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Although fungi can occur in uncontrolled pharmaceutical processing environments, the levels would need to be considerably high for fungi to be regarded as a major source of glucan in pharmaceutical products.

Glucans as a Marker for Fungal Exposure

The detection of fungal glucan is, as indicated above, of great importance for clinical diagnosis [11]. Of the different sources, cellulosic depth filters remain the primary source due to their ubiquity in pharmaceutical manufacturing [12]. Filters are devices usually a membrane designed to physically block certain objects or substances while letting others through, depending on their size. Several types of biological products are clarified using disposable depth filters. Depth filters are the variety of filters that use a porous filtration medium to retain particles throughout the medium rather than just on the surface of the medium.

These filters are commonly used when the fluid to be filtered contains a high load of particles. This is because they can retain a large mass of particles before becoming blocked, relative to other types of filters. Such filters are commonly manufactured from a mixture of diatomaceous earth containing metal ions such as aluminum, iron, copper and other metals and cellulose, to provide the filtration media this provides two dynamic mechanisms for the removal of contaminants, electrokinetic charge and size exclusion.

Cellulose is used to construct the initial filter matrix to which filter aids and other additives are impregnated into, as well as to improve contaminant removal and loading capabilities of the filter sheets. Pearson et al estimate that the molecular weight of filter extracted cellulosic glucan is around 24, Daltons [14].

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Some filter manufacturers attempted to neutralize the glucan using processes such as contacting the acid-soluble portion of the glucan with an alkali solution under conditions sufficient to dissolve the alkalisoluble glucan. In other cases, pharmaceutical manufacturers will have undertaken rinsing studies using water-for-injections to demonstrably remove glucan prior to the pharmaceutical product being passed through the filter. However, it stands that in many cases an examination of the filter has not been undertaken and thus during processing glucans can be leached from certain filter types and into the filtrate.

Where a change to process conditions occurs, the problem is often only detected through routine testing performed using the LAL assay on the finished product. For large volume parenteral products the LAL test is normally a mandatory test for finished product release.

Toxicology of 1 - 3-Beta-Glucans: Glucans as a Marker for Fungal Exposure - CRC Press Book

The presence of glucans also has a tendency to cause test interference that is when the test indicates the presence of endotoxin when none are in fact present. Therefore, interference from glucans could produce an out-of- Specification result [15]. Glucans, alongside some other substances, are commonly termed lAl Reactive Materials or alternatively lAl activators [16].

Beta Glucans - Immune Boosting and Heart Healthy Fiber - Dr. Weston

Glucans share a heat-resistant property with endotoxins, and both endotoxins and glucans will pass through sterilizing-grade filters. This is because glucans are generally regarded as non-pyrogenic [17]. The reason for glucan interfering with the lAl test relates to the biochemical basis of the test itself. The reagent used in the lAl test is generally produced from the blood of a horseshoe crab although some recombinant varieties of lysate are now available.

When the endotoxin of Gram-negative bacteria connects with the horseshoe crabs amebocytes, a series of enzymatic reactions begin. The pathway alters amebocyte coagulogen into a fibrinogen-like clottable protein, which forms a coagulin gel. The defense mechanism is also effective against fungi; hence a similar reaction occurs in response to a fungal infection, which triggers the clotting cascade [18].

This is illustrated in Figure 1. Some manufactures of lysate used in the lAl assay produce variants which will not detect glucan that is, they are endotoxin-specific. This difference in lysates is considered later. Studies have shown that the degree of lAl test interference increases with the amount of glucan present one study showed that by increasing the amount of curdlan the degree of lAl test enhancement increased. However, this itself is unreliable and whilst the lAl assay in the unmodified form will detect endotoxin, more specific tests or variants of the lAl test are required to quantify glucan levels.

There are three approaches to testing for glucan. The first approach involves attempting to destroy any endotoxin in a sample using base hydrolysis. This is through the addition of sodium hydroxide to a sample and holding the sample at 37oC for between twelve and fifteen hours.

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After adjusting the pH through the addition of hydrochloric acid, the lAl assay is run. The basis for the method is that by mixing the final product with sodium hydroxide, any endotoxin in the sample will be destroyed. The second approach is the subtraction method. This uses two assays for endotoxin, one of which has a blocking substance added to the lysate to reduce the response of the assay to glucan in the sample; and the other with an unmodified type of lysate. The difference between the two results is proportional to the amount of glucan present [21].

This is a quantitative, specific, and much more accurate approach than the subtraction method. The monoclonal antibodies are produced by a fusion with myeloma cells and mouse spleen cells immunized with lpS-Factor C complex. From this, monoclonal antibodies that react with the complex but not with lpS or Factor C are selected.

When an appropriate amount of the monoclonal antibody is added to lysate, the lysate will respond to glucan but not to lpS [23].

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