Sequential isolation of plasma proteins aids immunoglobulin yields

The demand for immunoglobulin is high and is predicted to rise. John Curling reports that current goals are to improve yield of immunoglobulin while maintaining product quality.

Immunoglobulin (IgG) is in high demand and projections only point to an increase in the need. Polyclonal IgG, or IVIG (Intravenous Immune Globulin), is used to treat inherited and acquired antibody deficiencies.

Infection with the human immunodeficiency virus (HIV) causes deficiencies in both cellular and humoral (circulating antibodies) immune systems. Administration of IVIG temporarily restores protective levels of antibody and reduces infection related morbidity and mortality. There is thus a very large patient population and dose levels are both high and frequent. In primary immunodeficiencies dose levels range from 0.2­0.8 grams/kg/month placing demands on both availability, or output from the plasma fractionation industry, as well as product purity and convenient posology. Most modern products are formulated as 10 or 12 per cent solutions.

During the period between 1984, at the onset of the AIDS pandemic, and 2000 the worldwide demand for IVIG increased by a factor of 6.4 from 7.4 tons to 47.4 tons making IgG the second largest therapeutic protein manufactured.

Regrettably for the disadvantaged populations of less developed regions the distribution of IVIG use has not changed much in 10 years and Europe and North America account for 72 per cent of the total demand although they account for only 19 per cent of the world population.

Immunoglobulins are manufactured by almost all plasma fractionators by ethanol precipitation technology developed by Cohn and his co-workers, in particular Oncley in the 1940s and later developed by the Swiss under Kistler and Nitschmann. IgG is precipitated at ca. 18 per cent ethanol at acid pH in one or other of the classical methods for the recovery of IgG. Cohn Fraction II + III, Precipitate A or Cohn-Oncley/Kistler-Nitschmann hybrid fractions such as A+I are starting points for a range ofre-precipitation steps using ethanol and PEG aswell as additional chromatography.

Development of sub-fractionation routines with the inclusion of low pH treatment, ion exchange and affinity chromatography has been driven by the need to enhance viral safety, eliminate IgG aggregates, reduce IgA, IgM and other protein impurities toallow initially i.v. administration of relatively large product volumes and, subsequently, stable liquid formulations.

Current goals are to improve yield while maintaining product quality through incremental process amelioration. From a processing and licensing viewpoint inNorth America, Europe, Japan and Australia,the trunk fractionation scheme should not be changed.

Yield improvements can be achieved by reducing the number of steps through to the final product, by increasing individual step yields and by an integrated processing approach, eliminatingainter-step' adjustments.

In countries with less developed fractionation infrastructures it is easier to implement new technologies, such as entirely chromatographic processes not using organic solvents or cold processing, and leap-frog into the future.

At the same time new technologies to ensure safety from prions, assumed to be the causative agent of variant Creutzfeldt-Jakob disease, can be integrated in the same way that heat and chemical inactivation and elimination procedures were introduced in the mid 1980's to reduce the riskof transmission of HIV by blood and plasma products.

There are many interesting technological developments in blood plasma fractionation and one ambitious programme has been initiated by ProMetic BioSciences, in partnership with the American Red Cross (www.redcross.org) who currently collect a large volume of plasma and are distributors of IVIG and other plasma products.

The outcome of this research will be new and fully integrated and intensified processes for individual plasma proteins, IgG andalpha1-antitrypsin being the initial targets.

The technology is based on the sequential isolation of the most important plasma proteins by adsorption using specifically developed ligands immobilised on solid supports as illustrated in Fig. 1 where the colour coded boxes illustrate different unit operations.

Chromatographic processes are partitioning systems between mobile and stationary phases and lend themselves to viral reduction when the target protein is bound to the adsorbent. The processes are a further development of existing technologies and will also include viral inactivation and proprietary prion reduction steps.

The starting material for IVIG will continue to be human blood plasma because of the very nature of the product and the requirement for a wide spectrum of antibodies to very many pathogens and because recombinant alternatives are not available. Although there are research companies targeting expression of polyclonal antibodies, for the foreseeable future the only source of immunoglobulin will be human blood plasma.

In addition there is at least 80 times more IgG manufactured per month than there is the largest volume monoclonal per year.

This indicates an expression technology gap that will not be easy to close except for the smaller volume hyperimmune globulins. IgG concentration in source plasma is in the range of 7­8 grams/litre whereas the highest reported expression levels in mammalian cell culture are approaching 2 grams/litre antibody and this together with the polyclonal nature of IgG will continue to be a limitation long into the future.

Enter 42 or at www.scientistlive.com/elab

John Curling is with ProMetic BioSciences Inc, Montreal Quebec, Canada. www.prometic.com

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