HOW BIOTECH MANUFACTURERS CAN IMPROVE
CHROMATOGRAPHY THROUGH CUSTOMIZATION
By Iggy Gyepi-Garbrah (Pall Europe Ltd.) & Ian Sellick (Pall Corporation)
Biotech manufacturers use chromatography for a number of steps during downstream
drug processing, from initial protein capture and separation, to final polishing. While it is the
most powerful purification tool available in the industry, it is not always used efficiently.
Manufacturers today are faced with new and changing production variables in developing new
drug therapies. Bio-molecules harvested for processing are becoming more complex, and they
come from increasingly diverse sources. At the same time, manufacturers are looking to
simplify purification steps in an effort to streamline production and improve process economics.
This column offers some recommendations to manufacturers hoping to achieve greater
process efficiencies. The best way to simplify and economize is to select a customized
purification protocol that works best for a particular system and process. For each novel and
variable production method there is a set of particular separation and purification challenges, and
manufacturers need to consider the whole spectrum of production options. Choosing
independent and knowledgeable suppliers is critical in order to get the widest variety of
platforms available.
DIVERSE OPTIONS FOR DIVERSE CELLS
New monoclonals are being produced from bacterial cells, mammalian and nonmammalian animal cells, in transgenic milk, and even from transgenic plant sources such as corn
and tobacco. If your system is designed for harvesting cells from Chinese hamster ovaries, you
need to use different processing methods to harvest cells from duckweed plants.
Just as there is a diversity of cells to process, there is a diversity of chromatography
resins, columns, and membranes available. Manufacturers need to have access to as many of
these options as possible in order to achieve the most cost-effective and efficient process.
The expertise of a chromatography manufacturer is important, from selecting the right
column, the correct resin, to efficiently packing resin in the column. New chromatography
technologies, including recent improvements in automated column packing techniques, are
helping to reduce cycle costs and improve throughput.
Disposable chromatography membrane capsules and cartridges offer another option when
used alongside column chromatography, particularly for removing residual contaminants such as
DNA and host cell proteins. Because disposability eliminates cleaning and cleaning validation,
this saves considerable time and labor. Membranes have also demonstrated effective virus
removal, and they offer high throughput processing that would otherwise require enlarging
column systems, at considerable capital expenditure.
INTEGRATING MULTIPLE CHROMATOGRAPHY STEPS
Manufacturers have long been integrating different types of chromatography into
processing. In some instances, up to six separate chromatography steps can be incorporated into
a single purification cycle.
Most monoclonal therapies are manufactured using a cell suspension in a reactor. In this
feed stream there is the potential to produce residual DNA, RNA, endotoxins, host cell proteins,
and also viruses. These contaminants need to be removed to the lowest detection levels required
by regulatory authorities for a finished product.
Figure One offers a schematic of the general steps required for processing a generic
monoclonal antibody. Chromatography is incorporated mainly into downstream processing,
which basically encompasses all steps following cell harvesting. Chromatography is used first
for clarification, capture and purification. It can also be incorporated in the final product
formulation stage.
The initial chromatography capture step may use a Protein A affinity resin. Protein A
separation binds the target antibody while significantly reducing the bulk of unwanted process
volume.
In monoclonal antibody purification, the industry trend is to use smaller columns for the
initial capture step, and to recycle Protein A resin for as many runs as possible. This is due to the
expense of Protein A resins, which can cost upwards of US$8,500 per liter; by contrast, ion
exchange resins used in later states of processing are a tenth of the cost, at upwards of $750 per
liter. A 200 liter column of Protein A resin can cost an operator approximately $1.7 million
dollars to pack. Recycling is therefore extremely important for process economics; in many
cases a Protein A column can be cycled up to 300 or even 400 times.
After the initial capture step, the monoclonal is eluted off of the Protein A column. The
solution may then go into a holding vessel prior to diafiltration (to remove salt), and then to
additional purification steps, which may be in reverse phase, or ion- exchange. The diafiltration
step can be eliminated if a hydrophobic interactive chromatography (HIC) resin is used
following initial capture. HIC resins are loaded at high salt concentrations, which are usually the
same conditions under which Protein A is eluted. This potentially saves thousands of dollars per
batch in processing costs, especially for large bulk systems.
RESINS, COLUMN PACKING AND SCALE-UP
For a truly customized solution, a chromatography column supplier should help
manufacturers select the best resin media appropriate for a given process. To maximize
flexibility, a supplier should furnish information on as many different resins as possible. This
should include analyses of flow rates, densities, slurry mixes, as well as thorough packing
instructions and results of previous packing studies. An independent column manufacturer can
usually provide the above information for a wide spectrum of resin options.
“Pack-in-Place” column packing technology is becoming more widely used as it helps
ensure consistent column packing and unpacking. By automating packing procedures, “pack-inplace” systems help eliminate the human variables involved in column packing. Recent
innovations in packing include the use of ultrasound technology to monitor the bed build as resin
is pumped into a column. Ultrasound can detect bed construction, formation of voids, bed
compression, and the position of the mobile phase in the column. Although the use of ultrasound
for chromatography has not yet been commercialized, it is currently being beta-tested at the
manufacturing scale, and it promises to provide manufacturers a novel method of controlling the
rate of bed formation, and optimized resin performance.
Another important consideration is linear scalability of the column design, which is
critical for moving a product from small to medium and large scale processing. The best column
design maintains all operational parameters at constant levels regardless of column size. It is
important for manufacturers to realize that many columns on the market have different designs as
the column size increases, and are therefore not linearly scalable. Columns and valves should be
engineered to scale together; as columns increase in size, packing valves need to scale up as well
in proportion. However, for many column manufacturers with a pack-in-place system, the
packing valve is the same size on differently sized columns. If a column is doubled in size for
processing, a packing valve needs to be scaled to accommodate a four- or even eight-fold
increase in flow rates. A valve set at a fixed flow rate cannot cope with such an increase.
MEMBRANE CHROMATOGRAPHY FOR DNA AND VIRUS REMOVAL
A relatively new step that has been integrated into processing is disposable ion exchange
chromatography membranes. Membranes have been particularly effective as a polishing step to
remove residual levels of large molecules such as DNA. Membranes offer enhanced processing
capabilities because adsorption and flow rates are not limited by diffusive flow into pores.
Because of this and their large (>0.8um) pore sizes, membranes offer higher capacities (>10X)
and higher flow rates (>50X) when compared to column chromatography. To achieve the same
flow rates as membrane cartridges, column sizes need to be substantially increased at great
capital expense. Membrane cartridges are disposable, which means that cleaning and cleaning
validation steps are eliminated, saving substantial labor time as well as buffer costs.
A more recent processing option has been the use of membrane chromatography for virus
removal. Viruses can be removed through either inactivation or removal, and while in many
instances effective removal of other contaminants can occur in a single step, acceptable titer
reductions can usually only be achieved by using a combination of technologies, including
chromatography, in an orthogonal process.
A Q-membrane chromatography cartridge will remove most undesirable contaminants,
including residual DNA, host cell proteins, and endotoxins. This same Q-membrane has also
demonstrated high levels of effectiveness in binding viruses for removal. The adsorption
capability of membrane chromatography has been well documented. For example, Q chemistry
membranes have demonstrated binding efficiencies of 1013 adenovirus particles per ml bed
volume of membrane.
Removal of virus from
flow-through
(log10)
Recovery of input virus*
MuLV


PRV




PPV


HAV


BVDV
(%)
As Figure Two shows, membrane chromatography has also demonstrated effective
removal of model viruses such as porcine, parvovirus, hepatitis A virus, murine leukemia virus
and pseudorabies virus at removal efficiencies between 104 and 107. Model viruses range in
size and complexities. If a manufacturer’s data demonstrate removal of these model viruses, it is
safe to assume that the same purification protocol will work for other similar viruses.
CONCLUSION
Manufacturers have a diversity of chromatography options at their disposal, and new
technologies can be utilized in a number of processing stages. When it comes to
chromatography, one size does not fit all, and process economics today require operators to
integrate customized steps into their production. In choosing a chromatography vendor,
manufacturers need to consider the full range of options, and should select a partner who offers
the widest flexibility and expertise possible.
Author Contact Information:
Iggy Gyepi-Garbrah
European Marketing Manager for Chromatography
Pall Europe Ltd.
Europa House
Havant Street - Portsmouth
Hampshire, England PO1 3PD
Tel: +44 23 9230 2542
Fax: +44 23 9230 2510
Email: Iggy_Gyepi-Garbrah@pall.com
Ian Sellick
Director of Marketing
Pall BioPharmaceuticals
2200 Northern Blvd.
East Hills, NY 11548-1289
Tel. 516.801.9497
Fax 516.801.9548
Email: ian_sellick@pall.com.