Advances in Monoclonal Antibody Purification - New technologies and adaptations of existing technologies can improve platform processes. - BioPharm International

ADVERTISEMENT

Advances in Monoclonal Antibody Purification
New technologies and adaptations of existing technologies can improve platform processes.


BioPharm International Supplements


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
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
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
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
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,


blog comments powered by Disqus

ADVERTISEMENT

ADVERTISEMENT

AbbVie to Acquire Shire for $54.7 Billion
July 18, 2014
AstraZeneca Reveals Design for New Global R&D Center and Corporate Headquarters
July 18, 2014
Particulate Matter Prompts Baxter's Recall of IV Solutions
July 17, 2014
Mylan to Acquire Abbott's Non-US Businesses in $5.3 Billion Stock Deal
July 14, 2014
Shire and AbbVie Discuss Possible Deal
July 14, 2014
Author Guidelines
Source: BioPharm International Supplements,
Click here