by Philip Chapman, Protein Purification Specialist, Bio-Rad Laboratories, Co-authored by Katie Schaefer, Protein Purification Specialist, Bio-Rad Laboratories
Conventional downstream processing for protein purification begins with a protein capture step, commonly using a Protein A resin, followed by two or more polishing steps to remove product- and process-related impurities. Although Protein A resins demonstrate high specificity and affinity, there are several limitations associated with their use, such as the cost — using Protein A resins can account for over 50% of the entire process. Additionally, they are unable to differentiate between functional and aggregated proteins, which may impact purified sample quality during the acidic elution step. These issues are often heightened by low protein expression, pH sensitivity, and limited stability.
The nature and sequence of the polishing steps depend on the protein of interest and the type of impurities that must be removed before isolating the product, such as host cell DNA and proteins, endotoxins, viruses, and protein aggregates. Considering the physicochemical properties of solutes, these steps may include the sequential use of different modes of interaction, such as affinity, size exclusion (SEC), hydrophobic interaction, and ion exchange.
However, given the need for a faster, more cost-effective purification process that can remove a range of impurities, a traditional single-step chromatographic process is suboptimal for many research, process-development, and laboratory-scale applications. Scaling-up chromatography processes from laboratory to manufacturing scale can also be challenging due to differences in column size, flow rate and resin behavior.
“Looking back over many years of a career in science, it is interesting to see how chromatography applications and technologies have evolved and improved, leading this old(ish) man to think ‘Kids nowadays? They’ve never had it so good!’” said Philip Chapman, protein purification specialist at Bio-Rad Laboratories. “Over the years, technology improved and chromatography systems became more robust and reliable. Software struggled to keep apace though and being a chromatography expert able to program and run a method made you as rare as a unicorn and worth your weight in gold.”
Automated Multi-step Chromatography — a Sophisticated Combination of Hardware and Software
With traditional sequential chromatography, user intervention increases the probability of introducing errors, impacting the reproducibility of each run. The solution? Automated liquid chromatography systems. By combining software advances with expanded hardware capabilities, purification workflows that once took many days of intensive manual input can now be automated and achieved in a matter of hours.
“With the continued evolution and introduction of chromatography platforms the mysterious dark art of protein purification has become democratized and much more accessible to anyone in the lab,” said Chapman.
These systems are typically preconfigured or have custom-configuration options to meet a range of throughput and application needs for research, process-development, and laboratory-scale chromatography. Valves and accessories can be supplied by the vendor and easily added to expand, scale, and customize purification capabilities. Additionally, automated platforms support multidimensional (Multi-D) chromatography workflow setup — this approach allows continuous, optimized multi-step purification, providing a “one-push” functionality for efficient and more reliable protein purification. Compared to traditional sequential chromatography, Multi-D chromatography yields equal protein purity but with more consistent and reproducible runs, allowing the robust screening of multiple samples.1 Not only is the workflow faster, but the user can start a method and then walk away to focus on other work.
“Instead of fearing to turn your back, you can now program your automated chromatography system, ensure your buffer bottles are full and degassed, click start and go home relaxed in the knowledge that your system’s air sensors, system pressure monitors, and automatic column selection will keep things running smoothly. No need to spend all night with your fingers crossed hoping your fractions didn’t skip a tube and form a puddle on the bench,” said Chapman.
Automated chromatography liquid systems are often integrated with a software platform to support lab-scale protein purification, including instrument setup and calibration, method development, real-time monitoring and system control, chromatogram comparison, and peak analysis. Process conditions and parameters can all be rapidly redefined, providing the flexibility for process optimization. Intelligently designed to ensure functional simplicity and support straightforward method development, software platforms often include method templates for simple, single-mode techniques as well as more advanced multi-column, multidimensional chromatography.
“Gone are the days of using a column and protocol handed down from post-doc to post-doc over the years because ‘that’s the way we’ve always done it.’ Now you can easily scout multiple column chemistries and determine ideal binding and elution conditions,” Chapman explained.
Demonstrating the versatility of these systems, a recent study describes an automated tandem immunoglobulin (IgG) antibody purification with inline pH neutralization and SEC. Following initial antibody capture by Protein A and subsequent acidic elution step, the two-column purification technique facilitated immediate inline neutralization of the eluate prior to loading onto the SEC column. Using automated tandem purification reduced the exposure of antibodies to low pH, preventing aggregation and minimizing sample loss from dialysis. Consequently, this process resulted in increased yield and purity of the final product at the tens-of-milligrams scale — especially key to biologics production.2
Mixed-mode Chromatography — Optimal Protein Purity and Recovery in a Single Step
Stepping away from automated solutions for multi-step purification, mixed-mode chromatography has emerged as another robust tool to overcome the challenges presented by traditional chromatography applications. By using resins capable of at least two modes of interaction, mixed-mode chromatography can selectively remove a broad range of impurities with high target recovery in a single step, improving process productivity and economy. This approach achieves optimal protein purity and recovery, superior to single-mode resins even when used sequentially. For example, combining the properties of anion exchange (AEX) and hydrophobic interaction chromatography into a single hydrophobic AEX mixed-mode resin allows users to effectively purify both basic and acidic antibodies in either bind-elute and flow-through mode.
Additional benefits of mixed-mode chromatography include a broad range of binding and elution conditions for optimal process scale-up thanks to a wide design space, easy parameter manipulation for complex feed stream purification, and straightforward method development to purify even the most complex molecules.
Challenges Shared by Traditional and Advanced Chromatography Workflows
Column packing and preparation is another key challenge in protein purification that permeates traditional chromatography techniques and innovative new approaches, such as Multi-D and mixed-mode chromatography.
“We would usually have to prepare and pack our own columns, pour our own gels, dialyse our samples and stain/destain our gels overnight. Such labour intensive and fraught workflows made many fear chromatography and consider it a necessary evil better done by someone, anyone else but yourself,” said Chapman.
Although in-house column packing gives users the flexibility for custom applications and process optimization, it requires significant time, effort, and resource investment. A typical packing process involves multiple steps, such as calculating the slurry concentration needed for a desired column bed volume, column packing, testing, cleaning, unpacking and resin storage after use (Figure 1). The complexity of this process creates several hurdles, including column preparation, resin availability, hardware availability and storage, user expertise and training, column quantification and failure.
Figure 1: Typical column packing and preparation.
Limited by space and storage needs, in-house column packing can quickly create a bottleneck in the laboratory workflow when larger or multiple columns are needed. Loose resin packing, slurry and buffer preparation, and column cleaning also rely on specialist expertise and knowledge, which can vary from person to person, potentially introducing variability in column quality and performance between batches. Working with resins from different suppliers adds another degree of variability. Process variability can result in inconsistent data, making it challenging to ensure the required quality standards are followed consistently, and ultimately cause project failure. Moreover, manually packing resins and cleaning columns by hand can increase the risk of contamination.
Using prepacked chromatography columns can overcome many of the issues associated with in-house resin packing. Prepacked columns are ready-to-use, Good Manufacturing Practice (GMP)-compliant columns, accompanied by tailored instructions and documentation. They are available in different sizes, prefilled with a variety of different resins, and compatible with most common chromatography systems. Unlike in-house packing, prepacked columns do not require specialist knowledge or expertise, and they also minimize the risk of cross-contamination, making them an attractive option for biopharmaceutical manufacturing. The reproducibility and reliability of data is also ensured thanks to their manufacture under controlled conditions. Prepacked columns offer an innovative tool to streamline project timelines, increase productivity, and enable process scale-up.
“Any chromatographer knows that the ‘magic’ happens in the column. Pouring your own column is less common now with people often preferring to select the chemistry with the desired flow characteristics, selectivity, and binding capacity from a broad range of pre-packed columns,” Chapman said.
Conclusion
Researchers, process developers and laboratory-scale chromatography users are continuously exploring new and innovative methods to streamline the purification process and increase efficiency. By combining sophisticated software platforms with expanded hardware capabilities, automated liquid chromatography systems ensure consistent, reliable, and customizable protein purification. Mixed-mode chromatography adds an additional layer of optimization to these workflows, providing a faster, more cost-effective way to remove a range of impurities and purify target molecules than sequential single-step approaches. Using prepacked columns can reduce the risk of column-to-column variability and deliver a consistent and reliable purification outcome, contributing to the quality and consistency of the final product.
In a commercial setting, leveraging these innovations could accelerate time to market for biotherapeutics, help to ensure the efficacy, patient safety and accessibility of therapeutic products, and improve overall cost-efficiency.
References
- Becker, W. et al. (2019) “A fully automated three-step protein purification procedure for up to five samples using the NGC chromatography system,” Protein Expression and Purification, 153, pp. 1–6. Available at: https://doi.org/10.1016/j.pep.2018.08.003.
- Hilario, J et al. (2022) “Automated Two-Column Purification of Trastuzumab on the NGC Chromatography System with Modifications for Inline Neutralization”, Bio-Rad Laboratories, Bulletin 3435. Available at: https://www.bio-rad.com/Bulletin_3435.pdf