Considerations for Protein Pilot Study Scale-up

Abstract

You’ve developed a new candidate therapeutic protein, and you’d like to scale up its production for a pilot study. Transitioning the workflow from research project to either pilot or production scale is not a trivial task. “Often, lab-scale scientists underestimate what their lab-scale work translates to in a pilot scale-up or a manufacturing process scale-up, in terms of matters associated with a purification workflow, for example,” says Xuemei He, senior process chromatography applications manager at Bio-Rad Laboratories. Here we discuss some essential considerations as well as available tools for researchers when scaling up expression and purification of candidate therapeutic protein targets for pilot studies and analysis.

What is scalable?

To more efficiently transition the workflow from lab-scale, discovery-phase research to production scale, researchers should consider, early on, which tools or processes are scalable. One example is size exclusion chromatography (SEC), a common protein purification tool for small samples—but not an appropriate large-scale tool, because it’s comparatively slow, and the column capacity is relatively small. “SEC has a limit for sample loading; if you load too much, you will lose the resolution,” says He. Hence SEC columns are great for running a few milliliters of sample in the lab, but they can’t be scaled up to pilot runs.

To more efficiently transition the workflow from lab-scale, discovery-phase research to production scale, researchers should consider, early on, which tools or processes are scalable

Luckily, there are many other types of resins available for column chromatography. Bio-Rad Laboratories offers a wide range, and the company works with customers to optimize their scale-up efforts. “We look at whether all the process steps are necessary—or can we combine steps, or re-arrange the order of the columns so our customers don’t have to do as much buffer exchange, for example,” says He. “How you do this is going to affect the future product quality, production efficiency and the cost of making this molecule.” Sometimes efficiency improves by using another type of resin, with different properties, for column chromatography. “A resin that has better salt tolerance, for example, may result in the researcher having to do less or even no dilution,” says He. “That’s going to save a lot of time in a larger-scale production.”

At the lab scale, disposable pre-packed columns are common, so many researchers need help with large-scale columns that typically must be packed and re-used. “We offer assistance with packing and column qualification, because the industrial, large-scale columns have different configurations,” says He. Researchers also need to monitor whether the pressure of the packed column is stable throughout each run, and from one run to another. “After a few cycles, you might have impurities build up during runs,” says He. “You need to evaluate changes in column performance over time with repeated use, so you know how many cycles you can use the packed column, and how stable the resin is.”

Use the right devices at the right times

Also with an eye to scale-up, it’s essential to choose steps and devices that are suited to cGMP quality control practices, says Joe Makowiecki, global technical manager at GE Healthcare’s Life Sciences business. For example, it’s important to plan into the experimental design any required viral clearance steps. GE’s XDUO mixing systems are used, for example, to lower pH or mix detergents in virus inactivation operations. “If you don't have appropriate viral clearance steps built into your process, that can prevent you from entering the clinical trials stage,” says Makowiecki. “And it can be painful, because then you will have to go back and redesign your process, which adds to the cost and delays time lines.”

Devices to monitor the health of your cells upstream can be invaluable. Biotherapeutics processes, especially, often use metabolite screening and substrate analysis to verify cell health during scale-up and optimization. Roche’s Cedex Bio and Cedex Bio HT are benchtop analyzers that measure essential nutrients, metabolites and IgG titer from cell culture or fermentation samples. Assays for 21 parameters, such as galactose, LDH, iron, optical density and acetate, help researchers monitor cell health. Recently, Roche partnered with Flownamics to add auto-sampling capabilities. “The timely and accurate information enables our customers to make high-confidence decisions faster, offering the potential to reduce cycle time and production costs,” says Miranda Kheradmand, group marketing manager at Roche Custom Biotech.

Identify process parameters and product attributes

Another key factor in scale-up is identifying and maintaining critical process parameters. Appropriate documentation is also necessary, so you can monitor if and where the product changes as it progresses through various scales. “You need to understand those parameters in order to scale them,” says Makowiecki. “That’s critical when you’re scaling up the process and moving it towards cGMP manufacturing.”

It is equally important to know the quality attributes of your product—for example, how long your target protein can be stable under specific conditions. “When you start to scale up, certain things might take a little bit longer, and your product may or may not be able to handle those extended times,” says Makowiecki. “It’s important to identify those steps as early as possible.”

Sometimes a protein’s qualities can complicate its intended use in biotherapeutics—for instance, different glycosylation patterns can change a protein’s biological activity

Sometimes a protein’s qualities can complicate its intended use in biotherapeutics—for instance, different glycosylation patterns can change a protein’s biological activity. Roche Custom Biotech has enabled the use of in vitro glycoengineering (IVGE) as a downstream application, giving researchers greater control over the glycosylation profiles of their target proteins. The company offers a range of galactosyltransferases and activated sugars for changing the glycosylation patterns of proteins independent of cell lines. “[This reduces] the time and effort needed for glycan profiling during clone selection and [allows] time to focus on other critical quality attributes, viability or yield instead,” says Kheradmand. “Until recently, application of IVGE as a downstream application has been cost-prohibitive.”

GE’s Fast Track services offer assistance and education to researchers transitioning from lab scale to pilot or manufacturing scale, including process development and manufacturing support, education and training, for example, on the scale-up process. Eppendorf also offers in-depth support at its facility in Enfield, Conn., where customers can spend two weeks learning scale-up technology transfer from Eppendorf’s scientists and bioengineers, says Ma Sha, director of technical applications at Eppendorf.

Use scale-down modeling for optimization

Using scale-down modeling is an essential part of scaling up. Scale-down modeling tools, such as benchtop bioreactors, are valuable for identifying process parameters and make optimization less costly. These systems mimic more traditional, larger-scale growth chambers but enable researchers to manage and monitor multiple cell lines and experiments simultaneously. Examples include Eppendorf’s scalable BioFlo 320 benchtop bioprocess system and recently released BioBLU® 3f Single-Use Fermentor; GE’s ReadyToProcess WAVE™ 25 bioreactor and Xcellerex™ XDR-10 systems; and Sartorius Stedum’s widely used ambr® 15 and ambr® 250 bioreactor systems.

MilliporeSigma’s scalable FlexReady system is modular, with options for chromatography, tangential flow filtration, media and buffer preparation, clarification and virus filtration. In protein scale-up protocols, the order of events and the tools utilized can be critical to the optimization process. Workflow optimization is hard to predict and requires some trial and error testing, but scale-down modeling can give researchers a glimpse into the workflow process without a heavy investment of time on the instruments and costly reagents. Sequencing and integration of unit operations is essential,” says Bala Raghunath, director of global manufacturing sciences and technology in process solutions at Merck KGaA, Darmstadt, Germany. “Unit operations are often equipment- and operation-dependent and may not be easy to estimate, based on operations at a small scale.” Scale-down modeling can help to optimize the sequence of unit operations.

Makowiecki urges researchers to view the scale-up process holistically, from beginning to end. This perspective will help them avoid common pitfalls, such as choosing unnecessary process steps, not identifying appropriate process parameters and integrating devices that are not cGMP-compliant. “Quality by design begins early, at the bench scale—designing in the appropriate tools for the appropriate job,” he says. Arm yourself with information early, and your scale-up process will be relatively smooth.