Bio-Rad Laboratories, Inc. (BIO) Earnings Call Transcript & Summary

Hello, everyone. Welcome to Bioprocess International Spring Digital Week brought to you by the producers of the face-to-face Bioprocess International events, with visits in Santa Clara in August, Boston in September and Milan in October this year. Bioprocess International Europe will be delivered as a 100% virtual conference and exhibition on July 13 to the 17, 2020. My name is Catherine Simpson, and I'll be your host for today's session titled: Purifying Biotherapeutic Molecules with Unique Chromatography Selectivities. First, I'll cover some quick housekeeping items. If you experience difficulties with audio or advancing slides, refresh your screen with F5. If you are experiencing up issues, hit the question mark button to receive assistance. At any time during the presentation, submit your questions in the Q&A window on the left-hand side of your screen. In 24 hours, you'll receive a link to watch the recording of this session. You can also download a few featured white papers in the resource list box on the right-hand side of your screen. Now let's begin by introducing our speaker from Bio-Rad Laboratories: Mark Snyder, R&D Manager, Process Chromatography Application, from Bio-Rad Laboratories. Thank you for joining us today, Mark. Now I'll hand it over to you to begin the presentation.

Well, thanks very much for that kind introduction. Today, I'm going to be talking to you about purifying biotherapeutic molecules, some of the unique selectivities that Bio-Rad resins possess. Let me just say at the outset that I always number my slides. So if you're getting bored, you don't have any more, you have to go until I'm finished and take questions. Okay. I'm going to be talking about 3 subjects: first is monoclonal antibody purification using our hydrophobic anionic exchange resin, Nuvia aPrime 4A; second is aggregates and process impurities removal and virus purification with our ceramic hydroxyapatite media; and finally, large molecule purification for things such as IgA or IgM, viruses and VLPs using our Nuvia HP-Q resin. On this slide, I've just shown the suite of resins which Bio-Rad offers. And in green are the ones that I'm going to talk about today. So first Nuvia aPrime 4A. In the center of this slide, you see the structure of the Nuvia aPrime 4A ligand. Again, it is an anion exchange hydrophobic mixed-mode interaction ligand. And biomolecules will interact with this ligand, depending upon their pI and their hydrophobicity as well as the buffer conditions. Basic species those with a high pI tend to flow through the resin while acidic species those with a low pI tend to bind to it. Accordingly, chromatography can be performed in flow-through or bind-elute mode. However, buffer pH and conductivity impact charge and hydrophobic interactions and as a result of selectivity and recovery of target molecules can be fine-tuned. So this slide shows the results of some flow-through studies that we did on an antibody with pI of about 9.1. What you see in the left-hand panel is that the HCP level in the flow-through of Nuvia aPrime 4A remained consistently low, about 100 or 150 nanograms per mg, while those with a competitor resin rose and remained significantly higher than Nuvia aPrime 4A. Right-hand panel shows the purity data from these flow-through fractions. And what you see again is that Nuvia aPrime 4A remained consistently at about 96% or so percent pure, while those of the competitor resin decreased dramatically over the course of the flow-through study. Here we have the results of some DoE screening, a purification of a basic monoclonal in flow-through mode. And I want to point out a number of things. We have monomer purity on the left, step yield on the right. And you can see by the Blue dots, we've chosen a position on the left-hand side of both plots. Now it may look like this is opposite of what you might want. After all, monomer purity is best over on the right-hand side, while step yield is best on the left-hand side. However, if you look at the spread of these values, you see that the purity across this entire range varies only by about 0.5%, which isn't very much. While the step yield varies much more going from less than 50% to greater than 85%. Accordingly, taking the point at a pH of 6.5 does give you the best step yield with only a minor sacrifice in monomer purity. When this experiment was run at pH 6.5, albeit at a fairly modest loading for flow-through, you see that the peak for Nuvia aPrime 4A rose and fell quite sharply. With a very small strip peak, therefore, at the end of the step. Competitors resin by comparison showed a significant amount of tailing. When the quality outputs from the experiments on the previous slide were examined, what you see is that the monomer content from the experiments were all about the same at about 99.3%, give or take. And in addition, the host-cell protein and DNA clearance was the same. However, the recovery on Nuvia aPrime 4A was significantly higher, about 94% compared to significantly lower levels with the other resins. Now I'm going to turn our attention to using Nuvia aPrime 4A bind-elute mode. What I'm showing here is a third-party case study with an existing process of protein A, ion exchange and HIC compared to a 2-step process using just protein A and Nuvia aPrime 4A. If you look at the step results for Nuvia aPrime 4A, what we see is a quite reasonable yield of at least 75% and often significantly higher with some clearance of aggregates as well as host-cell proteins. When the cost of these 2 processes was examined, customers found that the overall downstream cost using Nuvia aPrime 4A workflow was significantly reduced compared to the 3-step workflow, driven by a number of factors, including a 50% reduction in process time as well as an increase in process yield. When the quality outputs were examined, host-cell proteins and host-cell DNA were well within the required limits. In addition, the customer found that there was about 5.5 log reduction in viruses. This then led to the replacement of these 2 steps, ion exchange and HIC with Nuvia aPrime 4A. I want to talk specifically for a moment about purification of slightly acidic mAb with a pI of about 6.9 in blind-elute mode. What this DoE analysis shows is that there's efficient binding in the presence of modest amounts of sodium chloride. What's not shown on this slide is that the monomer was eluted at a fairly mildly acidic pH. DoE conditions were then transferred to small-scale column formats with binding and wash at pH 7.8, followed by elution at pH 6 and stripping of the column at pH 4. Analysis of the load fraction indicated that the material contained about 93% monomer, about 6.5% dimer and a small amount of tetramer. When the eluate was analyzed, no dimer or tetramer could be detected. And hence, the eluate concentration was approximately 100%. I should point out 2 additional things not shown on this slide. The first is that the DBC of Nuvia aPrime 4A for this antibody was greater than 50 mg/ml at 300 centimeters per hour. In addition, when these conditions were repeated using Capto adhere, at pH 6, there was no monomer eluted, it all came off in the strip. And even when the pH was dropped to 5 for the elution, there was still no monomer coming out in the elution. Everything came out of the strip, and hence, there was no purification achieved. I want to touch briefly on the ability of certain mobile phase modifiers to tune the selectivity of mixed-mode resins. Here I've shown 5 of the more common ones. In addition, the interactions with, say, modulate as well as interaction either increasing or decreasing. One interesting side note shown in this slide. Many people have used propylene glycol eluates in chromatography. However, what we found was that it enhanced the binding of human serum albumin with Nuvia aPrime 4A. We believe that this is because propylene glycol is enhancing the hydration on the HSA surface, preventing hydrophobic interactions between HSA and the resin and therefore allowing better presentation for charge interactions of the various charge site groups on the protein. Now I'd like to turn to another mixed-mode resin, ceramic hydroxyapatite. Ceramic hydroxyapatite or CHT is a mineral. It's composed of a particular form of calcium phosphate, and I've shown the composition at the top of the slide. It's also a mixed-mode media and has 2 kinds of interactions. The left-hand panel shows that positively charged groups saw the services of proteins or other biomolecules, interacting in anion exchange fashion with a negatively charged phosphate groups on the service of the resin. This is classical cation exchange and can be associated with sodium chloride or with a buffering salt, such as phosphate. And this interaction could be weakened or removed by increasing the pH. The center panel shows that the calcium groups on the surface of CHT can act as a metal affinity site for clusters of negatively charged groups on the surface of proteins or other biomolecules. This calcium chelation is modulated by, again, classical polydentate coordination. It's much stronger than ionic interactions and cannot be associated even at very high salt concentrations. In this case, desorption from these sites requires phosphate as a competitive inhibitor. Finally on the right, I just want to highlight one other sort of interaction with highly negatively charged species, such as DNA or endotoxin or retroviruses. Here, the binding of these biomolecules to the surface of CHT is mediated almost exclusively by calcium chelation. Again, it's much stronger than ion exchange. And in fact, adding sodium chloride will lead to increased binding of these species because there's some shielding of the negatively charged phosphates, which will tend to repel the negatively charged, in this case hydroxyl groups on the surface of these species. Again, this kind of interaction requires phosphate to dissociate. In fact, it requires very high concentrations of phosphate, typically much higher than where the biomolecule of interest would come off. This table shows the observed log clearances of a variety of impurities typically found in process streams. I want to point out one thing, in particular, on this slide, and that is that DNA, endotoxin and retroviruses, such as XMuLV, almost always show these very high log clearances. This is because these species, as I indicated on the previous slide, bind very tightly with CHC and therefore, really only come off when you strip the column long after your target protein or target biomolecule of interest has been eluted. This slide shows the ability of CHT to elute high levels of aggregate. In the left-hand panel, we have an HPLC SEC of an aggregate from our UNOsphere SUPra protein A resin. And what you can see is that the total level of high molecular weight species as well as some low molecular weight species was about 40%. Right-hand panel shows that after elution of CHT, the level of aggregates was below detection, in this case, below 1%. Here we have the same process with a different antibody. In this case, UNOsphere SUPra eluate had dimer levels at about 1.4% and aggregate levels at about 0.6%. After CHT, you see that the levels of these species was below the level of the capacity of the system, which in this case was about 0.03% for each one. I want to turn for a couple of minutes to the use of CHT in the vaccine market. Vaccine market size is expected to be almost $80 billion by 2024, with a pretty hefty annual growth rate of about 10%. A comparison of the gene therapy market size is worth about $6 billion by 2026 and has an even larger annual growth rate of above 30%. Right now, there are almost 250 studies ongoing in the U.S. alone. And what I've shown below this are the various areas in which gene therapy is currently being used. And as we all know, there are a variety of gene therapy vectors that can be used. So what I'm showing here is the purification of influenza virus on 2 of our CHT Media, CHT XT on the left and CHT Type II on the right. Infectivity is shown in Orange, HA titers in red, A280 is in blue and A260 is in green. And what you can see is that the virus was well separated from the bulk of the A280 and A260 absorbing material. When the eluate pools were analyzed, what we found was that the final yield of infectious buyers in both cases was greater than 75%, and the final purity of the pools was greater than 90%. This slide shows the uses CHT drawn from publicly available data. It's known that CHT is using the purification of Gardasil or Cervarix depending upon what part of the world you're in. It's used as a polishing step for all 4 of the monomers and reduces HCP and nucleic acid. On the right, I've shown use of CHT for the purification of an HPV L1 protein. I want to point out couple of things. The first step actually uses another one of our resins, Macro-Prep High S, which is cation exchange resin. And of course, it's eluted in fairly high salt. The second step is CHT. And what they found is that the load did not need to be diluted. It had about 1 molar salt. This species is acidic. And you may remember I said that acidic species binds metal chelation which is not affected by high concentrations of salt. So what they ended up doing was eluting this with a gradient up to 1.5 molar salt in a very high phosphate concentration. This very high concentration of salt in addition to the high phosphate concentration was enough to elute the HPV protein from the resin. Step recovery of CHT off of CHT was about 80% with a purity of 98%. I want to touch on 2 other examples of CHT being used for VLP purification. The top part of the slide shows the E protein of West Nile Virus conjugated to some VLPs made from bacteriophage Qbeta. It's captured in a low amount of sodium phosphate and a modest amount of sodium chloride and then eluted with higher concentrations of both sodium phosphate and sodium chloride. In this case, because the VLPs were made from bacterial sources, this step was used for depletion of endotoxin. You remember that I said that endotoxin was one of the other species that binds very tightly the CHT and hence the media is capable of very good removal. Second example is the plant-produced malaria vaccine. This is Pfs25 from P. falciparum fused to the Alfalfa mosaic virus coat protein, and this is expressed in tobacco plants. Again the VLP fusions were loaded in fairly high concentrations of sodium phosphate at a neutral pH and eluted with even higher concentrations. In this case, it was followed by SEC and what was found was final product was 85% pure as well as very highly immunogenic, which is crucial for economic production of these therapeutics. Now I'd like to talk for a few minutes about our large pore anion exchange resin Nuvia HP-Q. Nuvia HP-Q is designed for large molecule purification. It's a strong anion exchange resin containing a quaternary of mean functionality. It has an optimized particle size of about 50 microns for resolution. It has optimized pore size and grafting ligand technology for purifying large amounts of large biomolecules. As a result of this optimization, the mass transfer kinetics are very fast. The rigid bead prevents collapse even at high flow rates of 300 centimeters an hour, in spite of the large pores. And it has very low nonspecific binding. This is a representation of what the inside of the Nuvia HP-Q bead looks like. The pores are covered with surface extenders that have been optimized for length and density, which contain the quaternary ammonium anion exchange groups. Nuvia HP-Q can be used for purification of a large variety of large molecules. It can be used for the fractionation of some plasma proteins that I've indicated here, viruses, PEGylated proteins, VLPs, some large recombinant proteins, such as factor VIII and von Willebrand factor as well as plasmid DNA. We compared the binding capacity of Nuvia HP-Q for thyroglobulin, which is a protein of about 700 kilodaltons and a diameter of 17 nanometers with several other resins at 2 different flow rates. What you see with the blue bars a binding capacity at 150 centimeters an hour and the green bars is the binding capacity at 300 centimeters an hour. At 150 centimeters an hour, the 10% breakthrough for thyroglobulin on Nuvia HP-Q was about 50 milligrams per ml, which was higher than any of the other 3 resins. The same result was found at a higher flow rate of 300 centimeters an hour. Similarly, we looked at the binding capacity of HP-Q and 2 other resins for Fibrinogen, which is a protein that has a smaller molecular mass of thyroglobulin but a much larger size because of a much more extended protein. And again, binding capacity of Nuvia HP-Q was about 25 mg per ml, which is 5x higher at least than the other 2 resins that we studied. I want to talk for a couple of minutes about the purification of IgM on Nuvia HP-Q. Many of you know IgM antibodies are candidates for both diagnostic and therapeutic applications. They are large between 900 and 1,000 kilodaltons and are generally expressed at fairly low levels in cell culture. Their typically low DBC is due to the small pore size of commercially available resins. So here, I'm showing you some experiments that were run by a customer using Nuvia HP-Q at flow-through mode to remove IgM from a plasma process feed stream. And what you can see is that the binding capacity of Nuvia HP-Q for IgM is more than double as that of its 3 nearest competitors. So we designed the 2-step purification workflow for an IgM, capture Nuvia HP-Q from cell culture, followed by pausing on CHT. The top panel shows the nonreducing criterion XT SDS-PAGE gel. And what you can see in Lane 2 is the cell culture supernatant with IgM barely visible at top and a significant amount of process impurities down around 50 kilodalton. Lane 3 is a Nuvia HP-Q flow through, which shows that most of these process impurities flowed through the column and did not bind. Lane 4 is Nuvia HP-Q eluate and Lane 7 is a CHT eluate, and you see the presence of large amounts of IgM in both of these fractions, very low level of other host-cell impurities. Reducing gel shows much the same thing. And the HP-Q eluate is in Lane 4 and the CHT eluate is in Lane 7, where you see now the presence of both the light and heavy chains from IgM. HPLC SEC analysis of -- both of the Nuvia HP-Q eluate shows fairly large monomer peak with a significant amount of low molecular weight impurities, which are primarily serum albumin and transferrin as well as some other species. Analysis of the CHT eluate however shows very, very pure monomer, here's the IgM peak, with very low or almost nondetectable levels of these low molecular weight impurities. When these fractions were analyzed, purity of the HP-Q eluate was about 60% with an 80% recovery and a small amount of DNA removal, but a significant amount, probably greater than 10 logs of host-cell protein removal. CHT II -- CHT Type II eluate by contrast was greater than 99% purity, about an 80% recovery. Now DNA was not detectable. You recall that DNA was one of those species that binds very tight with a CHT and a host-cell protein level that was now 2 orders of magnitude or a bit more lower than the HP-Q eluate, which is pretty much in the reasonable range for therapeutic and certainly for diagnostic. This slide shows the purification of H1N1 virus from cell culture on Nuvia HP-Q. On the left, I've shown the overall process flow up to the purification on HP-Q. The binding capacity of HP-Q for this virus was about 6x 10^11 viral particles for milli resin. Chromatogram shows that these particles alluded at about 590 millimolar sodium chloride much smaller amount coming off in the strip. What you see is that the recovery of viral RNA and hemagglutination were both about equal, indicating that what we've recovered here is active virus rather than inactive virus. So this slide shows the results of some SDS-PAGE and Western Blot analysis of the HP-Q fractions for the previous chromatogram. As you can see on the left hand panel, by SDS-PAGE of the 590 millimolar eluate, clearly shows the HA protein pretty much free of all the other contaminants, which are present in the culture supernatant. Middle panel shows a blot of that gel using an anti HA antibody. And what you can see is the HA protein, again clearly lights up in the 590 millimolar eluate as well as a smaller amount in the 1.5 molar strip. In the 590 millimolar eluate there are also HA1 and HA2 bands, but unfortunately, there are a bit to light to really show up well on this presentation. The right-hand section shows an anti-influenza A Western Blot. And again, what you can see is the presence of the expected proteins NA and MNP proteins in the 590 millimolar eluate. Next slide presents some dynamic light scattering analysis of the material from the workflow that I just presented. What you see on the left is that the average size of the virus particles is about 130 nanometers, which is well within the reported size range for influenza, which varies depending upon a number of factors between 80 and 180 nanometers. On the right is a quantitative analysis. And what you can see is that the material that was monomeric is about 99.8% of the total, indicating that the vast majority of the material was single virions and not aggregates, which is exactly what you're looking for in a purification. The results of the purification are shown on this slide, I want to make a number of points. First of all, on the top panel, what you see is that the HA and viral RNA recovery were about equal, they were about 75%, 80%, which is a terrific recovery from the starting material. But I want to point out that nuclease treatment was not required for both efficient capture and purification. These experiments were performed both with and without nuclease, and it didn't seem to make any difference. The HCP and DNA levels were both very low as indicated in the middle columns. On the right, I've shown the log reduction of impurities, and we get about 1.5 log clearance of host-cell protein and about 4 logs clearance of host-cell DNA. Now you see that I've put the impurity levels in blue and orange, and if you look down below, what you see are those same impurity levels as reported from a variety of previously tried workflows. And you see that this single step purification is significantly better than any of the previously published processes. So let me just briefly summarize what I've talked about over the last 15 or 20 minutes. Nuvia aPrime 4A resin is a mixed mode anion exchange hydrophobic resin with optimized ligand density and hydrophobicity of the ligand. I've shown that there's effective separation under pretty gentle conditions of monoclonal antibodies from a variety of impurities. You can operate this resin in either flow-through or bind-elute mode and the method development is straightforward. You can take what you already know about ion exchange and HIC developments and simply combine them. CHT media is used as a major purification step for a number of commercial subunit vaccines. It has demonstrated utility in the purification of a variety of live and attenuated viruses, dengue, influenza and a host of others. It has a long history of purification of conjugate vaccines. And it effectively removes both product- and process-related purities. And finally, Nuvia HP-Q resin, we've designed for the purification of large biomolecules, and I've shown you some very exciting results on the purification of influenza A with this resin. So in addition to products itself, Bio-Rad offers a variety of process chromatography resources. We have a global network of experts that will help you do methods development. We can, if you want, assist you in optimizing steps and/or the manipulations between steps. We provide continued support all the way from lab scale up through scale-up, process transfer and regulatory filings. And at the bottom of this slide, I've put 2 ways that you could contact us either to request samples or to contact your process specialists for further health or information. So with that, I want to thank you very much for the attention you paid to this presentation, and we will now throw the floor open for any questions that you might have at the moment. I should just conclude by saying that if you ever have any questions about any of our products, please let us know because that's a big part of our job, is helping our customers use our products. If you don't call us, we can't help. So with that, I'll take questions.

Thank you, Mark, for an excellent presentation. We've received a few questions already, but we'll give the rest of you a moment to enter your questions in the Q&A box on the left of the slides. Before we begin the Q&A, I'll run through some brief announcements. First, I'd like to thank Bio-Rad Laboratories for sponsoring this digital week. Next, I'd like to quickly draw your attention to our face-to-face BioProcess International events visiting Santa Clara in August, Boston in September and Milan in October, this year. Additionally, BioProcess International Europe will be delivered as a 100% virtual conference and exhibition on July 13 to 17, 2020. Also be sure to check out the resources list to the right of your screen where you can download a few featured white papers. Now back to Mark to begin the Q&A. Mark, so we had a number of questions come in. The first one is what are the strategies and technologies to separate empty and full viral vector?

Right. So in other talks, I've talked about some of our other resins, notably Nuvia cPrime, which is a cation exchange hydrophobic, multimodal resin. And during elution, we actually see a split peak. We haven't looked at that in great detail, but we think that, that could very well be empty versus full capsids. So if you want, you should talk to your local process specialist, so they could give you more information on that. But occasionally on that resin as well as some other resins, we see split peaks, that's probably an indication that we are separating empty from full.

Perfect. And for our pH sensitive asset sensitive mAb, what resin would be a good solution in the capture step from cell supernatant? Would you need buffer exchange to low salt buffer?

So it depends somewhat on the resin, what I would suggest trying with that is probably an anion exchange HIC resin, for example, our Nuvia aPrime 4A, which is typically operated at PH values above -- at or above neutrality. And so you could work directly with that. Their salt -- Nuvia aPrime 4A is salt tolerant. So you probably would not need to do a buffer exchange. You may have to do some minor buffer dilution, but maybe not even that. Or for that matter before we move on to the next question, you can actually use CHT. CHT has operated from pH 6.5 up and that could be a step for capture, but we should talk about that further off-line.

Perfect. And can you remove a purification step when using one of these mixed mode resins?

We have seen that. We know that with several of our mixed mode resins that our end users have said that they can combine 2 steps into 1 using the mixed mode resin. And that obviously has benefits in terms of cost savings, time saving, yield and so on. So the answer is yes, sometimes.

Perfect. And has CHT been using viral vector purification?

CHT has been used a lot in viral vector purification. Depending on what you mean by the word vector. So if you're talking about the actual viruses, yes, we have a lot of resources on purification of viruses on CHT. It works really, really well. If you were talking about the plasmid used to derive any of the components, then yes, CHT has also been used for plasmid purification.

Brilliant. And what is the difference between virus purification and viral clearance?

So they are opposite sorts of things, it depends upon what you're trying to do if you're actually purifying a viral vector for use as a therapeutic in and of itself, then that is virus purification. If you have anything derived from mammalian fermentations, then there are both endogenous viruses that are present in the fermentation and sometimes you can get viral contamination. And in those cases, you want to remove the viruses, that's viral clearance. And I could give an entire talk on viral clearance. But suffice it to say that there are guidelines that all the regulatory agencies worldwide have published in terms of how many logs of clearance you need to show for a virus removal for a therapeutic to be approved.

It sounds like a talk for another day. And the next question is, can Nuvia aPrime 4A replace protein A as the capture step?

Yes, we have a number of workflows, in which Nuvia aPrime 4A has replaced protein A. So it's typically a 3-step workflow with aPrime 4A followed by perhaps CHT directly or CHT as a polishing step with a number of different options for the intermediate second step.

Sorry, Mark. The next question is, what resin can be used for purification and separation of modified proteins like scFc?

We had an experience with a number of our resins, purifying modified protein. CHT is a good choice. Also, either of our mixed mode anion exchange resins, Nuvia aPrime, Nuvia cPrime has actually proven to be pretty useful for that.

And is CHT preferred over other purification methods for virus purification?

That is a good question. In my opinion, I think the answer is yes, because some of the other methods, for example, ultra certification and so on, don't scale well particularly with all due apologies to the strategic manufacturers out there. CHT has been used for probably purify, at this point, a dozen different viruses, and the yields typically are above 80%. And the purification seems quite good. So I think it's probably the best resin out there for virus purification.

And how does clean-in in place and durability compare with mixed mode media and traditional media?

Is the question meaning for CHT or for other resins? Well, assuming it's for CHT, CHT could be sanitized in up to -- we've gone as high as 2 normal sodium hydroxide, which is well above where most people use anyway. So it's quite stable to any CIP, which has a condition that you want to use. And if you need to use detergents or urea or guanidine, it's also very stable to that as well -- to those as well, sorry.

Excellent. And when should I use an ion exchange resin over CHT for vaccine development?

So I typically prefer ion exchange resins as the first step, especially with some of the newer generation resins like our Nuvia S and Nuvia Q because their capacity is the highest for -- really for almost any resin out there. And since the major contaminant in feed streams is water, you want to try and de-water as much as you can in the first step. So I would -- for those reasons, I would probably pick an ion exchange resin as the first step.

And if my molecule works best at mild conditions, what resin should I work with first?

Well, if it's both stable under mild conditions, it doesn't really matter whether it's first, second or third, you're going to want to pick resins where both binding and elution could be performed at a neutral pH. And so I would say -- let me think about that for a second. So CHT is a good resin because that is designed to operate at neutral conditions and most people are using CHT between 6.5 and, let's say, 8. Either of our mixed mode resins, Nuvia aPrime 4A or Nuvia cPrime can be operated at or near neutrality, so that would be another place to start. We have 2 HIC resins, which I haven't talked about at all in this talk, obviously, and those can be operated at neutral pH so those would be a starting place. And if you want further information, talk to your process specialists or contact me. And I guess, maybe I should take this opportunity to say, if you ever have any questions please, please, please contact us so that we can help. Because if you don't call, we can't help, and this is my job. So please let us know. Okay. That's it.

Thank you, Mark. And what resin should I begin with for purifying a plant-based biologic?

Yes, plant-based biologics tend to have a dirtier feed stream just because of the way they have to macerate the leaves to get stuff out. So I would probably start again with an ion exchange resin, typically, the volumes tend to be a bit larger at the beginning and ion exchange resins, probably more than any other resin sometime stand up to harsher CIP conditions but probably start with ion exchange, pure ion exchange.

Excellent. And how Nuvia HP-Q being used for plasmid purification?

So Nuvia HP-Q works really well for plasmids. I have a whole separate talk on that, and we have a nice workflow for Nuvia HP-Q with plasmid. As pore size is designed for large molecules, and it has -- we've tested it against a number of other resins, and it has at least doubled or tripled the binding capacity of other resins for plasmid, again because its pore size is so large.

Excellent. And do you have these resins in prepacked columns?

Yes. These resins are all available prepack columns. We call them our foresight line, and they are available as prepack 1 in 5 milli columns or in 96 well plates if you want to go that route. So just contact your process specialists and they can get them to you.

And do you have any recommendations for purifying large RNA molecules from their fragments?

We have not worked extensively with RNA. But I would say Nuvia HP-Q is a good place to start as is CHT because CHT as at the -- history with CHT binding and separate nucleic acid is very long. That goes back probably 40 years at this point. That would be a good place to start and also Nuvia Q, again, because of its large pore size. Just remember, if you're working with RNA, you have to be careful not to let any nucleases in there. So you're going to want to sanitize everything pretty hard ahead of time.

And does Nuvia aPrime have the same matrix composition as the competitors you mentioned?

No. Our Nuvia resins are all derived from -- and that's also a separate talk in and of itself. Nuvia is -- the Nuvia resins are derived from a UNOsphere backbone. UNOsphere has been in the market now for a couple of decades, it's extraordinarily hydrophilic. So you don't have to worry about any protein binding and then denaturing because of a maybe a slightly hydrophobic surface. And there have been some papers published on that, that I can direct people to if they're interested.

Excellent. And are there any watchouts with regards to sanitization with CHT?

No. But again, you can sanitize CHT easily at one normal sodium hydroxide or 2 normal, if you want. And as I said, it's impervious to other cleaning reagents that people sometimes they add. So I don't think there are any watchouts.

Excellent. And there is a whole family of CHT resins, including some newer members. Could you briefly explain what each type is most applicable for in terms of biomolecules?

So that answer is going to take a little too long, I think, to address here. If you look at some of our published literature, we have a flow chart which describes how to prioritize, which CHT resin to try first based upon the size and the PI of your protein. So I would direct you there rather than me trying to answer it here.

No problem at all then, Mark. And then do you have any suggestions for addressing the current pandemic for biologic development and purification?

So it depends upon -- well, actually, it doesn't depend on what you're trying to do. I mean, if you're trying to address the current pandemic, we do have some publications on purification of another coronavirus MHV, and if you e-mail us or call us, we can direct you to that publication. That is -- again, that's not the same as COVID-19, but it is another coronavirus. As a general statement, if you're trying to purify viruses or vaccines, again, I think any of the resins that I've mentioned in this talk are a reasonable place to start.

Excellent. And just one final question here is, does how -- how does CHT XT compared to the original CHT when scaling up?

I'm not sure what they mean by comparison in terms of what sense. But as a general statement, it scales up exactly the same way. It is also a 40 micron bead. It is still hydroxyapatite. So it should scale the same way, and while I'm thinking about it, anyone who tells you that CHT cannot be scaled up really needs to perhaps contact me for some information. A couple of summers ago, I packed a 1.8 meter column with CHT and it went absolutely fine. It took us about 45 minutes. So really, that is easy. And I have a whole separate talk on packing CHT, which I'm happy to give to anyone who would like me to talk to them.

Thank you very much, Mark there. Well, that's all the time we have for questions today. Thank you again, Mark, for a great session.

My pleasure.

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