April 1, 2008 (Vol. 28, No. 7)
Using Inexpensive Resins Upstream Improves Purification and Downstream-Column Life
Within the pharmaceutical and biopharmaceutical industry, there are many process streams that require purification by some means or another. Typically, this requires that a specific impurity is removed or that a range of impurities are eliminated. In the latter case, these impurities are never fully characterized and are generally called color-species.
In all cases, the color is regarded as an impurity that must be removed in order to avoid any adverse quality problems downstream with the final pharmaceutical product. The removal of upstream color also serves to improve the resin lifetime of expensive chromatography media that is used in downstream purification.
Color in a process stream can be derived from a variety of different sources but there are two major sources. The first is the fermentation from which the product, or a bulk intermediate, is derived. Those colors that are derived from the carbon source used in the fermentation and are often similar to those encountered during the processing of sugar tend to be large molecules.
The second source is derived from degradation products. They are likely to be different according to the type of product being studied. The molecular weight of these degradation products vary greatly but will probably have a carboxylic functionality, which is commonly present in products such as antibiotics.
Despite the fact that a particular solution can be quite colored, the concentration of the species responsible for the color can be quite low. The most common method of measurement is by UV absorbance at a defined wavelength, although HPLC and other techniques can sometimes be used.
One of the biggest challenges in biopharmaceutical production today is the reduction of costs. Increased production titres in products such as mAbs have now created bottlenecks in downstream processing. High titre (>60 OD) bacterial cultures can be very darkly colored, and this color can bind tightly to strong quaternary amine anion exchange media. These anion exchange media can be fairly expensive, ranging from $700 to $1,500 per liter.
For larger capture columns, the installed cost of a packed column could be hundreds of thousands of dollars. Therefore, it is important to ensure that the media lifetime is sufficient to provide good process economics. One strategy for improving chromatographic column lifetime is the use of inexpensive, large particle-size anion-exchange resins for the removal of column-fouling color bodies.
Amberlite™ FP resins from Advanced Biosciences are priced in the range of tens of dollars per liter for large volumes, so they are economical choices for upstream color and contaminant removal. These resins function essentially as a guard column for the more expensive resins and reduce the need for more frequent column repacking and media replacement. Of particular interest in this type of application are the anionic ion-exchange resins.
In general, the colors that are present in typical fermentation processes are similar to those seen in the food industry. This is hardly surprising as the carbon source for the fermentation tend to be molasses or some other form of natural products such as rapeseed oil. Within the sugar industry, these colors have been studied and classified into four groups: caramels, melanoids, sugar degradation products (Maillard reactions), and phenolic-iron complexes.
As mentioned earlier, most degradation products tend to be polymeric species containing one or several carboxylic acid groups. These colors are inclined to be large organic molecules that have a weakly negative charge. The types of resins that are typically used in these cases are ion-exchange resins that have a defined pore structure (macroreticular) or gel matrix and a positive charge derived from either a quaternary amine or tertiary amine function.
The Table includes a list of ion exchange resins that are widely used for decolorization, but other similar products are available.
A filtered, crude vancomycin broth was adjusted to pH 8, and the broth was then pumped over a column of Amberlite FPA40 Cl using a Rainin Rabbit Pump. The column effluent was monitored with a Linear Model 200 detector (Grace Davison Discovery Science) and fractions were collected in one tube every five minutes using a Gilson 201 Fraction Collector. The experiment involved a column of 2.0 cm ID x 30 cm L and a flow rate of 1.63 mL/minute (1 CV/Hr). Detection was performed with a Thermo Fisher Scientific Biomate 3 Spectrophotometer at 400 nm.
The broth was pumped over the column for a total of five column volumes (470 mL) before the experiment was terminated and then reactions of the effluent were analyzed.
The average color removal was 50% (Figure 1) after the first column volume had passed through the column. Figure 2 shows a close-up of Rohm & Haas’ Amberlite™ FPA40 Cl column with the color adsorbed on the top of the column.
Jon Fisher is technical service manager, Americas, Amie Gehris is technical service scientist healthcare, process chemicals and biocides, and John Maikner is senior scientist healthcare, process chemicals and biocides, at Advanced Biosciences, a unit of Rohm & Haas. E-mail: firstname.lastname@example.org. Web: www.advancedbiosciences.com.