Many solutions have been published to overcome the problem of leached Protein A (Gagnon P 1996 Fahrner R L 2001). It was calculated that for the downstream processing of a 10 m 3 cell culture the cost for the Protein A affinity chromatography is about 4-5 million USD (Farid SS 2009). A Protein A resin is approximately 30 times more expensive than an ion exchange resin. The other disadvantage is the high cost of this type of resin, particularly at the industrial scale necessary to purify therapeutic antibodies. One drawback is the undesired leaching of Protein A or fragments of Protein A which are known to be toxic (Gagnon P 1996). However, besides its advantages, two disadvantages should also be mentioned. This capture offers exceptional selectivity for Fc-bearing molecules, thereby removing more than 99.5% of contaminants in a single step.
![capto mmc pi cation interaction capto mmc pi cation interaction](https://static.fishersci.com/images/F296696~wn.jpg)
One of the most frequent capture steps used for mab purification is affinity chromatography with Protein A. Such a universal process, with more or less product-specific adaptions, can be applied to many mabs, especially for those immunoglobulins of the same class or subclass, e.g. Since mabs are a well-defined class of glycoproteins possessing common physicochemical properties, the use of a generic platform process is reasonable (Kelly B 2009). In recent years platform approaches have been successfully established in the field of mab purification. The classical procedure for purifying cell-culture derived polypeptides follows the sequence of capture-intermediate-polishing chromatographies, accompanied by filtration, concentration or dialysis steps at various positions of the downstream sequence. Only very low residual amounts will be tolerated. The presence of these impurities is a potential health risk for patients, and hence their absence from the final product is a regulatory requirement.
![capto mmc pi cation interaction capto mmc pi cation interaction](https://www.pharmamanufacturing.com/assets/Media/0511/pm0511_chromatography_gehealthcare-lg.jpg)
The mabs in crude fractions are typically associated with impurities such as host cell proteins (HCP), host cell DNA, viruses, aggregates, other undesired product variants, and various leachates from process materials.
![capto mmc pi cation interaction capto mmc pi cation interaction](http://patentimages.storage.googleapis.com/US20140193876A1/US20140193876A1-20140710-C00001.png)
Usually, the downstream process accounts for a major part of the total manufacturing costs of therapeutic antibodies. The entire downstream process has to: (i) manage an increased mass of product, (ii) efficiently remove increased process- and product-related impurities to below defined acceptance criteria, and (iii) maintain economic yields and sufficient quality of the mab. The increasing concentrations in the culture fluids of product and contaminants set higher demands on the capture chromatography, on its preceding sample clarification steps and on the subsequent polishing chromatographies. In the recent past the need for large scale purification processes for monoclonal antibodies (mabs), due to their exceptionally high therapeutic dosages in medical use, has been further intensified with the use of improved cell culture methods resulting in higher cell densities and higher expression rates. The selection of efficient and economic downstream sequences for purification of polypeptides produced by recombinant DNA technology is a crucial step in the development of every new biopharmaceutical intended for therapeutic use.