Gel filtration chromatography as a method for removing bacterial endotoxin from antibody preparations

The removal of bacterial endotoxins from biological samples is critical to avoid the potentially fatal pyrogenic response possible when introduced to mammalian systems. Endotoxins have a variety of specific characteristics that can be exploited to target their isolation and subsequent removal, but one that has not been extensively characterized is their difference in size from that of monoclonal antibodies. Here, we present a study which utilizes gel filtration chromatography as a method for endotoxin removal from both aggregated and nonaggregated antibody preparations, outlining a mechanistically simple method for removal of this impurity. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1497–1501, 2014     

Membrane cartridges for endotoxin removal from interferon preparations

The suitability of membrane cartridges for the removal of endotoxin from both distilled water and interferon preparations was examined. The endotoxin concentrations were reduced to 4.0 and 7.3 EU/ml, respectively, when about 4000 ml of distilled water with 20 and 28 EU/ml were passed through the deoxycholate and chitosan immobilized membrane cartridges. When 200 ml of interferon preparation with endotoxin concentration more than 80 EU/ml and pH 3.9 were applied to a deoxycholate immobilized membrane cartridge at a flow-rate of 9 ml/min, the endotoxin concentration was reduced to less than 10 EU/ml. However, if an interferon preparation of 450 ml, with more than 80 EU/ml of endotoxin and pH 3.9 was applied to the chitosan immobilized membrane cartridge at a flow-rate of 18 ml/min, the endotoxin concentration was reduced to less than 10 EU/ml.     

Removal of tightly bound endotoxin from biological products

The method for endotoxin removal described in this paper is useful for separation of tightly bound endotoxin from biological products, particularly those produced in Escherichia coli in the form of inclusion bodies for which a denaturation step is required to solubilise the product. We employed guanidine hydrochloride and ammonium sulphate in combination with hydrophobic interaction chromatography (HIC). These conditions enable binding of the endotoxin to the matrix, giving unbound product in the column flow-through. This makes the method generally applicable to biological products. An endotoxin reduction of about 3.7 logs was achieved; from as much as 1,100,000 EU mg(-1) in the solubilised material to about 200 EU mg(-1) in the product purified by this method. The method was developed for a cervical dysplasia vaccine, a fusion protein comprising L2, E7 and E6 from Human Papilloma Virus type 16, because both conventional and commercially available methods of endotoxin removal were ineffective in removing the tightly bound endotoxin from this product.     

Effective use of gamma irradiation for pathogen inactivation of monoclonal antibody preparations.

Gamma irradiation has been used for decades as an effective method of pathogen inactivation of relatively inert materials. Until recently, its application to biologicals has resulted in unacceptable losses in functional activity. In this report we demonstrate that the damaging secondary effects of gamma irradiation can be controlled while maintaining the pathogen inactivation properties due to damage by primary effects. Control is achieved by a combination of protection from free radical damage to a monoclonal antibody through the use of the antioxidant ascorbate and by freeze-drying to minimize the potential for generating free radicals. The data demonstrate a synergy of these two approaches that results in quantitative recovery of functional activity while maintaining the ability to inactivate greater than 5 logs of porcine parvovirus infectivity.     

Removal of biologically active admixtures from immunoglobulin preparations

Inclusion of an additional treatment of the products obtained at centrifugation stages b1 and b13 with activated bentonite and aluminium hydroxide into the alcohol method for the production of immunoglobulin from placental and abortion blood permits obtaining preparations with lowered content of proteolytic enzymes and thermostable acid phosphatase, free from chorionic gonadotropin and blood pigment. The treatment of the final preparation with DEAE cellulose removes blood group antigens from immunoglobulins. The preparations obtained by this method have been shown to meet the requirements for immunoglobulins imposed by technological specifications.     

Removal of heme from preparations of human chorionic somatomammotropin

The operations involved in the extraction of chorionic somatomammotropin from human placentas cause hemolysis of blood which is present in placental tissue and contamination of crude extract with large amounts of hematic pigments. Most of the pigments are removed by the purification procedure. The portion of purified hCS with high heme content was subjected to treatments with acid acetone and neutral butanone on the basis of procedures generally used for the separation of heme and protein portions in hemoproteins. Both treatments resulted in the removal of heme from hCS; the treatment using butanone was more efficient than the one using acetone. After treatment with both organic solvents, the immunological activity measured with radial immunodiffusion was entirely retained while binding activity on rat ventral prostate particles was slightly decreased.     

Removal of restriction enzyme inhibitors from lambda phage preparations

Inhibitors of restriction endonucleases and other enzymes are found in large quantities in λ phage solutions prepared by growth on agar plates. Chromatography on Sepharose-6B column of the phage suspension will eliminate these inhibitors. The DNA extracted from this purified phage suspension is suitable for genetic manipulations in vitro.     

Removal of the sphingolipid impurity from preparations of yeast phosphatidyl inositol

A complex sphingolipid containing inositol and mannose, present in lipid extracted from toluene-autolyzed baker's yeast, was eluted from silicic acid columns immediately after phosphatidyl inositol, and was the main nitrogenous impurity in crude preparations of this phospholipid. Nitrogenfree phosphatidyl inositol was obtained by rechromatography on alumina. Modifications to the chromatographic procedure also gave diphosphatidyl glycerol containing the theoretical 4.29% P.     

Removal of endotoxin in the purification of histone H1.5 from Escherichia coli

Triton X-114 and cation-exchange chromatography, SP-Sepharose FF, removed endotoxins from solutions containing recombinant histone H1.5. Dissociated endotoxins were removed but fractions containing histone H1.5 were enhanced in the elution step. The final concentration of endotoxins, measured by a limulus amoebocyte lysate (LAL) assay, was below 0.05 EU mg^–1 histone H1.5. The recovery of protein was above 95%.     

Removal of endotoxin from water by microfiltration through a microporous polyethylene hollow-fiber membrane

The microporous polyethylene hollow-fiber membrane has a unique microfibrile structure throughout its depth and has been found to possess the functions of filtration and adsorption of endotoxin in water. The membrane has a maximum pore diameter of approximately 0.04 micron, a diameter which is within the range of microfiltration. Approximately 10 and 20% of the endotoxin in tap water and subterranean water, respectively, was smaller than 0.025 micron. Endotoxin in these water sources was efficiently removed by the microporous polyethylene hollow-fiber membrane. Escherichia coli O113 culture broth contained 26.4% of endotoxin smaller than 0.025 micron which was also removed. Endotoxin was leaked into the filtrate only when endotoxin samples were successively passed through the membrane. These results indicate that endotoxin smaller than the pore size of the membrane was adsorbed and then leaked into the filtrate because of a reduction in binding sites. Dissociation of 3H-labeled endotoxin from the membrane was performed, resulting in the removal of endotoxin associated with the membrane by alcoholic alkali at 78% efficiency.     

Autoradiolysis of iodinated monoclonal antibody preparations

We investigated the effects of ionizing radiation on the immunointegrity of antibody fragments (Fab) because large amounts of high specific activity ¹³¹I may damage the proteins. We found that 1000 Gy of external ¹³⁷Cs γ radiation was sufficient to destroy 80–90% of the immunointegrity of the initial preparation. This effect was also produced by internally added [¹³¹I]NaI in a quantity sufficient to provide the same radiation absorbed dose. Since radioiodinated monoclonal antibodies labeled to high specific activity are being evaluated for radioimmunotherapy, the above observation is significant since high levels of internal radiation occur with therapeutic doses of ¹³¹I-labeled antibody. Human serum albumin in low concentration (2%) added to the iodinated antibody solutions was successful in preventing loss of immunoreactivity and can be used to protect and stabilize therapeutic quantities of radiolabeled monoclonal antibody preparations.     

Removal of toxic preservatives in pharmaceutical preparations of insulin by the use of ultra-stable zeolite Y

Ultra-stable zeolite Y and mordenite with pore sizes of 0.75 and 0.70 nm, respectively, showed the capacity to bind phenol and m-cresol rapidly. Phenol and m-cresol are used as preservatives in pharmaceutical preparations for humans and, with the use of a 15 mg filter of ultra-stable zeolite Y, 98 % of the preservative in a 200 l pharmaceutical insulin preparation, Actrapid®, was removed after 5 s of passage through the filter. The insulin content in the filtrate was not disminished. © Rapid Science Ltd. 1998     

Removal of catalytic activity by EDTA from antibody light chain

Gp41 peptide antigen of the HIV-1 envelope (TP41-1:TPRGPDRPEGIEEEGGERDR, a highly conserved region) was enzymatically degraded by the antibody light chain 41S-2-L after an induction period. The peptide bond between Glu¹⁴ and Gly¹⁵ was cleaved early in the reaction. When EDTA was added in the induction period, it inhibited the degradation of TP41-1 thus ceasing the catalytic activity of 41S-2-L. In contrast, when EDTA was added after the induction period, only a small reduction in the catalytic activity was observed. These observations suggest that metal ions are important in stimulating catalytic activity early in the reaction.     

Removal of endotoxin from water by microfiltration through a microporous polyethylene hollow-fiber membrane.

The microporous polyethylene hollow-fiber membrane has a unique microfibrile structure throughout its depth and has been found to possess the functions of filtration and adsorption of endotoxin in water. The membrane has a maximum pore diameter of approximately 0.04 micron, a diameter which is within the range of microfiltration. Approximately 10 and 20% of the endotoxin in tap water and subterranean water, respectively, was smaller than 0.025 micron. Endotoxin in these water sources was efficiently removed by the microporous polyethylene hollow-fiber membrane. Escherichia coli O113 culture broth contained 26.4% of endotoxin smaller than 0.025 micron which was also removed. Endotoxin was leaked into the filtrate only when endotoxin samples were successively passed through the membrane. These results indicate that endotoxin smaller than the pore size of the membrane was adsorbed and then leaked into the filtrate because of a reduction in binding sites. Dissociation of 3H-labeled endotoxin from the membrane was performed, resulting in the removal of endotoxin associated with the membrane by alcoholic alkali at 78% efficiency.     

Method for reducing endotoxin in Moraxella catarrhalis UspA2 protein preparations

The UspA2 protein from the bacterium Moraxella catarrhalis is a potential vaccine candidate for preventing human diseases caused by this organism. Before a vaccine can be administered parentally, the level of endotoxin must be reduced as much as possible. However, in this case the endotoxin was very tightly complexed with the UspA2 protein and could not be dissociated with Triton X-100. It was found that it dissociated from the protein with the zwitterionic detergents Zwittergent 3-12 and Zwittergent 3-14. The endotoxin could then be separated from the protein by either ion-exchange or gel filtration chromatography. Using the limulus amoebocyte lysate assay for quantitation, the endotoxin was reduced approximately 20 000-fold. The removal of residual endotoxin from UspA2 preparations had no detrimental effect on the immunological properties of the protein. Mouse antisera raised against UspA2 prior to, and following endotoxin reduction exhibited comparable antibody and bactericidal titers against the tested strains. Further, mice immunized with both preparations, followed by pulmonary challenge with either a homologous or a heterologous isolate, exhibited comparable levels of clearance.