Simplified and more robust EBA processes by elution in expanded bed mode.

This paper illustrates the feasibility of eluting EBA columns in the expanded bed mode as an alternative to the generally used method of packed bed elution. It is shown that at linear flow rates of 1-3 cm/min the difference in total elution volume between expanded bed elution and packed bed elution is less than 20%. It is suggested that expanded bed elution offers a range of significant advantages, while the drawbacks will be insignificant in most applications. The key to the success of this method seems to be the use of EBA matrices with a relatively low degree of expansion (i.e. a high density) at the linear flow rates employed for elution of bound product.

Capture of human Fab fragments by expanded bed adsorption with a mixed mode adsorbent.

A novel group of mixed mode adsorbents has been developed for purification of monoclonal and polyclonal antibodies from a broad range of raw materials such as hybridoma cell culture, ascites fluid, animal sera, milk, whey and egg yolk. The aim of this study was to determine whether such mixed mode adsorbents were also useful for the recovery of recombinant proteins from microbial feedstocks. This paper describes the performance of one of these adsorbents for expanded bed capture of a human Fab fragment from recombinant E. Coli cell extracts. It is concluded that the mixed mode adsorbent binds the Fab fragment efficiently from crude extracts without any requirement for preconditioning the extract by for example de-salting or dilution. The capacity of the mixed mode adsorbent is approx. 12 mg Fab/ml matrix. The novel mixed mode adsorbent can be useful during production of highly purified Fab fragments as the first step in a purification scheme. In this respect the mixed mode adsorbent is advantageous over alternative commercially available ion-exchange materials which require pre-conditioning of cell extract for Fab' capture. Together with the concentration and clarification effect a significant enrichment of the Fab fragment is obtained in one single high yield operation.

Sulfone-aromatic ligands for thiophilic adsorption chromatography: purification of human and mouse immunoglobulins.

New thiophilic matrices and new procedures were used for the purification of immunoglobulins both from human serum and from hybridoma cell cultures containing fetal calf serum. A range of aromatic and heteroaromatic ligands containing hydroxyl or amino groups have been coupled to divinyl sulfone-activated agarose. The resulting affinity matrices have the general formula M-O-CH2-CH2-SO2-CH2-CH2-X-Y, where M is the agarose matrix, X is oxygen or nitrogen, and Y is an aromatic or heteroaromatic compound. Contrary to earlier expectations these matrices showed pronounced thiophilic binding patterns when tested for the selective binding of immunoglobulins from human serum. The binding is influenced by the structure of the aromatic part of the ligand, the ligand concentration, and the concentration and type of lyotropic salt. 2-Hydroxypyridine coupled to divinyl sulfone-activated agarose was used to purify murine monoclonal antibodies (IgG1 and IgM) from hybridoma cell cultures containing fetal calf serum. Compared to previous methods, significantly increased binding capacity (300-1500%) was obtained by using 1.0-1.2 M ammonium sulfate. Purity of the monoclonal antibody may be optimized for each individual clone by washing the column with either a low concentration of ammonium sulfate or polyethylene glycol before elution.

Thiophilic adsorption chromatography: the separation of serum proteins.

Human serum proteins were separated on a matrix obtained by reaction of beta-mercaptoethanol with divinyl sulfone-activated agarose (the so-called T-gel). Binding of almost all serum proteins was observed at high concentrations of ammonium sulfate. Elution was achieved by gradually lowering the concentration of salt in the washing buffer. Fractions obtained during elution were analyzed by fused rocket immunoelectrophoresis. Proteins were recovered in high yields and with an excellent separation in this one-step procedure ("Thiophilic adsorption chromatography"). A rapid and straightforward procedure giving essentially pure immunoglobulins from crude rabbit serum with at least 80% yield by the T-gel is also presented.

Biochemical characterization, localization and immunostimulating properties of a soluble glycoprotein, Ag1, isolated from in vitro cultures of Plasmodium falciparum.

The soluble amphiphilic glycoprotein, Ag1 (gp60), purified from supernatants of in vitro cultures of Plasmodium falciparum has a molecular mass of 60 kDa and did not exhibit size variation in the different P. falciparum isolates tested by immunoblotting. Ag1 was shown to interact with the lectin Erythrina christagalli agglutinin, which is specific for carbohydrates bearing beta-D-galactose(1-4)-D-N-acetylglucosamine. Indirect immunofluorescence studies showed that Ag1 is located on the surface of trophozoites and schizonts but not on the surface of merozoites. Ag1 is recognized by human immune sera from six different malaria-endemic regions. Ag1 induces in vitro proliferation of lymphocytes from malaria-immune individuals in an antigen-specific manner.

Real time monitoring of acylations during solid phase peptide synthesis: a method based on electrochemical detection.

Monitoring of acylation reactions during solid phase peptide synthesis is important to ensure high coupling yields in all steps of the synthesis. We describe in this paper a simple and reliable method for monitoring the time course of the acylation steps as well as the washing and deprotection steps during computer-controlled solid phase peptide synthesis. The method is based on the continuous measurement of electrical conductivity in the reaction vessel. It is shown that there is a close correspondence between the degree of acylation (as determined from the amount of 9-fluorenylmethoxycarbonyl- (Fmoc) groups released during deprotection) and the conductivity profile obtained during coupling of the amino acids to the growing peptide chain. The measurements are fed back to the computer providing data for software control of the duration of the acylation, deprotection and washing steps. The method is demonstrated with pentafluorophenol esters, but is equally applicable to dihydroxybenzotriazole esters and symmetric anhydrides using the Fmoc-polyamide strategy in a continuous flow set-up with dimethylformamide (DMF) as the general solvent.

Acute phase reaction, heterogeneity, and microheterogeneity of serum proteins as nonspecific tumor markers in lung cancer.

The acute phase proteins, orosomucoid, ceruloplasmin, antitrypsin, and haptoglobin were measured in serum from 54 patients with lung cancer, 16 patients with benign lung inflammation, and 30 healthy individuals. A statistical correlation was found between tumor size and acute phase protein level, which, however, was ascribed to nonspecific inflammation in the tissues surrounding the tumor. The patients who subsequently could not be radically treated by surgery had higher concentrations of orosomucoid and ceruloplasmin than the radically treated patients. No difference in acute phase protein concentration was found between benign and malignant disease. The glycan-dependent microheterogeneity of orosomucoid and ceruloplasmin was analyzed by crossed affinoimmunoelectrophoresis with lectins, and the patterns of the patients with benign inflammation and malignant disease were different. The heterogeneity of ceruloplasmin was also analyzed by crossed immunoelectrophoresis without lectin. This analysis, combined with the total serum concentration of ceruloplasmin, made it possible to discriminate the 54 cases of malignancy from the 46 cases of nonmalignancy with a sensitivity of 78% and a specificity of 93%. It is suggested that the simple electrophoretic analyses of (micro-)heterogeneity is a valuable supplement to the acute phase profile in isolating high-risk patients and in monitoring radically treated cancer patients for relapse.

Divinylsulphone-activated agarose. Formation of stable and non-leaking affinity matrices by immobilization of immunoglobulins and other proteins.

Divinylsulphone-activated agarose is an attractive alternative to several of the activated supports usually used. Unlike CNBr-activated gels, it does not leak the immobilized protein at high pH. It reacts readily with proteins at near-neutral pH (unlike the epoxy-activated supports). Generally, divinylsulphone-activated agarose reacts with amino, hydroxyl, and sulphydryl groups, thus allowing immobilization of a wide spectrum of ligands. Moreover, it is available in an aqueous suspension free of organic solvents and neither requires time-consuming swelling nor washing.

Complexity of lectin-mediated reactions in bacteria-induced histamine release.

We have earlier suggested that bacteria-induced histamine release is caused by different mechanisms, including allergic and non-immunological mechanisms, and that the latter probably depends on lectin-mediated reactions. Two possibilities of lectin-mediated reactions were examined in this study, bacterial surface lectins bind to sugars on the basophil cell membrane leading to histamine release, and the reverse reaction where bacterial aminosugars react with lectins on the basophil cell surface. In the bacterial histamine release caused by the Staph. aureus strain Wood 46 it was possible to demonstrate a reverse reaction, but not a bacterial lectin-mediated reaction. The reaction seems to be complex, as lower concentrations of sugars might potentiate the release of histamine by binding to the target cell or bacteria, while the release is inhibited by higher concentrations.

Lectin-mediated reactions in histamine release caused by bacteria.

The bacteria-induced release of histamine was studied in human basophil leukocytes and in isolated rat mast cells. Whole bacteria of Staph. aureus caused release in a 98% pure population of peritoneal mast cells from germ-free rats, indicating a non-immunological mechanism and a direct interaction between the bacteria and the target cells. Probably the bacterial cell wall interacts with the cell membrane, since a preparation of the bacterial cell wall caused a dose-dependent release of histamine from basophil leukocytes similar to that induced by whole bacteria, and repeated washing of whole bacteria did not change the release. Inhibition studies by lectin-binding sugars indicate that aminosugars on the bacterial surface of Staph. aureus interact with lectins on the basophil cell membrane leading to histamine release.