Automated IgG Purification using IMCStips® and Hamilton Microlab® STAR™
Presenters and Moderator
Stephen D'Eri, MS
Janna Liptak, MS
Carter Mitchell, PhD
Executive Summary
This case study describes how Sanofi harmonized its automated Protein A purification workflows across multiple sites using Protein A IMCStips® (ProA IMCStips) and SizeX IMCStips on the Hamilton Microlab STAR. ProA IMCStips enable simultaneous purification of harvest samples in a 96-well plate, and consistently high recovery and quality of protein A eluates have been demonstrated. SizeX IMCStips enable simultaneous desalting of a full 96-well plate for denatured, reduced, and alkylated samples prior to tryptic digestion. The inter- and intra-plate reproducibility are comparable to manual approaches. This automated method can detect trending modifications without bias and has passed qualification showing suitable accuracy, precision, linearity, and repeatability for known modifications quantified via targeted LC-MS analysis.
Challenges
Although Sanofi operates this method across several sites globally, there were many challenges such as redundancy in analytics as well as labor-intensive and variable sample preparations. In the early stages of developing and optimizing the automated tryptic digest method, laboratory results show artificially high and inconsistent levels of asparagine deamination due to cysteine reduction conditions associated with the method. Using the PENNY peptide serving as a global indicator of deamination, absolute levels of deamination were observed to be very high. Automated method yielded levels 9-11% deamination compared to 2-5% deamination from a manual run and varying levels of deamination were shown across different sites. Additionally, some man methods require long run times to achieve chromatographic separation of analytes of interest and visual inspection of data strongly suggests peptide degradation and increase in oxidation as a function of autosampler dwell time. Sanofi hoped to develop a fully automated, harmonious workflow for protein A purification, A280 analysis, and tryptic digest across multiple sites.
How IMCS Helped
Overall, IMCS helped Sanofi to transition from a semi-automated to a fully automated workflow. The IMCStips were the enabling technology for automated protein A purification workflow that allowed for low variability, high reproducibility, and high recovery. And the SizeX IMCStips were used during the buffer exchange step and allowed Sanofi to transition from a low throughput manual tube-base digest to a high throughput automated plate-based digest. Implementing this fully automated workflow allowed Sanofi to also save a lot of time, requiring the analyst to only be there at the beginning to set up the deck and at the end to grab their sample.
Results
Quotes from Presentation
"This automated workflow takes about an hour to set up the deck [and] that’s really the only hands-on time for the analyst. If you think about just the digestion when we were doing it manually, an analyst could probably do around 24 samples a day, and [often] would take them a full 8 hours: 3 hours of hands-on time, but they would have to be there the whole time. And that’s just for 24 samples. If you wanted to do 96, you are talking about 3 to 4 days. So, implementing these IMCStips helped to fully automate that workflow, and make sure that the user only had to be there at the beginning to set up the deck and at the end to grab their sample. They might not even have to be there to retrieve their sample at the end. They might just have their data output to them."
"We don’t really know of other tips that are like the SizeX tips to do that buffer exchange step. So, that’s really where IMCS stood out for us. Showing all this data of IMCS, we were happy with the results we got. The workflow that we had was really working out for us."
"In developing and optimizing the Protein A tips in house, we consistently monitor where all the samples have gone to in different wash and elution steps. By process of elimination, we saw that the IMCS Protein A consistently captured, if I had to put a number on it, greater than 92%. We can always account for close to 100% of our sample, whether it ended up in the wash based on our buffers, in the elution, or whether it remained on the tip when we were optimizing the elution procedure. Indirectly, we measured that they captured a very high harvest."
"IMCS provided the basic scripts; these were sub-methods that we could just plug right into our entire Hamilton method. From there, we could plug in different inputs so we could change the number of aspirate and dispense cycles or the volume that we’re aspirating and dispensing each time. It has flexibility that we could change any of those inputs. The base method was already there. There were also firmware controls. The tips are cut at the bottom, so you need to make sure the Hamilton method knows that it’s using these different types of tips. It made it easy and flexible for us to continue to optimize based on what’s needed for our group."
