Biotech Processes.

]
David M. Fetterolf, Coordinator

Column Chromatography
Biotech Processes discusses fundamental information about biotechnology manufacturing useful to
practitioners in validation and compliance. Reader comments, questions, and suggestions are needed to
make this column a useful resource for daily work applications. Suggestions for future discussion topics
are most welcome. The key objective for this column: Useful information.
Contact column coordinator David Fetterolf at dfetterolf@biotechlogic.com or journal coordinating editor
Susan Haigney at shaigney@advanstar.com with comments, questions, or suggestions for future
discussion topics.

KEY POINTS
The following are key points discussed in this article:
Column chromatography is the most common biotech purification process used to separate the target molecule(s) from
product and processrelated impurities
Example types of column chromatography used include anion/cation exchange, reversed phase, and gel filtration
Chromatography columns are constructed of glass, acrylic, or stainless steel
C hromatography columns must not react with manufacturing process materials or reagents and must be cleanable
Column packing is the process by which the chromatography bead resin is transferred into the column and prepared for
use in the separation process
Column chromatography processes include sanitization, equilibration, loading, washing, elution, regeneration and
cleaning, and storage
The validation package for column chromatography steps includes critical parameter determination, sanitization and
storage studies, resin lifetime assessment, and process and productrelated impurity removal evaluation
The ultimate goal of column chromatography processes is to purify the target molecule from product and process-related
impurities, while optimizing product recovery and yield, and to do so in the fewest number of steps.

INTRODUCTION
Previous articles in the Biotech Processes column (1, 2, 3) discuss the means by which a product is expressed and
recovered in biotechnology processes. We now turn to the primary means by which a crude product stream is purified
to the level appropriate for human use. Column chromatography is the most common biotech purification process
used to separate the target molecule(s) from product and processrelated impurities. For the purposes of this article,
column chromatography is, simply put, any type that uses a cylindrical tube to hold chromatography resin (or media)
in place. The following are the most common types of column chromatography used in biotech manufacturing:
Affinity
Anion/cation exchange
Gel filtration (size exclusion) Hydrophobic interaction
Reversed phase.

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Biotech Processes.
For more Author ABOUT THE AUTHOR

information, [ David M. Fetterolf is the column coordinator of Biotech

Processes. David is a consultant with go to BiotechLogic, Inc. of Glenview, IL and other locations. David may be

reached at dfetterolf@biotech-

gxpandjvt.com/bios logic.com.
This article is intended to provide a general overview of pilot- and manufacturing-scale chromatography. Entire books
could be (and have been) written based on each individual section of this article;
Figure 1: Flow in a packed chromatography column.

however, this article provides the reader with insight into the typical steps used during column chromatography,
along with their purpose. Because much of the equipment and general operations are similar between the various
types of column chromatography mentioned previously, the sections that follow are high-level overviews and
generally apply to all types of column chromatography. Specific chromatography modes will be discussed in more
depth in future articles in this series. Helpful references are provided at the end of this article and are recommended
for further reading on the various sub-steps discussed.

CHROMATOGRAPHY EQUIPMENT AND MEDIA

Chromatography columns are typically constructed of glass, acrylic, or stainless steel. These materials are used
because they will not react with the most common solutions used in chromatography and are cleanable materials used
throughout biotech processes. The materials of construction are usually selected based on the pressures expected. It
is nice to see the chromatography resin and the packed bed while performing chromatography (i.e., with a glass or
acrylic column), but when higher pressures are used, moving to a stainless steel column is sometimes the only
choice. Because columns are cylindrical, they are characterized by their diameter and height expressed in
millimeters or centimeters. With those two dimensions, the column volume (CV) can be calculated, which is used to
determine amounts of buffers and solutions needed. Column diameters most often employed in large-scale biotech
manufacturing are between 20cm and one meter, although sizes outside that range are not uncommon. Typical
volumes range from one liter to several hundred liters.
Solutions and product are most often applied to the resin-containing column using a bioprocessing/ chromatography skid.
While chromatography skids appear to be complex, they are really just a set of pumps, gauges, valves, etc., all connected by

44 Journal of Validation technology [Spring 2009] ivthome.com

tubing, that facilitate the quick and controlled addition of various solutions and product to the column. While skids are the
norm today, chromatographers in the past (and to this day at some manufacturing plants) simply used a peristaltic pump to
deliver solutions and product onto chromatography columns. Skids are sized to deliver and withstand a defined range of flow
rates and pressures.

Column Packing
The first step in column chromatography is column packing, which is the process by which the chromatography resin is
transferred into the column and packed for use. Chromatography resin (also referred to as media) is the material that
interacts with the product and/or impurities and aids in the purification. Resin typically consists of uniformly sized beads that,
when packed into a column, still allow liquid to flow through and around them, as demonstrated in Figure 1. The space in
between the resin beads is commonly referred to as the void volume.
The basic column packing principle is the same for all types of chromatography. Resin is packed in a manner that allows for
as many beads as possible in the column, while not over-compressing them. When too much pressure is put on the beads, they
can compress, because they are made out of nonrigid materials. Columns are packed at conditions that exceed the conditions
used during processing. For example, if the maximum flow rate and pressure used during processing is 2 L/min and 3 bar
pressure, then the column is packed at a flow rate greater than 2 L/min and a pressure greater than 3 bar. The method used
depends on how the column is packed (i.e., flow vs. pressure) and the maximum allowable pressure on the resin stated by the
manufacturer. In either case, the idea is to ensure the column bed is compressed enough during packing so that it wont further
compress during the run.
Column packing is an art form in itself. Essentially every different chromatography resin has its own optimum packing
conditions. In almost every case, the instruction manual for each resin includes conditions to be used for packing. The
manufacturer will indicate best solutions to use, maximum operating flow rates and pressures, and often will recommend
criteria by which to judge how well the column was packed. In general, the resin is mixed by gentle agitation and poured into
the assembled column. The resin is allowed to settle, or packing solution is applied via flow or pressure that forces the resin to
the bottom of the column. The top of the column (i.e., adaptor) is then lowered to the top of the resin bed. It is important to
ensure air is not introduced onto the column during packing or at any time during processing.
Testing packed columns. Height equivalent to theoretical plate (HETP) and asymmetry testing is widely used to test the
integrity of the resin bed and is a direct measure of how well the column is packed. After the column is packed, a solution is
applied to the column to remove the packing solution. This solution (Solution X) is the equilibration solution for the
HETP/asymmetry test. Next, a small amount of testing solution (usually, about 1 3% of the column volume) is applied to the
column. Various test solutions can be used; however, one with a high/low conductivity or high/low ultra-violet (UV)-
absorbance compared to Solution X is typical. For example, if Solution X is 10 mM NaCl, the test solution could be 2 M
NaCl, which has a much higher conductivity. Other solutions commonly used are 2M NaCl/water (conductivity difference)
and water/acetone or water/caffeine (both have a UV difference). Solution X is used to push the small volume of test solution
through the column. Upon exiting the column, the test solution creates a conductivity or UV peak, and the traits of that peak
are measured and compared to manufacturer-recommended values or historical values from routine production. Once a
column is packed, the column is often used multiple times (i.e., cycles) as long as integrity is maintained. HETP/asymmetry
testing is typically performed after a defined number of runs, or more frequently if deemed necessary. If the results do not
meet the acceptance criteria, the resin should be removed from the column and repacked until passing results are obtained.

COLUMN CHROMATOGRAPHY PROCESSES

Column chromatography processes are comprised of a defined series of steps. These include sanitization, equilibration,
loading, washing, elution, regeneration and cleaning, and storage. Each of these steps is briefly described as follows.
David M. Fetterolf, Coordinator.

Sanitization
To ensure the chromatography system (i.e., the bioprocessing skid, column containing resin, and tubing and piping) is
free of bioburden and endotoxin prior to processing the product stream, a sanitization step is performed. A sanitizing
agent is applied to the system to inactivate any bioburden and remove any endotoxin that may be present. The most
widely used sanitization agent used in column chromatography is sodium hydroxide (concentrations vary between
100 mM and 1 M). The manufacturers instruction manual for the resin should be consulted to ensure compatibility
with the sanitization agent selected. If the sanitizing agent has not been chosen, the sanitization agent and operating

gxpandjvt.com Journal of Validation technology [Spring 2009] 45

Biotech Processes.
conditions selected should be based on what the manufacturer recommends. Oftentimes the manufacturer will suggest
a total contact time or static hold in the sanitization agent. After the defined volume and/or time, the sanitization
agent is flushed from the system with water. Many manufacturers then take samples to confirm the absence of
bioburden and endotoxin.
The sanitization regimen selected should eventually be validated for the reduction of a wide variety of organisms,
especially those typically isolated within the respective manufacturing facility.

Equilibration
Chromatography uses the physicochemical properties of the product and impurities to effect separation. The purpose
of the equilibration step is to bring the conditions of the chromatography column to those required for product
application (i.e., load). In most cases, the relevant conditions specified are pH, conductivity, and temperature. The
column is flushed with the equilibration solution until the specified ranges for these parameters are met and the
outputs are stable. Although chromatography skids widely used throughout the industry today have probes and online
monitoring of each of these parameters, samples are typically taken and tested offline to confirm the online values.

Load
Once the column is properly equilibrated, the product is applied to the column. In bind/elute chromatography, the
product is applied to the column and the target molecule binds (adsorbs) to the chromatography resin. The portion of
the product stream that does not bind to the resin is called the flowthrough. Depending on the equilibration
conditions and conditions of the product stream itself, the flowthrough could contain product or process-related
impurities, or just buffer. If the intent is to maximize product binding, finding any product in this flowthrough stream
indicates an issue with the conditions.
One of the many parameters that most significantly affects the ability of the product to bind to the column is the
flow rate. In general, the higher the flow, the less material binds because the contact time with the resin, also called
residence time, is shorter. Dynamic binding capacity studies are usually performed during process development to
determine the flow rate that maximizes product binding and become part of the overall chromatography validation
package.
Because the chromatography resin can only hold so much of the target material (based on the number of beads
packed in the column and the number of binding sites on each bead), studies are also performed to determine resin
capacity. Then, during production, calculations are performed so that this maximum product load amount is not
exceeded. For example, a batch record will state that the resin can be loaded up to 10 grams of product per liter of
resin. A calculation is then performed using the column volume and the concentration of the product in the load to
determine the maximum volume of product that can be loaded.

Wash
After the product stream is loaded onto the column, a wash step is performed to remove the loosely bound impurities
and prepare for elution. This step is sometimes called wash unbound, or just simply wash, as the intent is to
remove the unbound material. The solution used for this step is typically the same solution used for the equilibration
step, so as to maintain the conditions under which the product was initially bound to the resin while washing away
impurities. As in the equilibration step, the outlet is monitored and the step is not complete until the outputs (usually
pH and conductivity) are stable. However, because product-related impurities are being washed from the column,
UV is also monitored and ensured to be at baseline prior to continuing on with elution.

Elution
The elution step refers to the process by which the bound material is removed from the chromatography resin. In
bind/elute chromatography, the product is eluted from the resin by changing one or more of the physicochemical
properties (e.g., pH, conductivity, temperature) of the column. For example, in ion-exchange chromatography, the
most widely used method of removing the product is by increasing the salt concentration in the column. If the
equilibration/wash solution is 50 mM Tris buffer, the elution solution may be 50 mM Tris, 200 mM NaCl. Once the
elution solution is applied to the column, the product is released from the resin, enters the void volume, and flows
through the outlet of the column.
One of the properties of peptides, proteins, oligonucleotides, etc. is that they are UV-absorbing materials. Therefore, in the
majority of chromatography processes, a UV detector is used to determine when the protein is eluting from the column. The
UV trace is monitored, and product collection is initiated when it starts increasing. When the UV trace returns to baseline on
the backside of the peak, collection is stopped.
The elution step is studied during process development to determine the optimum conditions for product elution. The overall
goal is product purification, so various avenues are explored that could potentially lead to increased purity and/or yield of the
target molecule. Some examples are single vs. multi-step elution, gradient vs. step elution, and multiple product collection steps

46 Journal of Validation technology [Spring 2009] ivthome.com

(i.e., fractionation) during product elution. Some chromatography steps are designed solely to remove process-related
impurities, such as DNA, endotoxin, or host-cell protein (HCP). Others are designed to remove product-related impurities,
such as aggregated, mis- folded, denatured, etc. forms of the target molecule. However, many are designed to do both.
Depending on how closely these impurities elute from the target molecule, the more important separation efficiency becomes.
An example chromatogram is shown in Figure 2. Fractionation would most likely be used in this scenario to separate HCP
(Fraction A) and the product-related degradant (Fraction C) from the main product peak (Fraction B).

Regeneration And Cleaning

Because chromatography resins are a major contributor to overall cost of goods, they are typically reused (i.e., cycled) within a
batch or during subsequent batches, if possible. Therefore, like any reusable component or piece of equipment, it must be
properly cleaned before it is stored and used again. Once the product is eluted from the column, a step is performed to remove
remaining material (i.e., anything that didnt elute from the column during elution). This is oftentimes performed by using a
combination of extreme elution conditions and/or vendor recommendations. Using the ion-exchange example, if the elution
solution was 50 mM Tris, 200 mM NaCl,

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 David M. Fetterolf, Coordinator

Biotech Processes.
Volume or
Time

a regeneration
solution could
be 50 mM
Tris, 2 M
NaCl. The
salt
concentration
Sample elution chromatogram demonstrating fractionation. is increased to
remove as
much material
as possible.
Then, the
vendor may
recommend,
for example,
0.5 M NaOH
to clean the
column. In
Figure 2: the ion-
exchange
example, the
high pH
would remove
any remaining
impurities, as
well as
effectively
sanitize the
column prior
to storage. If
the

Degradant

Fraction A Fraction B Fraction C

48 Journal of Validation technology [Spring 2009] ivthome.com

 chromatography column is to be reused within a batch, the sanitization through regeneration and cleaning steps are repeated,
as necessary.
Due to the harsh nature of the cleaning agents typically used, chromatography column efficiency decreases over time. So, in
addition to validating the cleaning of the resin, the number of reuses must also be validated. Cycling studies can be performed
to determine the lifetime of the chromatography resin, which is always specific to the respective manufacturing process.

Storage
Between batches, chromatography resin is usually stored as a packed column. It can also be stored, unpacked, in a separate
storage container between manufacturing campaigns. Regardless of how it is stored, the storage solution is applied while the
column is still packed. The storage solution is flushed through the column to remove the cleaning agent used in the previous
step. This solution is most often recommended by the vendor, but in all cases, it should be a bacteriostatic agentone that will
not allow bioburden to grow and proliferate (e.g., low concentration of NaOH, 20% ethanol). Since the column is cleaned and
sanitized in the previous step, this storage solution is used to make sure it stays that way during storage.
HCP
VALIDATION OF COLUMN CHROMATOGRAPHY PROCESSESS
Several pieces make up the validation package for column chromatography steps. For all sub-steps described in the
UV sections,
previous Absorb ance
critical operating parameters are determined via a risk assessment, followed by studies designed to
assess the relationship between the normal operating range and the proven acceptable range (4). These critical
operating parameters become part of the process validation protocol. Additionally, studies are performed to validate
the sanitization and storage of the chromatography resins. These studies are done at laboratory scale, as they involve
spiking used resin with bioburden to evaluate the effectiveness of the sanitization and storage procedures. Resin
lifetime studies (also called column cycling studies) are performed to ensure step performance is not significantly
affected by repeated uses. The results of these studies, if performed at laboratory scale, are confirmed using a
prospective protocol at commercial scale. Furthermore, spiking studies can be used to assess the ability of the step to
remove process and product-related impurities, depending on the intended purpose of the column chromatography
step. Removal of these process and product-related impurities can also be included as part of the process validation
protocol.
Figure 3: Typical column chromatography flow.
Column Packing

Sanitization

Equilibration

Load

Inter
- Batch
Intra-Batch Wash Re-Use
Cycling

Elution

Regeneration/
Cleaning

Storage

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Biotech Processes.
CONCLUSIONS
Column chromatography is a powerful separation tool used in biotech purification processes. There are many types,
but in general, all types utilize the same general flow of sub-unit operations (see Figure 3).
The ultimate goal of column chromatography is to purify the target molecule(s) from product and process-related
impurities, while optimizing product recovery and yield. Manufacturers commonly use a combination of different
types of chromatography steps in their purification processes. Similar to what was mentioned in the Recovery
Process article in this series, the goal of chromatography is to utilize the fewest number of steps to achieve the
highest level of purity, while attaining adequate yield. Because each product and process is different, the various
sub-steps are modified, as appropriate, to achieve that goal.

REFERENCES
1. Epp, Kurtis A., Biotech Processes: Fermentation, Journal of Validation Technology, Volume 14, #4, Summer, 2008.
2. Houp, Rachel C., Biotech Processes: Cell Culture, Journal of Validation Technology, Volume 14, #5, Autumn, 2008.
3. Epp, Kurtis A., Biotech Processes: Recovery Processes, Journal of Validation Technology, Volume 15, #1, Winter 2009.
4. Parenteral Drug Association, PDA Technical Report no. 42: Process Validation of Protein Manufacturing, Bethesda, MD: PDA; 2005.

RECOMMENDED RESOURCES
Miller, J., Chromatography: Concepts and Contrasts, (2nd ed.), New Jersey: John Wiley & Sons, 2005. Jonsson, J. A., Chromatographic
Theory and Basic Principles, New York: Marcel Dekker, Inc., 1987.
Sofer, Gail K., Handbook of Process Chromatography: A Guide to Optimization, Scale-up and Validation, London, UK: Academic Press,
1997.
Hagel, L., Jagschies, G. & Sofer, G., Handbook of Process Chromatography: Development, Manufacturing, Validation and Economics, (2nd
ed.), London, UK: Academic Press, 2008.
Robards, K., Jackson, P. & Haddad, P., Principles and Practice of Modern Chromatographic Methods, London, UK:
Academic Press, 1994.
LC Support (n.d.), Chromatography Glossary [online], Available: http://www.lcsupport.com/glossary.htm [accessed
12 March 2008]. JVT

ARTICLE ACRONYM LISTING

CV Column Volume
HCP Host-Cell Protein
HETP Height Equivalent To Theoretical Plate
UV Ultra-Violet

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