Traditional polyclonal antibody preparations

Polyclonal antibody preparations have been used for several decades to induce passive immunization against infectious diseases and other harmful agents, particularly toxins. The antibody preparations are usually administered by direct i.v. injection. While this affords immediate immunological protection, its effect is transitory, usually persisting for only 2-3 weeks (i.e. until the antibodies are excreted). Passive immunization can be used prophylactically (i.e. to prevent a future medical episode) or therapeutically (i.e. to treat a medical condition that is already established). An example of the former would be prior administration of a specific anti-snake toxin antibody preparation to an individual before they travel to a world region in which these snakes are commonly found. An example of the latter would be administration of the anti-venom antibody immediately after the individual has experienced a snake bite.

Pharmaceutical biotechnology: concepts and applications Gary Walsh © 2007 John Wiley & Sons, Ltd ISBN 978 0 470 01244 4 (HB) 978 0 470 01245 1 (PB)

Antibody preparations used to induce passive immunity may be obtained from either animal or human sources. Preparations of animal origin are generally termed 'antisera', and those sourced from humans are called 'immunoglobulin preparations'. In both cases, the predominant antibody type present is IgG.

Antisera are generally produced by immunizing healthy animals (e.g. horses) with appropriate antigen. Small samples of blood are subsequently withdrawn from the animal on a regular basis and quantitatively analysed for the presence of the desired antibodies (often using ELISA-based immunoassays). This facilitates harvesting of the blood at the most appropriate time points. Large animals, such as horses, can withstand withdrawal of 1 or 2 l of blood every 10-14 days, and antibody levels are usually maintained by administration of repeat antigen booster injections.

The blood is collected using an aseptic technique into sterile containers. It can then be allowed to clot with subsequent recovery of the antibody-containing antisera by centrifugation. Alternatively, the blood may be collected in the presence of heparin, or another suitable anticoagulant, with subsequent removal of the suspended cellular elements, again by centrifugation. In this case, the resultant antibody-containing solution is termed 'plasma'.

The antibody fraction is then purified from the serum (or plasma). Traditionally, this entailed precipitation steps, usually using ethanol and/or ammonium sulfate as precipitants. The precipitated antibody preparations, however, are only partially purified and modern preparations are generally subjected to additional high-resolution chromatographic fractionation (Figure 13.1). Ion-exchange chromatography is often employed, as is protein A affinity chromatography. (IgG from many species binds fairly selectively to protein A.)

Following high resolution purification, the antibody titre is determined, usually using an appropriate bioassay, or an immunoassay. Stabilizing agents, such as NaCl (0.9 per cent w/v) or glycine (2-3 per cent w/v) are often added, as are antimicrobial preservatives. Addition of preservative is particularly important if the product is subsequently filled into multi-dose containers. Phenol, at concentrations less than 0.25 per cent, is often used. After adjustment of the potency to fall within specification, the product is sterile filtered and aseptically filled into sterile containers. These are sealed immediately if the product is to be marketed in liquid form. Such antibody solutions are often filled under an oxygen-free nitrogen atmosphere in order to prevent oxidative degradation during subsequent storage. Such a product, if stored between 2 and 8 °C, should exhibit a shelf life of up to 5 years.

Although specific antisera have proven invaluable in the treatment of a variety of medical conditions (Table 13.1), they can also induce unwanted side effects. Particularly noteworthy is their ability to induce hypersensitivity reactions; some such sensitivity reactions (e.g. 'serum sickness') are often not acute, whereas others (e.g. anaphylaxis) can be life threatening. Because of such risks, antibody preparations derived from human donors (i.e. immunoglobulins) are usually preferred as passive immunizing agents.

Immunoglobulins are purified from the serum (or plasma) of human donors by methods similar to those used to purify animal-derived antibodies. In most instances, the immunoglobulin preparations are enriched in antibodies capable of binding to a specific antigen (usually an infectious microorganism/virus). These may be purified from donated blood of individuals who have recently:

• been immunized against the antigen of interest;

• recovered from an infection caused by the antigen of interest.

Figure 13.1 Overview of the production of antisera for therapeutic use to induce passive immunization. Refer to text for specific details

Although hypersensitivity reactions can occur upon administration of immunoglobulin preparations, the incidence of such events is far less frequent than is the case upon administration of antibody preparations of animal origin. As with all blood-derived products, the serum from which the immunoglobulins are due to be purified is first assayed for the presence of infectious agents before its use.

The major polyclonal antibody preparations used therapeutically are listed in Table 13.1. These may generally be categorized into one of several groups upon the basis of their target specificities. These groups include antibodies raised against:

• specific microbial or viral pathogens;

• microbial toxins;

• snake/spider venoms (anti-venins).

Table 13.1 Polyclonal antibody preparations of human or animal origin used to induce passive immunity against specific biological agents

Antibody

Source

Specificity

Anti-D immunoglobulin

Human

Specificity against rhesus D antigen.

Botulism antitoxin

Horse

Specificity against toxins of type A, B or E Clostridium

botulinum

Cytomegalovirus immunoglobulin

Human

Antibodies exhibiting specificity for cytomegalovirus

Diphtheria antitoxin

Horse

Antibodies raised against diphtheria toxoid

Diphtheria immunoglobulin

Human

Antibodies exhibiting specificity for diphtheria toxoid

Endotoxin antibodies

Horse

Antibodies raised against gram negative bacterial LPS

Gas gangrene antitoxins

Horse

Antibodies raised against a-toxin of Clostridum novyi,

Clostridum perfringens and Clostridum septicum

H. influenzae immunoglobulins

Human

Antibodies raised against surface capsular

polysaccharide of H. influenzae

Hepatitis A immunoglobulin

Human

Specificity against hepatitis A surface antigen

Hepatitis B immunoglobulin

Human

Specificity against hepatitis B surface antigen

Leptospira antisera

Animal

Antibodies raised against Leptospira

icterohaemorrhagiae (used to treat Weil's disease)

Measles immunoglobulin

Human

Specificity against measles virus

Normal immunoglobulin

Human

Specificities against variety of infectious and other

biological agents prevalent in general population

Rabies immunoglobulin

Human

Specificity against rabies virus

Scorpion venom antisera

Horse

Specificity against venom of one or more species of

scorpion

Snake venom antisera

Horse

Antibodies raised against venom of various poisonous

snakes

Spider antivenins

Horse

Antibodies raised against venom of various spiders

Tetanus antitoxin

Horse

Specificity against toxin of Clostridium tetani

Tetanus immunoglobulin

Human

Specificity against toxin of C. tetani

Tick-borne encephalitis immunoglobulin

Human

Antibodies against tick-borne encephalitis virus

Varicella-zoster immunoglobulin

Human

Specificity for causative agent of chicken pox

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