INTRODUCTION
As the biopharmaceutical industry continues to grow at a rapid pace, there is a greater need for high-throughput workflows to meet the demand for novel biotherapeutics. Automated liquid handlers (ALH) have gained popularity for sample preparation and purification, but the time-consuming off-deck buffer exchange step remains a bottleneck for many workflows.
We have recently shown that SizeX IMCStips® enables rapid and automated buffer exchange of 96 mAb samples in as little as ten minutes, saving significant processing time for end users without adversely affecting sample product quality attributes (PQAs).[1][2] These findings have important implications for affinity purification and plasmid purification workflows, demonstrating the broad potential of this approach. As scientists seek to streamline their workflows and accelerate time-to-market for new products, automation technologies powered by IMCStips will undoubtedly play a critical role.
Our latest publication in SLAS Technology highlights IMCS’s successful incorporation of automated buffer exchange into two fully automated workflows: affinity purification and anion exchange. By using pipet-based dispersive solid-phase extraction (dSPE), these workflows are highly adaptable and can address production constraints for biotherapeutics. The inclusion of buffer exchange as a last step in both workflows enables further integration for downstream processing, saving valuable time and increasing efficiency. These findings demonstrate the benefits of incorporating IMCStips into high-throughput protein and plasmid purification, paving the way for a more streamlined and effective biotherapeutics discovery process.
METHODOLOGY
The study utilized Ni-IMAC IMCStips for affinity purification and SizeX IMCStips for the automated protein purification workflow and µPure LE IMCStips with SizeX for the automated plasmid purification (or automated miniprep) workflow. The combined affinity and buffer exchange workflow was also used to test the effect of time post-induction on protein production. The methods were automated on a Hamilton Microlab STAR.
The pipette-based dispersive solid-phase extraction (dSPE) approach optimizes sample binding to resin by utilizing repeated aspiration and dispensing cycles via an automated liquid handler (ALH).
WORKFLOW
RESULTS
Automated Protein Purification
Combining the workflows for affinity and buffer exchange resulted in clean, usable protein in less than 80 minutes. Recovery rates were a little bit higher than the sum of the two separate phases. As anticipated, the pattern exhibited in distinct affinity purifications was reflected in the overall yield. Although the yields were 15–20% lower than with IMAC-only purification, exchange into the suitable buffer had already been carried out after the initial purification phase, offering numerous advantages over dialysis or spin-column. The combined chromatography methods of affinity purification and buffer exchange on automated liquid handlers can be used for high-throughput workflows.
Affinity purification followed by buffer exchange on an automated liquid handler for high-throughput workflows. Polyhistidine-labeled proteins (GFP, ArSulf, βGlc) were added to bacterial lysates and purified using the Ni-IMAC method in a pipette format followed by SizeX150 desalting. Percent recoveries (A) and recovered amounts (B) correspond to the initial amount of protein added to the lysate. (C) SDS-PAGE of the proteins at different quantities correspond with NanoDrop and fluorescence measurements. (D) Enzyme activity assay using p-nitrocatechol sulfate (pNCS) indicates ArSulf remains active after undergoing the combined automated affinity and buffer exchange workflow. ArSulf converts pNCS into red, indicating enzyme activity. Yellow wells contain no ArSulf, demonstrating no well or tip carryover.[3]
Automated Plasmid Purification
The study demonstrated a new method for automated plasmid DNA purification (or automated miniprep purification) that results in low-endotoxin samples suitable for transfection and sequencing. Unlike many commonly used silica-based purification methods, this new method removes lipopolysaccharides that can trigger immune responses and reduce transfection effectiveness. The purified plasmid yields were sufficient for downstream use and similar for plasmids of different sizes. The inclusion of Triton X-114 in the purification process reduced endotoxin content. This method is suitable for up to 96 samples simultaneously and can purify ≥10 µg of low-endotoxin pDNA/sample in under 60 min, making it a fast and efficient alternative to other purification methods.
The amount of plasmid recovered in µg varied based on the size of the plasmid construct. (A) Recoveries for pCRS158 were significantly higher than those for pCRS166 and pCRS240.3. (B) The final concentration of plasmid in the elution well varied by plasmid construct. The concentration of pCRS158 was significantly higher than that of either pCRS166 or pCRS240.3. (C) The volume eluted from the SizeX100 tip varied slightly by construct. The volume of the pCRS158 eluate was significantly lower than in pCRS240.3. *p<0.05; ns: not significant.[3]
CONCLUSION
One of the earliest and most common phases in biotherapeutic characterization and discovery is the purification of a biological agent. IMCS introduces the idea of tip-based buffer exchange using a zero-pressure pipet pick-up step and precise air displacement controls on the Hamilton Microlab STAR. Affinity enrichment and anion exchange chromatography were used with this approach to achieve downstream-ready protein and plasmid DNA purifications, respectively. By enabling researchers to prepare their purified product for following procedures without manual intervention, the development of an automated buffer exchange method contributes to the alleviation of a significant bottleneck in the production of biotherapeutics.
REFERENCES
- Sitasuwan, P., Powers, T. W., Medwid, T., Huang, Y., Bare, B., & Lee, L. A. (2021). Enhancing the multi-attribute method through an automated and high-throughput sample preparation. MAbs, 13(1). https://doi.org/10.1080/19420862.2021.1978131
- Ogata, Y., Quizon, P. M., Nightlinger, N. S., Sitasuwan, P., Snodgrass, C., Lee, L. A., Meyer, J. D., & Rogers, R. S. (2022). Automated multi‐attribute method sample preparation using high‐throughput buffer exchange tips. Rapid Communications in Mass Spectrometry, 36(3), 1–10. https://doi.org/10.1002/rcm.9222
- P.A. Kates, J.N. Cook, R. Ghan et al., Incorporation of automated buffer exchange empowers high-throughput protein and plasmid purification for downstream uses, SLAS Technology, https://doi.org/10.1016/j.slast.2023.01.005
