December 1, 2009 (Vol. 29, No. 21)

Crucial Step in Process Development Can Be Aided with Smaller-Scale Technologies

Assessing the performance of a cell line is a crucial step in process development, and it can be done faster, cheaper, and more effectively by employing high-throughput small-scale technologies. A seminar on the benefits of scaling down was recently sponsored by Applikon. According to Sharon Brownlow, Ph.D., sales and marketing manager, the company is now actively moving toward small-scale screening of cell lines with its Micro 24 bioreactor system, which can rank constructs and conditions.

“High-throughput technology should be simple. It does not need all the capabilities of a stirred tank reactor, but it must be able to predict the performance at a larger scale.”

Working with shake flasks—the traditional approach—does not always predict what happens in the larger stirred tank reactor used for production. “Scale-down technology aspires to change what larger fermentors are used for—that is, use is more targeted and, ultimately, more molecules will go through these larger vessels.”

Jim Mills, Ph.D., director of operations at Xenova Biomanufacturing, now known as Cantab Biopharmaceuticals said that there was a need for increasing throughput in upstream processing, with many companies wanting high-throughput protein expression for rapid process development. “High throughput allows for statistical experimental design and cell-line selection under relevant conditions.” Quality by design (QbD) is a huge driver too. “This will be the way all processes are done in the future. QbD demands a lot of work up front giving quality information. Miniaturization and statistical design make this possible.”

According to Dr. Mills, traditional small scale—less than 2 L—has a limited throughput of just a few hundred experiments per annum. In the traditional approach, smaller-scale vessels were geometrically similar to large-scale ones. “It is better to look instead at what the cell needs and not worry about geometry.”

Dr. Mills noted that service providers need to do more work up front on small-scale systems to help customers get the most out of them. Issues requiring more attention include the need for predetermined accuracy of these systems with respect to larger-scale operations, and also, a need to integrate scale-down upstream processing with scale-down downstream processing. On-line or low volume analytic techniques are also an important requirement.

The challenge is how to scale-up to different systems, Dr. Mills explained. “The knowledge to do this is not there yet. However, this area is developing extremely fast, and it is really the way forward.”

Gareth Lewis, Ph.D., senior research scientist at MedImmune, now part of AstraZeneca, reported that cell-line selection is often done in a platform that is not a good model for manufacturing. Good clones can be missed or, conversely, the wrong cell line might be selected. “This is where scale down is coming forward.”

MedImmune wants to screen more cell lines earlier on and in a better model. It has evaluated various small-scale systems and shaking-well plates (the latter with a team at University College London). These experiments have revealed that mixing time is important, and shaking speed needs to be optimized for this.

The work done at the university has now been brought in-house and compared to conventional techniques. The shaking-plate system allows the screening of 150 clones in 6–8 weeks, compared to 50 clones in 10–12 weeks by the usual approach. “This makes it easier to pick out the top clones,” Dr. Lewis said.

High-throughput shaking plates speed up process development, especially for media and feed, he concluded. “We now want to implement this over multiple programs.” However, although both scale-down systems offer advantages over the conventional approach, data handling and analytics are still an issue, but the analytical companies are catching up with these new demands.

Gernot Thomas John, Ph.D., product specialist at PreSens, described the company’s precision sensing technology, which is suited to smaller-scale bioreactors. Optodes are chemical-optical sensors that allow for oxygen and pH sensing. PreSens has developed plastic shake flasks with sensor spots that give a WiFi readout to PCs. These have been used in a wide range of experiments. For instance, a comparison of different closures on flasks showed how oxygen levels drop during sampling.

Applikon’s Micro 24 bioreactor, a high-throughput fermentation and cell culture device, is composed of 24 controlled bioreactors on a microtiter plate.

Baculovirus Experiments

Jared Cartwright, Ph.D., head of protein production at the technology facility at York University, discussed the integration of the Micro 24 and the Appliflex single-use bioreactor in baculovirus experiments. At York, this system is used for parallel expression screening so that 10–50 mg samples can be obtained for customers’ structural biology needs in protein target and biocatalysis work.

Insect-cell production involves infecting the cells with the Autographa californica multiple polyhedral virus. The York team has been using the flashBAC system to improve its transfer vectors and can now produce recombinant baculovirus—but it still wants to screen and optimize protein expression.

One issue in this work is how adherent and suspension cell culture compare. The team has found found the best results when it optimizes in suspension, rather than adherent, culture, using the Micro 24 system. “This gives us near direct scale-up conditions we can work with,” Dr. Cartwright said. He added that multiplicity of infection (MOI) is very important.

Tiffany Rau, Ph.D., principal scientist, microbial and cell culture development at GlaxoSmithKline (GSK), described validation of scale up and the incorporation of the Micro 24 screening reactor in process development, which was, traditionally, all done in shake flasks.

“We were finding issues, and we needed a better way of screening cell lines.” For instance, an apparently low titer clone in shake flasks can become a winner in the bioreactor, so good cell lines may be thrown away. The reverse may happen, with those cell lines that are great in shake flasks not being good in process optimization.

Now GSK process development scientists screen in a controlled environment and then identify those clones that respond to feed earlier. Investigation of design space, pH, temperature, and oxygen in the system is a big part of tech transfer. “We are redefining how we do cell-line selection using microreactors, to carry out rank ordering of clones.” 

The Micro 24 can also be used for troubleshooting—for instance with clones that have growth issues in shake flasks. The microreactor is used to see if oxygen and pH control helped, which cannot easily be done in a shake-flask system. pH issues are therefore more easily identified using the microreactor. According to Dr. Rau, more process factors can be investigated with the microreactor and, in the future, GSK will be implementing them in cell-line selection.

Andreas Schneider, Ph.D., director, global sales at Innovatis, now part of Roche, described how to keep up with sample analysis from high-throughput screening systems. In 2005, Innovatis began looking into online monitoring of many parameters, like optical density and pH, but in some cases it could not readily measure cell count or metabolites. Then, working with Bayer, it developed the BaychroMAT®, which is a steam-sterilized sample port for auto sampling.

MedImmune has evaluated a number of small-scale systems and shaking-well plates in its quest to find a better model for manufacturing.

Susan Aldridge, Ph.D. ([email protected]), is a freelance science and medical writer specializing in biotechnology, pharmaceuticals, chemistry, medicine, and health.

Previous articleLink Technologies Licenses University of Manchester’s Platform to Reduce Background Fluorescent Signal
Next articleReport Highlights Biotech Strengths, Opportunities, and Weaknesses in Newest EU Countries