Sample Preparation

The purpose of sample preparation is to modify the chemical and physical state of the crude product into a state compatible with the initial capture step. From a chemical standpoint, this could involve changing the ionic strength, pH, or type of ion in the product solution. In the simplest cases, this may involve dilution or the addition of salt as a solid or concentrated solution. In a current GMP (cGMP) manufacturing setting, such methods are not particularly desirable. Addition of volume means increased costs. Solids that may be added will generally not be sterile, thus requiring an additional sterile filtration of the modified sample.

A more typical process at manufacturing scale would be a buffer exchange by size-exclusion chromatography (SEC) or by ultrafiltration or diafiltration. Because of its time-and volume-intensive nature, the related technique— dialysis—is typically used only on a laboratory scale.

From a physical standpoint, there are two typical processes carried out. One is concentration by ultrafiltration. The second is removal of particulate matter, by microfiltration or centrifugation. A very dirty feedstock (e.g., a batch fermentation) would clog a filter rapidly, making cen-trifugation the choice. However, for a relatively clean feedstock, the simplicity of filtration makes it the economic choice.

On a small scale, precipitative techniques such as ammonium sulfate, capyrilic acid, and polyethylene glycol (PEG) are also in common use. These techniques actually have at least modest purification power and generally have a concentrating effect as well (12-15). These techniques are less common at large scale, especially sterile operations, due to engineering considerations. Also, recovery from these operations can be low (15-17).

In addition to the above very common sample preparations, there are a number of other possibilities. It is sometimes desirable to remove certain contaminants early in the process, even though their concentration is low. Their removal is needed because their presence could affect subsequent purifications step(s). Some examples of these contaminants are DNA and RNA, endotoxins, and cell culture media dyes such as phenol red, which binds tightly to certain chromatography resins and could diminish their capacity. Bound dyes may also change the selec tivity of a chromatographic resin (18,19). Many of these are removed when antibody is prepared by the precipitative methods noted earlier. Other techniques are possible, but these are generally used as secondary purification steps and will be described as such later. Another example of a troublesome contaminant is lipid, which can clog columns, affecting their performance and lifetime. Bulk lipids, especially as found in small volumes of ascites, may be partially removed by high-speed centrifugation. As before, the precipitative methods for antibody preparation will generally leave lipids in solution, thus effecting their removal. In addition, there are a number of extractive and chro-matographic techniques that can remove lipids (16,20-21).

Brew Your Own Beer

Brew Your Own Beer

Discover How To Become Your Own Brew Master, With Brew Your Own Beer. It takes more than a recipe to make a great beer. Just using the right ingredients doesn't mean your beer will taste like it was meant to. Most of the time it’s the way a beer is made and served that makes it either an exceptional beer or one that gets dumped into the nearest flower pot.

Get My Free Ebook

Post a comment