Endotocxin Definition And Removal

Endotoxin Introduction

Lipopolysaccharides (LPS), also known as lipoglycans and endotoxins, Endotoxins are part of the outer membrane of the cell wall of Gram-negative bacteria. Although the term "endotoxin" is occasionally used to refer to any cell-associated bacterial toxin, in bacteriology it is properly reserved to refer to the lipopolysaccharide complex associated with the outer membrane of Gram-negative pathogens such as Escherichia coli, Salmonella, Shigella, Pseudomonas, Neisseria, Haemophilus influenzae, Bordetella pertussis and Vibrio cholerae.

The relationship of endotoxin (lipopolysaccharide) to the bacterial cell surface

The biological activity of endotoxin is associated with the lipopolysaccharide (LPS). Toxicity is associated with the lipid component (Lipid A) and immunogenicity is associated with the polysaccharide components. The cell wall antigens (O antigens) of Gram-negative bacteria are components of LPS. LPS elicits a variety of inflammatory responses in an animal and it activates complement by the alternative (properdin) pathway, so it may be a part of the pathology of Gram-negative bacterial infections.

From: http://textbookofbacteriology.net/endotoxin.html

Structure of Endotoxins

Endotoxins are mostly found in the outer membrane of Gram-negative bacteria. They are the integral part of the outer cell membrane and are responsible for the organization and stability of the bacteria. The general structure of all endotoxins is a polar heteropolysaccharide chain, with three distinct domains: the O-antigen region, a core oligosaccharide part and a Lipid A part.

Lipid A is the most conserved part which is responsible for the toxicity of endotoxins, while, the effect of polysaccharides is negligible. The Lipid A structures were first studied based on Enterobacteria. The common architecture of Lipid A is a disaccharide, with glucosamine being the monomer. The two glucosamine monomers are linked between position 1 and 6, and both of them are phosphorylated to produce bisphosphorylated β-(1-6)- linked glucosamine disaccharide. Furthermore, there are fatty acids ester-linked at positions 3 and 3 and amide linked at positions 2 and 2. The position 6 is attached to the oligosaccharide region.

From: Chromatographic Removal of Endotoxins: A Bioprocess Engineer’s Perspective

Endotoxin Removal

BIC offers one-stop endotoxin removal service including gene synthesis, protein expression and purification, and endotoxin detection and removal. More details from: http://www.biologicscorp.com/blog/bacterial-endotoxin-definition/

High Cell Density Fermentation Definite

High Cell Density Fermentation VS Stand Large Scale Fermentation

High cell density fermentation by fed-batch strategies is one of the most cost-effective means of achieving high yields for the production of large scale recombinant proteins in the bio-industry. In fed-batch cultures, cell mass and productivity are maximized by adjusting culture conditions, including temperature and pH, the composition of the feed media, and the substrate feed rate. As one of the most critical factors to the success of high cell density culture, various nutrient feeding strategies have been developed.

High cell density fermentation VS Large scale fermentation（Picture from: Biologicscorp）

Feeding methods in fed-batch culture

Without feedback control

• Constant feeding – feeding nutrient at a predetermined (constant) rate. As a result, the specific growth ratecontinuously decreases.
• Increased feeding – feeding nutrient at an increasing (gradual, stepwise, or linear) rate. The decrease in specific growth rate can be compensated within a range.
• Exponential feeding – feeding nutrient at an exponential rate to achieve constant specific growth rate.

Notes:

Specific growth rate is maintained at a constant level by exponential feeding in most cases, but may deviate when unexpected conditions arise during culture. When it occurs, feedback control is required in the process to achieve constant specific growth rate.

With feedback control

Indirect feedback control

• DO-stat – feeding nutrient when there is a rise in the concentration of dissolved oxygen (DO), which results from depletion of the substrate.
• pH-stat – feeding nutrient when the pH rises after depletion of the principal carbon source.
• Carbon dioxide evolution rate (CER) – the most frequently method to maintain the specific growth rate. The CER is roughly proportional to the rate of consumption of the carbon source using a mass spectrometer.
• Cell concentration – the nutrient feeding rate determined from the cell concentration is measured by a laser turbidimeter.

Direct feedback control

• Substrate concentration control – nutrient feeding is directly controlled by the concentration of the principal carbon source (e.g. an on-line glucose analyzer in the fermentor).

From: Lee SY, High cell-density culture of Escherichia coli [J], Trends in Biotechnology, 1996 March: 14(3):98-105.