Mark R. Etzel, Ph.D.
Membrane Chromatography R&D

FoodTechSource: What areas does your research focus on?

Mark Etzel, Ph.D.: My work is in protein purification and bioseparation—basically the separation of biological materials—through the use of membrane chromatography. We’re not performing membrane filtration, which is what most membranes are used for. We focus mainly on ion exchange and affinity membranes. It’s a new topic area.

FTS: What is membrane chromatography?

Etzel: Membrane chromatography is when you have microfilters that have absorption properties. Versus sieving and filtration, where there’s no absorption.

FTS: Which industries do you expect it to benefit?

Etzel: The biopharmaceutical industry; eventually the dairy industry, the nutraceutical industry and somewhat the biochemical industry.

FTS: Would you take a moment to explain the basis for your work? The potential applications?

Etzel: Of course. Right now we’re mainly focusing on the process of protein purification, for the biopharmaceutical industry—which is where we expect membrane chromatography to have its initial impact. You see, many biotechnology medicines that are being developed for commercial use are proteins. For example, things like monoclonal antibodies and aminoglobulins, and viral vaccines—even some non-protein products in the gene therapy area like DNA, RNA, and plasmids—those are the targets of chromatography.
         Typically these materials are produced in some kind of cell culture, and the solution from the cell culture contains hundreds of other proteins as well as cells and media components, and many of these drugs are injected. So they have to be purified to a very high degree—many, many 9’s in purity—so that the injectable drug is just that one molecule.
         Right now this purification is accomplished through process chromatography, as well as through membrane filtration. Typically they might take a cell culture broth, and they would remove the cells by tangential flow filtration or by depth filters. At that point they have a clear fluid that contains their protein. Then they’ll usually use a series of ion exchange chromatography columns—at process scale these columns may have 100 liters of chromatography resin, and it’s typically either an affinity resin, which means it uses biological recognition to bind to the target protein, or it’s an ion exchange resin. They use this to capture the target protein on a column, after which they wash away the contaminants. Then they might use another column to capture residual contaminants, in which case the target protein does not bind to the column. And they’ll use a series of these steps. It’s very time consuming and very costly. And they need a large number of these ion chromatography beads to be packed into very large columns.
         So, we’re working on using membranes for the chromatography part of it. Instead of using ion exchange beads packed into a column, we would have a microporous membrane with ion exchange groups on the internal surface area of the microporous membrane. They would capture the target molecule by absorption.

“With membrane chromatography it’s easier to scale up. It’s hard to make chromatography columns bigger and bigger and still get the same performance.”

FTS: What are the advantages of using membranes for chromatography?

Etzel: It offers an advantage over column chromatography because the membrane can allow higher flow rates, and you can use less membrane volume than you need column volume. For example, if you use a 0.1 micron membrane you can do cell separation and clarification and product capture all in one step. So it increases yield and productivity, and decreases capital cost.
         Plus, with membranes it’s easier to scale up for large-scale manufacturing. It’s hard to make chromatography columns bigger and bigger and still get the same characteristics and performance.

FTS: Typically, what size are they now?

Etzel: Oh, they might have a 100-liter column. And we might be able to do that with a 1-liter membrane.

FTS: What materials are these membranes made of? And how do they function?

Etzel: They’re generally polymeric membranes. There are different types: cellulose acetate, polyvinylidene difluoride. Typically they are flat sheets or hollow fibers. EM Science makes a nice hollow fiber ion exchange membrane that has 0.1 micron pores and it’s meant for both clarification of cellular debris and capture of the target protein. It allows tangential flow configurations. Millipore Corporation and Sartorius are the two big suppliers of the absorptive membrane. But they only offer a dead-end flow configuration product at the moment.

FTS: The hollow fiber is like tubing?

Etzel: It’s like a straw. Let’s say that you took a straw and it had microporous walls. As you pumped the fluid down the center, the cellular debris would not be able to pass through the wall so it would just emerge at the other end of the straw. The liquid itself would pass through the wall and it would contain the dissolved target molecules. And as the target molecule flowed through the wall of the straw it would be captured by the wall because of the ion exchange groups on the inside.

FTS: They absorb the molecules?

Etzel: Yes. Most membranes in use today lack absorption features—they’re used exclusively for filtration. With membrane chromatography you need absorption. Then you wash the membrane with a buffer and either use a high salt or a change in pH to allude the protein.

FTS: What properties of the membranes are you researching?

“We’re trying to understand the mass transfer characteristics and the binding kinetics of absorptive membranes, to make membrane chromatography more efficient and affordable.”

Etzel: What we’re doing is trying to understand the mass transfer characteristics and the binding kinetics of the membranes. Because once you eliminate pressure drop limitations, the actual kinetics of the binding of the molecules to the membrane become rate limiting—especially for affinity applications. Then, for ion exchange applications, once you eliminate the flow restrictions you can have mass transfer limitations, especially if the pore size of the microfilters are too big—say if they’re greater than 5 microns—then mass transfer limitations become rate limiting.

FTS: Why is that?

Etzel: What happens is the pores become so large that the protein shoots through the membrane faster than it has time to bind to the membrane. It needs to diffuse to the wall of the membrane and bind before it passes through.

FTS: Why haven’t researchers tried to do this before?

Etzel: Because the membranes are a new and emerging technology. And so it’s just now that the technology has been developed.

FTS: What is the history?

Etzel: What happened with chromatography is that people started using shorter, wider columns in order to use smaller beads that have more rapid capture without severe pressure drop. By making the column really short you eliminate the pressure drop; then, to get enough volume of resin you have to make the diameter bigger. Well, the extreme of a very short, wide column is a membrane where you would have a 100-micrometer bed height and a five-meter diameter. And, of course, you can’t do that with a chromatography column but you can do that with a membrane. You eliminate the pressure drop problem and yet have very, very small pores. That was the original thought.

FTS: Have you already seen a lot of interest from the biotechnology people?

Etzel: Sure. They face a big problem. As these gene therapy and vaccine products are accepted by the public and go worldwide, the amount of vaccine and gene therapy products that need to be manufactured are huge. And the chromatography systems that are out there now have low capacities. It gets to be a cost problem. And when you’re selling worldwide, your manufacturing costs can’t be sky high. So, they are looking for lower-cost manufacturing technologies.

FTS: What other industries would be able to make use of your research?

Etzel: Right now, the cost is too high for industries like wastewater treatment and food. Whenever you come out with a new technology it tends to go into the very high value-added products first. The dairy industry will be a much bigger application, but it will be much later. Right now dairy is just getting into the fractionation of protein for infant formula using chromatography methods that were developed for biopharmaceuticals 25 years ago.

FTS: How does that work? And why purify the proteins?

“Dairy is just now getting into the fractionation of protein for infant formula using chromatography methods developed for biopharmaceuticals 25 years ago.”

Etzel: Well, cheese whey, which is used to make infant formula in some cases, is made up of five or six major proteins. And one thing we’re interested in is making infant formula more like mother’s milk in protein composition. For example, mother’s milk contains 20 times more lactoferrin than cow’s milk, and lactoferrin, which is an iron-binding protein, has been shown to prevent ear infections in children—one of the most common reasons for doctor visits during infancy. By purifying lactoferrin from dairy products and adding it at higher levels to infant formula to more mimic mother’s milk we hope to increase the health of babies.
         Another example is that betalacticglobulin, the major protein in cheese whey, is essentially absent in mother’s milk. So, a lot of babies might develop an allergy to cow’s milk-based formula because of the betalacticglobulin. If we can purify that out from cheese whey, we can make the protein formula more like mother’s milk.

FTS: In the food area, what other applications are membranes being used for?

Etzel: They’re using microfiltration for clarification of fruit juices; and membranes are starting to be used for things that used to be done by other technologies, such as centrifugation. So what I think you’ll see first is the use of ultrafiltration for making whey protein concentrate and milk protein concentrate. Then you’ll see microfiltration and nanofiltration membranes making a big impact. For example microfiltration can be used to purify casein from milk or to remove fat from milk and whey. And the nanofiltration can be used to concentrate and demineralize lactose and other sugars. It’s an exciting new frontier for membrane filtration in the food industry.

FTS: Do you expect membrane chromatography to play a role in the nutraceuticals industry?

Etzel: Sooner or later, of course. A lot of these dairy proteins aren’t just a source of amino acids, they also have a physiological impact. For example, alfalactobulin is very high in tryptophan, which is converted into serotonin in the brain. And low serotonin levels in the brain can lead to obesity, migraine headaches and depression. Some people believe that by eating a diet high in tryptophan, say from alfalactobulin, that you might have an impact on some of these diseases. And some people believe that derivatives of lactoferrin have anticancer properties, which is another potential nutraceutical.

FTS: Is the technology financially viable for nutraceuticals?

Etzel: I think right now nutraceuticals is a $9 billion-a-year market, and it’s growing at 20% per year. So it’s a pretty profitable business. And the product is all produced by using column chromatography right now. So, as with all the other areas I mentioned, I believe here, too, membrane chromatography can be a major player in the near future.

For a more detailed description of the processes of membrane chromatography, Dr. Etzel suggests interested readers check out the Satorius web site at www.sartorius.com, focusing on their “Sartobind membrane absorbers.”