, respectively. The purified enzyme had a specific activity of 75 mumol min-1 mg-1. The preparation had a single protein band with a molecular weight of 67,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, evidencing its apparent homogeneity. Isoelectric focusing on granular gels revealed four molecular forms of the enzyme with pI values of 4.0, 4.5, 4.7, and 5.8 to 6.2. The purified enzyme hydrolyzed glucosyl ceramide and 4-methylumbelliferyl-beta-D-glucoside with Km and Vmax values of 0.6 and 2.5 mM, and 101 and 26.1 mumol min-1 mg-1, respectively. The enzyme also hydrolyzed octyl beta-glucoside, a linear mixed-type inhibitor of the enzyme. Binding constants (Ki) were determined for the inhibitors, sphingosyl-1-O-beta-D-glucoside (Ki = 20 microM) and its N-hexyl derivative (Ki = 0.3 microM). The enzyme had a half-life of 65 and 30 min at 50 degrees C and pH 5.0 or 6.0, respectively. In addition, two other classes of ligands were used for the purification of lysosomal beta-glucosidase, and their capacities and specificities were compared to those of the substrate analog affinity supports. These included (i) the alkyl amine inhibitors octylamine, decylamine, and tetradecylamine; and (ii) the inhibitors, 6-aminohexanoyl-beta-glucosylamine and aminododecanoyl-1-(2-N-sphingosyl-1-O-beta-D-glucoside). Compared to these other ligand columns, the substrate analog affinity supports had about 100- to 1000-fold greater capacities or afforded 8- to 40-fold greater purification of human lysosomal beta-glucosidase.