Adapting Existing Technologies
One potential purification challenge related to increased titer is the lack of capacity for Protein A resins. Several attempts
to address this by removing Protein A resin from MAb purification schemes have met with not much success. However, one promising
technology adapted from other industries is multicolumn countercurrent solvent gradient purification (MCSGP), which is a hybrid
between simulated moving bed technology used in the chemical industry and standard batch chromatography. The adaptation of
this technology for MAb purification has been explained in detail in multiple articles.9–11
This technology can be applied either to increase the capacity of standard affinity resins in antibody purification platforms,
or to increase the resolving power of non-affinity unit operations. This method potentially could facilitate the removal of
affinity chromatography from an antibody purification process. Using this technology in a nonaffinity two-chromatography step
process, we achieved a seven-fold increase in productivity with no reduction in impurity removal as compared with a standard
affinity three-column process (data not shown). Multiple companies are in the process of developing chromatography systems
and columns to optimize this technology.
A second type of technology used in other industries that has been adapted with some success is precipitation. This method
can be of two types: positive precipitation, in which the product of interest is precipitated, leaving the impurities behind;
and negative precipitation, in which impurities are precipitated, leaving the product of interest in the supernatant solution.
Precipitation generally is used early in the purification process, to remove large quantities of impurities for increased
column performance before polishing steps using resin chromatography. It has been classified as a technology with low resolution
potential but high industrial maturity.12 Chemicals such as ammonium sulfate, polyethylene glycol, and other polyelectrolytes for positive precipitation of antibodies
from crude broth have been used for several years. Although they have not gained a significant foothold against standard capture
chromatography methods, lately there has been renewed interest in developing them as alternatives to the standard antibody
purification process in which affinity resin is the workhorse.13,14
Recently coming into more extensive use are negative precipitants, designed to leave the product of interest in solution.
One of these is caprylic acid, a short-chain fatty acid that can be added directly to conditioned media out of the reactor
to remove host cell impurities such as DNA and protein. A significant reduction of DNA and HCP can be achieved using caprylic
acid at concentrations of 100–500 mM and pH values of 4.0–6.0.15 However, HCP removal was achieved only for CHO-produced antibodies, not for NS0-produced antibodies.15 Even with this limitation, the use of caprylic acid has been incorporated into manufacturing processes to facilitate the
use of a two-chromatography step process without Protein A resin.16
Advances in Filters
New advances in filtration, including both multilayer depth filters and nanofilters, have begun to address limitations in
throughput and purification by providing new tools that increase the flexibility of order for current unit operations. These
new filters also have the potential to replace standard chromatography unit operations.
A new depth filter under development by 3M Purification (St. Paul, MN), is being designed to increase flexibility in MAb purification
by enhancing the removal of DNA after centrifugation, and of both DNA and HCP removal after capture-step chromatography. This
filter, designated ZPQ020, is a hybrid purifier that contains both a size exclusion component and an AEX (charged) component.
Figure 4
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Figure 4 shows the excellent DNA removal capability of this filter after centrifugation of harvest broth. The graph presents
the log reduction values from five CHO-produced antibodies where a new filter was used after centrifugation. In two of the
five cases, the DNA was removed completely, and in the other three, the log reduction was significant. Other filters tested
showed minimal to no DNA reduction. Removing DNA before an initial Protein A capture chromatography step could potentially
extend the resin life of a very expensive raw material, or even allow the resin to be eliminated altogether and substituted
by a cheaper resin such as ion exchange.
Figure 5
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In addition, this filter also exhibits excellent impurity removal post-capture as compared with standard AEX technologies.
The results from two CHO-produced MAb tested post-affinity columns are presented in Figure 5. In both cases, these antibodies
exhibited higher than normal levels of DNA and HCP. The synthetic 3M filter exhibits better HCP removal for feed streams with
higher than normal impurity levels. In terms of DNA removal, the 3M filter showed better DNA and HCP reduction for the two
antibodies tested than the other AEX technologies evaluated. Future studies are needed to compare this filter with the new
salt tolerant membranes.
Figure 6
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Advances in nanofilters also have provided flexibility in the purification process, allowing for greater throughput resulting
in decreased membrane area, or reducing the processing time for comparably sized membranes. New nanofilters such as Asahi
Kasei's BioEX or Millipore's VPro can significantly increase flux. Figure 6 shows the flux data for two CHO-produced antibodies
comparing Asahi Kasei's Planova 20N to the new Planova BioEX. The flux for the BioEX is significantly higher than for the
20N, though with other antibodies we have seen greater flux decay for the BioEX (data not shown). The effect of this increased
flux on viral clearance could be a significant concern, although initial assessments show no effect.
Figure 7
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However, as we push the envelope toward higher concentrations and salt conductivities (such as in conjunction with the use
of salt-tolerant membranes), we may start to see a reduction in clearance values. Figure 7 shows the log reduction values
for Mouse Minute Virus (MMV) for two purified CHO-produced MAbs at a low pH and high pH value, at two higher salt concentrations
(>100 mM sodium chloride). The load antibody aggregate concentration for these antibodies was also relatively high (>5%).
These starting materials were filtered across a Planova 20N filter at standard operating conditions per the manufacturer's
recommendations. Complete removal of virus was only seen under one set of conditions—low pH and low salt (MAb 5). For all
other conditions, virus was detected in the filtrate. These data indicate that under certain conditions, the virus is extruded
through the nanofilter, a factor that must be considered when designing the purification process.
Summary
Expression titers and dosing levels for monoclonal antibodies (MAbs) continue to increase as medical needs are successfully
met with these products, placing pressure on both existing production capabilities and development decisions for downstream
purification processes. This means rising production costs because extra unit operations are needed to meet the regulatory
requirements for product purity and viral clearance. New technologies, such as disposable membranes and cassettes, along with
new depth filters and nanofilters, or the adaptation of existing technologies from other industries, can be used to meet these
production challenges. By providing flexibility not only in the order of unit operations, but in the design of the steps themselves,
these new products and methods will enable purification processes that are both cost efficient and effective in producing
the desired product.
Acknowledgements
The authors would like to thank the Cell Line Development and Process Development Analytics groups at Pfizer St. Louis for
their support, as well as Sartorius Stedim, 3M, and Asahi Kasei for providing product samples and technical expertise.
JUDY GLYNN is a senior principal scientist, DENIS BOYLE, PhD, is an associate research fellow, JAY WEALAND is a principal scientist, ERIN MILLER-CARY is a senior associate scientist, BRIAN CHEN is a senior scientist, and PAUL MENSAH, PhD, is an associate research fellow, all at Pfizer BioTherapeutics Research and Development, St. Louis, MO, 636.247.6519, judy.k.glynn@pfizer.com
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