Allan Otto Fog Lihme

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.

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.

A novel core fractionation process of human plasma by expanded bed adsorption chromatography

Current plasma fractionation technology combines ethanol precipitation with packed bed chromatography. We have developed a novel core fractionation process comprising five expanded bed adsorption (EBA) chromatographic steps on high-density modified agarose/tungsten carbide beads. Plasma was first chromatographed on two diethyl amino-ethyl (DEAE)-tungsten carbide agarose adsorbents (respective mean particle diameters of d(v)(0.5)=190 and 37 microm) to isolate at 50 to 80% recovery a fraction containing 4 to 7 IU/ml factor II (FII), factor IX (FIX), and factor X (FX) (specific activity >1 IU/mg) and another enriched in FVIII and von Willebrand factor (vWF) (approximately 1 IU/ml and 0.6 IU/mg, respectively). The flow-through was adsorbed on 4% agarose-10% tungsten carbide beads coupled with an acidic mixed-mode ligand to isolate an 80% pure immunoglobulin G (IgG) at a 93% step recovery. A highly purified alpha1-antitrypsin was isolated at 95% step recovery by adsorbing the flow-through on 4% epoxy-crosslinked agarose-10% tungsten carbide adsorbent material coupled with a cationic ligand. Isolation of 98% pure albumin was achieved at a 99% step recovery by pH 4.5 adsorption of the flow-through on 6% agarose-10% tungsten carbide beads coupled with an acidic mixed-mode ligand. EBA may represent a feasible alternative core plasma fractionation tool.

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.

Natural Pig Plasma Immunoglobulins Have Anti-Bacterial Effects: Potential for Use as Feed Supplement for Treatment of Intestinal Infections in Pigs.

There is an increasing demand for non-antibiotics solutions to control infectious disease in intensive pig production. Here, one such alternative, namely pig antibodies purified from slaughterhouse blood was investigated in order to elucidate its potential usability to control post-weaning diarrhoea (PWD), which is one of the top indications for antibiotics usage in the pig production. A very cost-efficient and rapid one-step expanded bed adsorption (EBA) chromatography procedure was used to purify pig immunoglobulin G from slaughterhouse pig plasma (more than 100 litres), resulting in >85% pure pig IgG (ppIgG). The ppIgG thus comprised natural pig immunoglobulins and was subsequently shown to contain activity towards four pig-relevant bacterial strains (three different types of Escherichia coli and one type of Salmonella enterica) but not towards a fish pathogen (Yersinia ruckeri), and was demonstrated to inhibit the binding of the four pig relevant bacteria to a pig intestinal cell line (IPEC-J2). Finally it was demonstrated in an in vivo weaning piglet model for intestinal colonization with an E. coli F4+ challenge strain that ppIgG given in the feed significantly reduced shedding of the challenge strain, reduced the proportion of the bacterial family Enterobacteriaceae, increased the proportion of families Enterococcoceae and Streptococcaceae and generally increased ileal microbiota diversity. Conclusively, our data support the idea that natural IgG directly purified from pig plasma and given as a feed supplement can be used in modern swine production as an efficient and cost-effective means for reducing both occurrence of PWD and antibiotics usage and with a potential for the prevention and treatment of other intestinal infectious diseases even if the causative agent might not be known.

EBA columns with a distribution system based on local stirring

A new type of liquid distribution system for expanded bed columns has been developed. The construction differs from traditional distribution designs by not having any small pores (like filters or distribution plates) in the flow path of the crude feedstock. A stirrer at the bottom of the column distributes the incoming feedstock. Due to the stirring, jet streams are prevented and a stable expanded bed is formed above a mixed zone. This article describes the new column design, and investigates the performance of the stirred distribution concept experimentally by measuring theoretical plate number and breakthrough profile. Furthermore, the possibilities of scaling up the concept will be discussed based on theoretical plate measurements.

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.