Biomedical subjects

D Petsch

Publications and source records attributed to D Petsch.

6 recordsLinked to original sources

PubMed2000

Endotoxin removal from protein solutions.

Endotoxins liberated by gram-negative bacteria are frequent contaminations of protein solutions derived from bioprocesses. Because of their high toxicity in vivo and in vitro, their removal is essential for a safe parenteral administration. A general method for the removal of endotoxins from protein solutions is not available. Methods used for decontamination of water, such as ultrafiltration, have little effect on endotoxin levels in protein solutions. Various techniques described in the patent literature are not broadly applicable, as they are tailored to meet specific product requirements. Besides ion-exchangers and two-phase extraction, affinity techniques are applied with varying success. Also, taylor-made endotoxin-selective adsorber matrices for the prevention of endotoxin contamination and endotoxin removal are discussed for this purpose. After giving an overview of the properties of endotoxins and the significance of endotoxin contamination, this review intends to provide an overall picture of the various methods employed for their removal. Avenues are pointed out how to optimise a method with regard to the specific properties of endotoxins in aqueous solution.

Adsorption↗

PubMed1998

Proteinase K digestion of proteins improves detection of bacterial endotoxins by the Limulus amebocyte lysate assay: application for endotoxin removal from cationic proteins.

Cationic proteins, such as lysozyme, ribonuclease A, and human IgG, impaired the detection of endotoxins with the Limulus amebocyte lysate assay (LAL assay) through formation of endotoxin-protein complexes, demonstrating pronounced masking of endotoxins. Methods, such as phenol extraction, dilution heating, and perchloric acid treatment failed to demask the endotoxins. Also, digestion with trypsin, chymotrypsin, or pronase recovered only 10 to 20% of the applied endotoxins. However, endotoxin recoveries up to 100% were obtained with proteinase K digestion of the samples prior to the LAL assay. This method was then applied to examine the impact of endotoxin masking on endotoxin removal from protein solutions by selective adsorption on membrane adsorbers. It was found that poly-L-lysine and poly(ethyleneimine) as endotoxin-selective ligands were able to pull endotoxins off the proteins studied, thereby guaranteeing successful decontamination.

Adsorption↗

PubMed1998

Endotoxin removal with poly(ethyleneimine)-immobilized adsorbers: Sepharose 4B versus flat sheet and hollow fibre membranes.

Poly(ethyleneimine) was immobilized on poly(vinyl alcohol)-coated nylon flat sheet membranes, poly(vinyl alcohol) and poly(ethylenevinyl alcohol) hollow fibre membranes as well as Sepharose 4B. The resulting poly(ethyleneimine)-immobilized adsorbers were used for removal of E. coli derived endotoxin from buffers and bovine serum albumin solutions. The efficiency of poly(ethyleneimine) proved to be constant over a wide pH range, including phosphate buffered saline. The performance depended upon the matrix type employed: endotoxin clearance factors varied from 100 to 120,000 in protein-free solutions and 40 to 33,000 in solutions of bovine serum albumin using 6000 EU/ml as feed concentration. The best adsorber was the flat sheet membrane-immobilized poly(ethyleneimine), followed by the hollow fibre-immobilized poly(ethyleneimine) and poly(ethyleneimine)-Sepharose. The factors influencing endotoxin clearance were the mass transport (convective systems were superior to the diffusive system), the chemical composition and the surface structure of the underlying matrix.

Absorption↗

PubMed1998

Selective adsorption of endotoxin inside a polycationic network of flat-sheet microfiltration membranes.

For the removal of remaining amounts of endotoxin, sorbents with high selectivity for endotoxin are required. Typically, particulate sorbents with positively charged ligands, such as histidine, polymyxin B poly-L-lysine and poly(ethyleneimine) (PEI), display moderate to high removal efficiencies in an environment of low ionic strength. It was found that polycationic ligands are most suitable to meet an endotoxin concentration which is below the threshold level required for parenteralia. Furthermore, protein recoveries close to 100% are obtained if the decontamination is performed at a pH close to the pI of acidic proteins. The high selectivity is probably caused by complexation of the polycationic ligand with the polyanionic endotoxin, leading to interactions with KD < 10(-9) M using PEI and assuming M(r) = 10 kDa for monomeric endotoxin; with BSA the same ligand reveals only KD = 4 x 10(-6) M. Using polymer-coated microfiltration membranes, immobilization of positively charged ligands leads to membrane adsorbers which are generally superior to chromatographic adsorbers and allow faster processing. Since immobilization takes place at polymer chains, low-molecular-weight ligands mainly add positive charges to the hydrophilic polymer. Consequently, membrane adsorbers with low-molecular-weight ligands, even DEAE, demonstrate similar selectivity to PEI or poly-L-lysine.

Adsorption↗

PubMed1997

Membrane adsorbers for selective removal of bacterial endotoxin.

Surface-modified flat-sheet microfiltration membranes were functionalised with poly-L-lysine, polymyxin B, poly(ethyleneimine), L-histidine, histamine, alpha-amylase and DEAE as well as deoxycholate. Their suitability to remove endotoxin from both buffers and protein solutions was examined using bovine serum albumin, murine IgG1 and lysozyme as model proteins. In protein-free solutions reduction from 6000 EU/ml to <0.1 EU/ml was achieved with all applied ligands; only alpha-amylase as well as L-histidine and histamine, when immobilized via the non-ionic spacer bisoxirane, exhibited low clearance factors at neutral pH. The adsorption of endotoxin is mainly ruled by electrostatic interaction forces. Thus in multi-component systems, such as endotoxin-contaminated protein solutions, competing interactions take place: acidic proteins compete with endotoxin for binding sites at the membrane adsorbers, basic proteins compete with the ligands for endotoxin and act as endotoxin carriers. With properly chosen conditions the membrane adsorbers presented here show exceptional effectiveness also in the presence of proteins. They are generally superior to functionalised Sepharose chromatographic sorbents and allow fast processing. They may contribute to reduce the risks in the application of parenterals and diagnostics.

Adsorption↗

PubMed1996

Purification of murine IgG1 on group specific affinity sorbents.

Murine IgG1 a CD4-antibody, was produced from the cell line MAX 16 H 5. DEAE-Sepharose CL-6B and group specific sorbents based on different ligands, such as protein A-Sepharose, AvidChrom, Thiophilic Resin (T-Resin) Fractogel EMD TA 650S, Sepharose 4B-immobilized metal chelate and histidine sorbents, were compared in terms of their suitability for the large-scale purification of this IgG. Evaluation criteria were selectivity for IgG1 protein capacity and recovery, stability under cleaning-in-place (CIP) conditions and the rate of IgG1 adsorption. Both thiophilic gels performed best under these conditions, with Fractogel EMD TA 650S demonstrating slightly higher contamination with other proteins compared to the T-Resin based on 6% beaded Agarose. Higher purities were obtained with these gels compared to conventional purification techniques employing a combination of ion exchange and hydrophobic interaction chromatography. Favorable adsorption kinetics were found on DEAE-Sepharose with the effective diffusivity in the pores of the sorbent being five-times accelerated compared to the diffusion coefficient of IgG1 in solution. Multilayer adsorption of the IgG1 was experienced from adsorption isotherms on DEAE-Sepharose and Fractogel EMD TA 650S. This was linked to an early breakthrough of this protein during frontal chromatography. Co-purification of pyrogens between two-(Thiophilic Resin) and five-times (DEAE-Sepharose) of the original level was significant with all sorbents except protein A-Sepharose and AvidChrom. Thus an additional chromatographic step is required in case of pyrogen contamination of the cell culture homogenate.

Animals↗