Protein A resins have significantly improved over the last 30 years in terms of capacity, productivity, and alkaline stability. 

However, even the most modern Protein A resins are not always amenable to newer IgG constructs that in many cases are prone to aggregation, are acid labile, or otherwise sensitive to low pH. 

This Question-and-Answer session excerpt taken from Purolite’s Bioprocessing Webinar Series hosted by Field Applications Scientist, Aaron Moulin – addresses questions regarding Purolite’s novel resin, Praesto® Jetted A50 HipH. 

Is there any change in porosity between Jetted and emulsified beads? 

I’d like to focus this response more generally around bead porosity. To begin, porosity is not a release criterion, meaning we don't explicitly measure the porosity. What we do, is monitor the apparent porosity, mostly by retention times – so apparent KAV for the resins.  

Much has been described in the literature about how to do this. That being said, we don't see any observable difference between Jetted and batch emulsified beads in terms of this sort of testing: retention time, KAV those sorts of things. So hopefully that answers that question. As far as we can tell, the porosity seems to be more a property of the agarose itself and the percentage of it (the agarose) rather than how the bead was formed explicitly by emulsification or jetting. 

Can Jetted A50 HipH be used for the purification of monoclonal antibodies that aren’t sensitive to low pH elution? Are there any benefits? 

The short answer – yes. Jetted A50 HipH can be used for any antibody that you would choose to use it for, even antibodies that are not sensitive to low pH levels.  

You may then ask, “what are the benefits of utilizing Jetted A50 HipH?” Well, that will depend on the antibody itself. We have seen with some of our customers that there is a palpable benefit to using HipH with antibodies that are prone to aggregation, at a low pH or any pH, especially antibodies that seem to have a lot of aggregate already present in the feed.  

Using a pH gradient with HipH has been able to nearly baseline separate high molecular weight species from monomeric content using HipH in a way that traditional Protein A resins haven’t been as successful at. That’s one of the main benefits.  

Of course, I also mentioned, that there seems to be potentially a trend that host cell protein (HCP) clearance is variable at different pH levels. During the webinar, I shared one set of data using the Fc-fusion protein; however, Purolite’s partner, Alvotech, has another set of data with an IgG2 that shows a similar trend with a much larger change in the log scale. The scale goes from 1.5 to 3.6 log reduction or so, providing one example of aggregate removal potentially helping to mitigate some problematic HCP clearance issues. 

These findings are certainly worth testing with HipH because you have a much wider range of pH that isn't available to you with traditional Protein A resins.  

Is there a guarantee this will mitigate issues? No, of course not. But it certainly gives you more options to try than with a traditional Protein A, in which you’re limited to pH levels between 3 and 3.7 at the upside. 

Can I use Standard Buffer Systems with Purolite’s Protein A resin, Jetted A50 HipH? 

In terms of chemical compatibility, all the standard buffer systems that you're using will be compatible with HipH.  

Of course, you just need to keep in mind the chemistry. Do you want to use citrate at pH 4.7? Probably not, because the buffering capacity is not great, but certainly acetate is probably the best suited buffer to use with Jetted A50 HipH due to its pKa being around 4.5. 

You have a really good buffering capacity in the pH 4 to 5 range with acetate, but of course you could use citrate. Glycine is probably not a great choice as you won't have good buffering capacity above a certain pH level – likely around three-point-something. But everything else you should be able to use. 

Does the high elution pH of Jetted A50 HipH mean that a wash buffer with intermediate pH (~5.5), occasionally used for traditional Protein A resins, cannot be used?   

The answer to this will be dependent on the feed streams used. Of course, we do have some data not presented in this webinar that shows you can see a significant amount of product leakage in washes at pH 6, for instance. A lot of this leakage can be mitigated by adding salt. We were able to mitigate almost all that leakage by adding one molar salt to the wash buffer.  

It’s important to note, if you're going to use buffer systems, especially washes at lower pH values between 5.5 and 6, you might want to evaluate those for use with HipH because you might see some reduction in recovery due to washing off some of the bound antibody. 

This question is driving it to a fair point – this ligand was designed to elute higher pH levels, so maybe it's not surprising that pH levels around 5 and 6 might show some elution. Beyond that, from a chemical standpoint, everything should be compatible. Your team should be sure to choose appropriately for your buffer system and perform some testing because Jetted A50 HipH won't behave exactly as traditional Protein A resins has behaved in the past. 

Do Purolite’s Protein A resins have any bonding for the VH3 domain? 

Excellent question, I alluded to this during the presentation.  

Purolite’s HipH Protein A resin has minimal binding to the VH3 domain. Currently, we don't have data delineating all the different isotypes of VH3 showing which do and which don't bind. In general, HipH binds to VH3 poorly.  

However, as part of our mAb purification toolbox, Purolite’s Protein A resin, Jetted A50 binds to the VH3 domain very well. 

We have some anecdotal evidence, including some of the salt mitigation in elution, pointing to Jetted A50 HipH being mostly Fc-driven, with very little VH3 binding. So, if you're looking to bind VH3 antibodies specifically or VH3-containing fab fragment, HipH is probably not the best choice going forward. However, Jetted A50 might be an alternative to explore. 

The above transcript was taken from Purolite’s recent “Advances in Protein A Chromatography” webinar. You can watch the full webinar On-Demand, here.