An Introduction to ChromatographyPatrick McKay

Senior Research Associate, Department of Recovery Sciences, Genentech, Inc.

A Personal Perspective

When people ask me about science, I try to explain to them that science is both fun and interesting. That was what drew me to choose a career in a scientific field many years ago. You don't want to be stuck in a job that is dull and boring for the rest of your life. As a scientist employed in biotechnology, I've never had the feeling of being bored, or that my scientific applications were dull. On the contrary, science to me is exciting and a tremendous challenge. My job involves the purification of recombinant DNA-derived proteins to a level of purity which will allow them to be used as therapeutic biopharmaceuticals. The whole design of the purification process which is needed to produce the protein is part of the challenge -- what step goes where, how do the steps interface, etc. Troubleshooting these processes is similar to playing a detective like Columbo -- you know that something is amiss, but what are the causes? Finally, being able to scale-up this purification process to enable our Manufacturing group to carry out these processes under GMP (Good Manufacturing Practices) conditions is a must if the FDA (Food and Drug Administration) is to approve the product. There's nothing quite like the satisfaction that a process chromatographer gets when his or her process is put into place, and the recombinant protein is ultimately used to treat patients, some of whom have life-threatening illnesses. Yes, science is fun and exciting. And, being a process scientist in the biotechnology industry provides a researcher with challenges above and beyond his or her wildest imaginations from what they learn in school. The following paper describes some of the techniques that I use in my daily job, and may give you an idea on how to integrate the field of chromatography into your biology classes.

What is Chromatography?

Chromatography is the science which is studies the separation of molecules based on differences in their structure and/or composition. In general, chromatography involves moving a preparation of the materials to be separated - the "test preparation" - over a stationary support. The molecules in the test preparation will have different interactions with the stationary support leading to separation of similar molecules. Test molecules which display tighter interactions with the support will tend to move more slowly through the support than those molecules with weaker interactions. In this way, different types of molecules can be separated from each other as they move over the support material.

Chromatographic separations can be carried out using a variety of supports, including immobilized silica on glass plates (thin layer chromatography), volatile gases (gas

chromatography), paper (paper chromatography), and liquids which may incorporate hydrophilic, insoluble molecules (liquid chromatography).

Chromatography and Biotechnology

This discussion of chromatography will focus on the separation of proteins into relatively homogeneous groups because proteins are often the target molecules which must be purified for use as "biopharmaceuticals" or medicines. It is important to remember, however, that chromatography can also be applied to the separation of other important molecules including nucleic acids, carbohydrates, fats, vitamins, and more.

One of the important goals of biotechnology is the production of the therapeutic molecules known as "biopharmaceuticals," or medicines. There are a number of steps that researchers go through to reach this goal:

identification of a "target protein" which may have therapeutic value identification of the "target gene" -- the gene responsible for encoding the target

protein isolation of the target gene insertion of the target gene into a host cell (such as E. coli) which will both grow

well, and continue to produce the protein product encoded for by the target gene separation of the target protein from the many other host cell proteins large scale production of the target protein under controlled manufacturing

conditions large scale testing for efficacy as a medicine marketing of a new medicine Many different disciplines, including microbiology, molecular biology, chemistry,

and others, are required to complete the steps listed above to bring a protein from the "scientifically interesting" state to that of a full-fledged drug to be used in treating a specific disease. This discussion will focus on the work and tools of the chromatographer.

Chromatographers use many different types of chromatographic techniques in biotechnology as they bring a molecule from the initial identification stage to the stage of a becoming a marketed product. The most commonly used of these techniques is liquid chromatography, which is used to separate the target molecule from undesired contaminants (usually host-related), as well as to analyze the final product for the requisite purity established with governmental regulatory groups (such as the FDA).

Some examples of liquid chromatographic techniques are described below:

Ion-Exchange Chromatography

Proteins are made up of twenty common amino acids. Some of these amino acids possess side groups ("R" groups) which are either positively or negatively charged. A comparison of the overall number of positive and negative charges will give a clue as to the nature of

the protein. If the protein has more positive charges than negative charges, it is said to be a basic protein. If the negative charges are greater than the positive charges, the protein is acidic. When the protein contains a predominance of ionic charges, it can be bound to a support that carries the opposite charge. A basic protein, which is positively charged, will bind to a support which is negatively charged. An acidic protein, which is negatively charged, will bind to a positive support. The use of ion-exchange chromatography, then, allows molecules to be separated based upon their charge. Families of molecules (acidics, basics and neutrals) can be easily separated by this technique. This is perhaps the most frequently used chromatographic technique used for protein purification.

Hydrophobic Interaction Chromatography ("HIC")

Not all of the common amino acids found in proteins are charged molecules. There are some amino acids that contain hydrocarbon side-chains which are not charged and therefore cannot be purified by the same principles involved in ion-exchange chromatography. These hydrophobic ("water-hating") amino acids are usually buried away in the inside of the protein as it folds into it's biologically active conformation. However, there is usually some distribution of these hydrophobic residues on the surface of the molecule. Since most of the hydrophobic groups are not on the surface, the use of HIC allows a much greater selectivity than is observed for ion-exchange chromatography. These hydrophobic amino acids can bind on a support which contains immobilized hydrophobic groups. It should be noted that these HIC supports work by a "clustering" effect; no covalent or ionic bonds are formed or shared when these molecules associate.

Gel-Filtration Chromatography

This technique separates proteins based on size and shape. The support for gel-filtration chromatography are beads which contain holes, called "pores," of given sizes. Larger molecules, which can't penetrate the pores, move around the beads and migrate through the spaces which separate the beads faster than the smaller molecules, which may penetrate the pores. This is the only chromatographic technique which does not involve binding of the protein to a support.

Affinity Chromatography

This is the most powerful technique available to the chromatographer. It is the only technique which can potentially allow a one-step purification of the target molecule. In order to work, a specific ligand (a molecule which recognizes the target protein) must be immobilized on a support in such a way that allows it to bind to the target molecule. A classic example of this would be the use of an immobilized protein to capture it's receptor (the reverse would also work). This technique has the potential to be used for the purification of any protein, provided that a specific ligand is available. Ligand availability and the cost of the specialized media are usually prohibitive at large-scale.

Other Methods

While the methods above are typically chosen for use in a purification process, there are in fact many others that can be used. Each of these methods or techniques takes advantage of a specific part of the protein being purified. The commonality is that all of the techniques employed are based on the protein's structure.

Chromatography : Then and Now

Chromatographic supports

The biggest changes in chromatography have been associated with the types of chromatographic supports used in protein purification. Many years ago, cellulose-based chromatography media were exclusively used for purification of proteins. These media were eventually replaced with carbohydrate-based supports, which offered better flow properties - "flow" is the term used to describe the progress of the test solution as it passes over the support. Carbohydrate based supports could also be more easily cleaned to allow for repeated use. The current generation of chromatographic supports incorporates synthetic, polymeric beads which have even higher flow rate capabilities, as well as new approaches in design of the support particle, or "bead."

Chromatographic columns

With the advent of superior chromatography media came the need for better "columns" - the containers which physically hold the support in place - to pack these media into. The columns used in biotechnology have been designed to permit very high flow rates at higher than atmospheric pressure (> 15 psi). Specialty columns, incorporating stainless steel, high density glass or acrylic components have taken the place of the standard plastic or glass columns that were previously used. New column designs have allowed development of new types of chromatography. For example, high pressure liquid chromatography (HPLC) was made possible by development of the steel columns and chromatographic media which are able to handle pressures in excess of hundreds to thousands of psi.

Process Development : Scaling it all up

In order to produce and purify large quantities of proteins for use as biopharmaceuticals, specialized equipment is necessary, as well as trained personnel to run this equipment. In addition, a special manufacturing facility, operating under GMP (good manufacturing practices) regulations, is required to bring the protein to market. The process of increasing the size of the initial experimental reactions to a level which makes large scale production, testing and marketing possible, is called "scaling-up."

A process development scientist will work closely with members of the research and manufacturing departments in order to develop a method (known as "a process") which will allow an economic and feasible scale-up of the purification of the target molecule.

Many techniques developed in a research environment simply cannot be used at a larger scale. The process development scientist will recognize these steps and will modify them to facilitate their incorporation at large scale. A second consideration is the scale-up itself. Biochemical operators/technicians who run the equipment in the manufacturing plant may not understand the critical aspects of the chromatography steps. By working alongside the operators, the process development scientist can answer questions as they come up, and avert any potential problems with the chromatography.

Bringing Chromatography to the Classroom

There are a lot of good ways to bring chromatography into the classroom. The best methods are those which encourage hands-on participation by the student, with minimal involvement by the teacher. Clearly, the experiments should be set up such that harmful reagents are excluded. The experiments should also be set up in a way which forces the students to think about what's going on in front of them -- what scientific principles are being tested, and what conclusions can and cannot be drawn from the results.

Two good choices for chromatography experiments are paper and liquid chromatography. In the paper chromatography experiment, for example, drops of water soluble ink are applied to a piece of toilet paper. The paper is dipped into a container of water, and the capillary action of the water migration spreads out the different colors into the constituent colors that made up the final ink color. Black ink works the best, and I've found that the markers used for transparencies ("Vis-a-vis") give great separations. Be careful on the papers that are used to spot on, however, as some paper towels don't provide the necessary resolution.

The liquid chromatography experiment included below involves the separation of two colored molecules on a small column of Sephadex G-25. These pre-packed columns ("PD-10 columns") aren't very expensive, and can be used for many cycles, if properly cared for. An outline of that experiment is listed below, with the explanation for how it works. Some of the equipment may not be readily available to the high school lab, but some improvisation can make it work successfully, time after time. If possible, please try this activity with your students so that we can discuss it during this science seminar.

Other types of chromatography can be done in the classroom. The use of thin layer chromatography ("TLC") can be great for looking at the different species in extracts of vegetables (carrots, beets, etc.), and the TLC plates can be obtained from suppliers. The one drawback might be the use of organic solvents, but if used in a fume hood, or in a contained environment, can be done successfully.

Access Excellence/Science Seminar

Science Demonstration--Chromatography

Pat McKay/ Genentech, Inc.

Introduction

The purpose of the demonstration is to allow the students to get a "hands-on" opportunity to do a scientific experiment. The focus of the experiment should be getting as many students involved as possible, and to have fun with the experiment.

In the experiment, Blue Dextran is mixed with a dilute Phenol Red solution in a buffer containing sodium chloride and sodium acetate at pH 5.0. At this pH, the phenol red turns yellow (right in front of the students' eyes!). Upon mixing with Blue Dextran, the solution turns green. Application of the solution to a Pharmacia PD-10 column separates the green back into blue and yellow.

Materials and Methods

The following solutions will be needed: Blue Dextran, 3 mg/ml, in water Phenol Red, 10 mg/ml, in water 150 mM NaCl, 50 mM NaOAc, pH 5.0 (Equil. Buffer)

Note: you can purchase Blue Dextran and Phenol Red from Sigma Chemicals. Call 1-800-325-3010 for more information. Sigma product numbers are as follows: Blue Dextran/D5751 (5g/$66.80); Phenol Red/143-74-8(5g/$7.75)

The following equipment will be needed:

Pharmacia PD-10 columns (at least 2) Note: You can purchase PD-10 columns from Pharmacia (1-800-526-3593/ product number 17-0851-01/ Box of 30/$112.00)

Powder funnels (1 per column) Ring stand with clamps P1000 and P20 Pipetmen/tips Disposable 12 x 75 mm test tubes Liquid waste container 25 ml or 50 ml plastic grad. cylinder

Experimental Procedure

1. Pre-equilibrate both PD-10 columns with at least 25 ml of equilibration buffer. This allows the second column to be used while with first column is being re-equilibrated.

2. Point out to the students beforehand that their eyes may play tricks on them. Let them think that magic is at work here. Add 1-2 ul of Phenol Red to 500 ul of equilibration buffer in a disposable 12 x 75 mm test tube. The solution turns yellow.

3. Ask the students if they know what color is made by the mixing of blue and yellow. Add 500 ul of Blue Dextran to the diluted Phenol Red solution to confirm their guess.

4. Transfer the green solution to the PD-10 column. Point out that the colors are separating from each other, but that only blue is visible at first.

5. Connect the powder funnel to the column. Add about 25 ml of equilibration buffer to the funnel and watch for the color separation. The students will notice complete separation of the blue from the yellow, with a white zone in between.

6. During chromatography, the students may have questions, and this is usually the best time to answer them, as well as to explain what is going on. A simple explanation of the theory is to compare the gel beads to "Wiffle Balls" and the difficulty or ease that big and small objects may have in fitting through the holes in the Wiffle Balls.