• Antifoams
• Antifoams Product Chart
• EZMix
• EZMix Product Chart
• Q & A

The time and effort expended on optimizing the growth conditions of recombinant organisms is amply rewarded with convenient fermentation and high yield of recombinant protein. After the expression system has been determined, and the preferred growth medium selected, it is critical to craft a set of conditions that results in low stress and high productivity.

It's not surprising: combine agitation with surfactant and you get ... foam.

In any suspension culture — whether your protein expression host is bacterial, yeast, insect, or mammalian — the growth medium needs to be agitated to aerate and suspend the cells. And many culture media contain proteins and other components with surfactant properties. Whenever these two conditions are met, foam is an almost inescapable complication.

Why should you care about foam? There are several good reasons:

• A head of foam seriously inhibits gas exchange at the surface, sickening the culture, especially as the cell density rises.
• If your expression system is designed for extracellular fusion protein production, the product is being sent into a hazardous environment; foam is notorious for denaturing proteins in solution.
• Finally, foam is just plain messy. It gets in the way of decanting, filtration, centrifugation — almost any fluid-handling operation you'll perform as you begin to isolate the product from your culture.

What to do?

Admittedly, this isn't rocket science. Truth be told, it's rather a low-tech answer to your high-tech problem. But it is an answer; it works; and it has the virtue of simplicity.

What's an antifoam?Antifoams have been used in foodstuffs, pharmaceuticals, and manufacturing processes for many years. They are silicone-based polymers, non-silicone organics, or mixtures of both. They work at very low concentrations to suppress foaming in aqueous media, typically in the range of 0.005 – 0.02%.

Here are a few things you should know when using antifoams in cell culture systems:

• If you intend to harvest cells using microfiltration or to concentrate extracellular protein with ultrafiltration, you should try to select a non-silicone antifoam. Silicones tend to foul the membranes used in these procedures.
• Microbes may actually adapt to use some antifoams as growth substrates. If so, the expression pattern of the bug can be affected, and expression of the desired recombinant protein may be reduced. Another concern: if the antifoam is metabolized, it can't continue to suppress foaming.
• It is possible for certain types of antifoams to inhibit the growth of the culture. You'll want to insure that the culture grows up to nearly the same final cell density with the antifoam as without it.
• Naturally, you'll want to add only sterile components to your fermentation. Choose an antifoam that can be sterilized without degradation.
• For controlling foam in large-scale fermenters, you'll want an antifoam that can be delivered by pump on an as-needed basis. Some are too viscous or heterogeneous for pumping. The addition can be automated if the pump is controlled by a foam probe in the fermentation vessel.

It's as simple as that. Sigma's antifoams (see Table 1) all have been tested with E. coli (ATCC #25922) and B. subtilis (ATCC #6051) in Terrific Broth. In all cases, the final OD600 in cultures with the antifoam is at least 80% of the value without it. We're in the process of extending testing to other expression systems and other media. If you check back here, on our Web site, you'll be among the first to know.

While we're on the subject of components for efficient growth of recombinant organisms, let's take a look at media.

For the best results, you must provide a rich, complete growth medium.This usually involves a standard-recipe broth with or without specific supplementation to meet the nutritional requirements of the expression host. Though it is one of the most fundamental techniques in microbiology, media prep can go awry.

Cleanliness is key. Contamination, wasted time and materials, and confused results are all but inevitable without it. Yet most microbial media components are dusty, sticky powders — hard to pour, hard to weigh, hard to dissolve — making it that much more challenging to meet the cleanliness commitment.

We'd like to propose a solution. At Sigma, we developed a proprietary process that converts standard microbial media components into EZMix™ free-flowing, non-dusting, dense, wettable powders. Each EZMix powder has the same growth characteristics as the untreated powder, with clear advantages in handling and dissolution.

These EZMix media and media components are the first in a growing family. Though the EZMix process is a benign one, we take nothing for granted. Each new formulation is tested before release so you can be assured that it will perform as expected. If your favored medium is not yet on this list or if you have some comments about your experiences with EZMix powders, drop us an e-mail. We want to hear from you.