Enhanced aggregate removal from bulk biologicals using ion...

Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Blood proteins or globulins – e.g. – proteoglycans – platelet...

Reexamination Certificate

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C530S390100

Reexamination Certificate

active

06177548

ABSTRACT:

FIELD OF THE INVENTION
The invention relates to removal of proteinaceous aggregates from bulk biologicals, especially pharmaceutical biologicals, using ion exchange chromatography.
BACKGROUND OF THE INVENTION
In the manufacturing of biopharmaceuticals using cell culture processes, one problem has been that proteins in the cell culture can form aggregates. The aggregates are usually formed by more than one molecule, or contain partially or completely denatured molecules. Aggregates are not usually removed in filtering or other preliminary purification processes because of their similarity to the biopharmaceutical product.
Aggregates can be a problem in biopharmaceutical manufacture. The aggregate level of the starting bulk must be kept below 0.1% in order to formulate high strength biopharmaceutical products, i.e., greater than 100 mg/ml, with an acceptably low aggregate concentration. Therefore, elimination of aggregates from the bulk product in an efficient manner is needed for successful production of high strength biopharmaceuticals.
A conventional method to remove aggregates from biological solution is by gel filtration, which is based on the molecular size difference between aggregates and product molecules. However, this method is difficult and costly for large scale production and when one wishes to concurrently remove other impurities, including endotoxins and nucleic acids, for production of high strength pharmaceuticals.
SUMMARY OF THE INVENTION
The invention includes methods and processes for removing aggregates from partially purified bulk product, in particular, from partially purified monoclonal antibody, using anion exchange chromatography. The invention is based on the principle that, because aggregates are larger and have a different conformation than biological product molecules, aggregates carry more charges near to the cell culture product's isoelectric point than do the products themselves.
When the working pH is close to the product's isoelectric point, the charge on the product molecules is low. Aggregates generally carry more charge(s) than the product at this pH range, because of the components and the conformation of the aggregates. Thus, when the pH is adjusted to near the isoelectric point of the products and the sample is loaded on an ion exchange column, more of the charged aggregates than product will be bound on the column, resulting in a greater degree of aggregate removal than using other chromatographic conditions.
Preferably, the pH of the loading sample would be adjusted to 0.2 logs lower than the pI of the product to achieve aggregate removal. Also, passing the partially purified product through the anion exchange matrix accomplishes removal of other impurities, including host cell DNA and endotoxins.
Making and Using the Invention
The methods and processes of the invention can be used in purification of any type of biopharmaceutical in a cell culture process, where one wishes to remove aggregates and other impurities, including nucleic acids and endotoxins, in a simple and efficient manner. It can be used in either small scale or large scale production, but it leads to greater increases in efficiency and greater cost savings in large scale production. These methods and processes are effective in purification in both mammalian and bacterial cell culture processes. Because mammalian cell culture is often larger scale than bacterial cell culture, however, it would be expected to be used more often in mammalian cell culture processes.
Antibody production frequently involves production of large quantities of product, and therefore, production on a large scale. It is, therefore, particularly well-suited for purification through the methods and processes of the invention. Examples of using the methods of the invention to remove aggregates from a production bulk for a humanized antibody (Hu-901) follows.


REFERENCES:
patent: 5110913 (1992-05-01), Coan et al.

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