Dealing with liquid chromatography (LC) separations across scales—say, from less than a liter to hundreds—requires changes in equipment and consumables. Many industries, especially biopharmaceutical producers, need methods that generate the same, or very similar, results at any scale of interest. As it turns out, scaling challenges go in some unexpected directions.
“There are two main challenges to scaling chromatography,” says John Liddell, chief technologist at CPI, a technology innovation center and founding member of the High Value Manufacturing Catapult. “The first of these is more expected, namely scaling up from laboratory to pilot or full commercial scale.” Then, he adds, “The second scaling challenge is less appreciated, which is the scaling down of chromatography to allow the application of high throughput–process development approaches based on the use of miniature columns run in multi-parallel automated equipment.”
Whether scaling LC up or down, scientists face challenges. A range of factors determines the complexity of the problem, and some personal preference comes into handling any obstacles.
Keep the cost down
At University College London, Daniel Bracewell, professor of bioprocess analysis in the department of biochemical engineering, uses various forms of chromatography, including affinity and ion exchange. “We scale these processes and consider the things that can and do go wrong in that process,” he explains.
One of the things that Bracewell and his colleagues keep in mind is cost. “Industry needs a certain number of cycles—say, 100—out of a column,” he says. That requires a resin in the column that holds up that long. “It can take months to run cycling studies at small scale to obtain an understanding of resin lifetime,” he notes, “but you need that data for regulators when working on a biopharmaceutical.” Plus, it can take more than $1 million of resin to pack a production-scale column. So, a company wants to run as many cycles as possible.
Image: Liquid-chromatography resin deteriorates with age. Image courtesy of Daniel Bracewell.
In thinking of how to scale up a process from lab scale to an industrial operation, developers need to consider other aspects of the resin. “You look at all of the reagents and the resin itself and make sure there are no issues,” Bracewell says, “like making sure that you can source enough of the correct grade.”
Prepare for a purpose
One size does not fit all in chromatography, although some scientists used to think otherwise. As Chris Pohl, vice president of chromatography chemistry at Thermo Fisher Scientific, says, “When I was first developing phases for biochromatography, the common story was that you needed a phase that was good for analytical purposes as well as preparative, but it’s better to make a phase defined for the purpose that you want to use it for.”
Otherwise, a process will always be based on compromises. For example, the most important feature of analytical chromatography is the resolving power, but for preparative chromatography it’s capacity. “And those two parameters are sometimes at odds,” Pohl explains.
When increasing the scale, a scientist needs to keep in mind how much material must be processed. Although many column makers specify the static loading capacity, Pohl points out that the dynamic loading capacity is the metric to keep in mind. As he asks, “How much can you inject under real conditions?” That’s what really matters.
Also, Pohl mentions carryover, which is the amount from a previous injection that can interfere with the next. “If there’s too much carryover,” he says, “then you won’t be able to successfully scale up media to an even larger scale.”
As Pohl concludes, “Dynamic loading capacity and carryover are the things that you focus on it the beginning.” Then, other features also come into play.
Image: At Thermo Fisher Scientific, researchers develop particles for analytical- and process-scale liquid chromatography.
Although Thermo Fisher Scientific started with a focus on analytical-scale chromatography, Pohl and his colleagues are working on particles that could be effective for process scale. In brief, they’re making 25 micron chromatographic particles coated with nanometer-scale particles with a very low nonspecific binding. “We’ve used it for analytical scale,” Pohl explains, “but we have work underway to make larger particle sizes for process scale.”
In any direction
Despite plenty of separation-specific problems to address, some similarities simplify a scientist’s life. “Fortunately, similar approaches help to ensure successful scale up and scale down based on some simple guidelines,” says Liddell. “The approach should include the use of the same bead size/grade of chromatography media across all scales—that is, the same type of media and bead size, basing flowrates on maintaining the same residence times for the steps across all scales, and all volumes for steps in the chromatography protocol—such as equilibration, elution, etcetera—relative to the volume of the column.”
When scaling up, Liddell notes that people would traditionally use a bigger column and a more powerful pump. “The scale-up protocol would include using the same chromatography bead, residence time and step volume related to column volume across all scales,” he says, but that’s not the only way.
At CPI, Liddell and his colleagues use other approaches. “The first of these has been continuous chromatography, where we have been working with a major U.K. consortium—which involves Pall Biosciences, GSK, AstraZeneca, Allergan, CPI, Fujifilm Diosynth Biotechnologies, and SCIEX—developing continuous chromatography processes,” he says. “Rather than using larger batch-based equipment to provide sufficient higher output of product, smaller continuous chromatography can provide the same amounts and quality of product.”
In another way to scale up, CPI is using “non-bead chromatography adsorbents with the ability to run at high flowrates so that a small device can be rapidly cycled to provide the same productivity as a large chromatography column,” Liddell explains. CPI—working with Cobra Biologics and GE Healthcare—applied this technology in a study on adeno associated virus (AAV)-based gene therapy being considered for treating single-gene genetic disorders, such as hemophilia. In this project, “CPI operated the complete process for AAV production with an upstream process linked to a downstream purification using the novel Fibro adsorbents—developed by Puridify, now part of GE,” Liddell says. “This proved the concept of rapid cycling with these novel therapies and confirmed that high productivities with compact and low-cost purification media could be achieved together with high product quality.”
So, whether scaling up or scaling down, scientists use a range of methods and tools to keep chromatography working in the most effective and efficient way. The process at hand will drive many of the decisions, and only solid expertise in this rapidly evolving field can produce the best results.
Hero image of Latex Agglomerated Supermacroporous Resin courtesy of Thermo Fisher Scientific