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Why Salt Active Nucleases Are Vital to Biopharmaceutical Manufacturing



The following is a transcript of the Q&A video below with Jennifer Hamilton, Ph.D (questions in bold) and Jørn Henrikson, Ph.D (answers).


"And it's surprising how many scientists working with Benzonase nuclease, try to adapt their process to that nuclease. Instead of picking a nuclease that is optimally performing in the conditions that is relevant for the process."

[music intro ♬]

Hi everyone. I'm Jennifer Hamilton with ArcticZymes and today we're discussing a paradox encountered when clearing DNA from viral vector manufacturing. The higher the salt concentration, the better the conditions for optimal clearance. Yet it's this very increase in salt that paradoxically impairs the function of Benzonase, the most commonly used enzyme for DNA digestion.

Joining us as Jørn Henrikson Sr Application Manager at ArcticZymes who will help us understand how to reconcile this contradiction to enhance de-aggregation without compromising enzyme efficacy.

Hello. Nice to be here.

Before we dive into it, I have a feeling we should first talk about:

Why salt is a kind of untapped potential for biomanufacturing?

You can't talk about that without introducing the concept of charge. When you are dealing with enzymes and proteins it's all about charge pluses and minuses. And every protein has a PI that decides which charge it has. When it comes to biomanufacturing, what you are doing is you're using a host cell to produce something, a biological product at high yield.

And the next step is removing all the contaminants so that you have one single product, that protein of interest or the viral vector that you are using as a therapeutic or in many other types of things. So biomanufacturing that is in essence to make a host cell produce a protein of interest.

Then the next step is to isolate that protein of interest to a single, pure high yield product that you can use in therapeutics or other types of workflows. And this separation. It's very based on charge.

Every protein has a PI that decides which charge it has in the formulation that they're using. So, if you have a high PI you have a positive charge at neutral. If you have a low PI, it is negatively charged. And these plusses and minuses can attract each other. So, if you are making this protein of interest and you're going to purify it, it is an advantage if you can separate everything.

What salt does is isolate the charges that you have on the surface of these proteins so you can reduce the aggregation or attraction that these contaminants and your protein interest might have towards each other. By increasing the salt, you disperse everything into smaller, isolated components, and it's easier to remove the contaminants and get your pure product in the end.

Can you briefly explain the critical role nucleases like ArcticZymes nucleases, and Benzonase play in the process of clearing DNA from viral vectors?

Host cell DNA is one of the contaminants that you want to remove when you are purifying a protein of interest or a viral vector. And host cell DNA is chromatin that is built up of many different components that has a various attraction profile. You have histones that is positively charged. You have DNA that is negatively charged, and the histones also have polar and non-Polar, areas of them that make them attached to different proteins, columns, equipment, and also of course the viral vector that you are trying to purify. So, by using a nuclease, you can chop up the DNA into smaller pieces so that they are easier to get rid of in the downstream processing.

And what challenges does the increased salt concentration present for the function of nucleases like Benzonase and DNA clearance?

Then we have to return back to this concept of charge. In order for the DNA to bind into the active site of a nuclease, you are dependent on charges. So you need a positive charge inside active site that fits to the negative charge of the DNA. And this is what attracts and keeps affinity of the enzyme to this DNA and allows it to do its function.

When you increase the salt. You isolate these charges, which again reduces affinity of the DNA to its active site of the nuclease. And you then start losing gradually more and more activity as you increase salt because Benzonase is an enzyme that is working best in the absence of salt. That's when it had its best attraction to this DNA.

How does this paradox affect the overall biomanufacturing process in terms of things like purity or cost or workflow efficiency?

When you add salt, you de-aggregate your protein of interest from contaminants. And at the same time, you are removing the affinity of the nuclease to its target.

Because even at its physiological salt conditions, Benzonase is losing significant activity, because of this. And if you go to the de aggregating levels, you want to really de aggregate things, it doesn't really work at all. Especially at the de-aggregating levels, you are forced to make some compromises. To maintain nuclease activity, you have to add less salt than you would like to.

Use salt to reduce the attraction between your protein of interest and the contaminants. And at the same time, you reduce the attraction between the enzyme and its target, the DNA.

So you are forced to make some compromises. And in order to get that, especially if you're using de aggregating levels of salt, like half a molar and above.

You need to either use more nuclease than you essentially need because you need to compensate for that loss of activity. And that can be significantly more expensive than it needs to be.

Otherwise, you need to shock-treat with salt to get a de-aggregating effect, and then remove the salt again, just to get the nuclease to work. And that's also something that just add costs to the entire process.

Could you introduce the concept of salt active nuclease and how they differ from conventional nucleases like Benzonase, when using a high salt condition?

Yeah, sure. ArcticZymes has been working with these salt active nucleases for more than a decade. And what we found is that by looking for nucleases that are pre-charged, that means that they are dependent on some salt in order to reach the optimum charge level in order to have that correct.

Affinity to its negatively charged DNA, to its positively charged active sites. These nucleases, they actually work less in absence of salt because the attraction to the DNA is too tight, it is less movable and it's not able to do its job properly. It needs some salt in order to work. And that allows you to find nucleases that is working in the conditions where they are needed.

In bioprocessing, that means at physiological salt or de-aggregating levels of salt, depending on what kind of process you are using. We have actually found two nucleases that allows you to do this. if you look at this slide, you can see how activity decreases as the nuclease isolated from Serratia marcescens.

That's Benzonase. It's Denarase. It is a lot of nucleases is out there and they all from the same organism. And you can see that the more salt you add, the less activity it has, as we described earlier. And with M-SAN HQ we have found an enzyme that is working optimally. It's optimally charged and ready for its digestion of DNA around physiological salt, corresponding to around 150 millimolar. And with SAN HQ, you can get up to this de aggregating levels of salt you might use if you're making where you go with 500 millimolar salt or above. This is an enzyme that need even more salt in order to reach that charge level where it works optimally.

It allows you to tailor-make enzymes to your particular conditions. And it's surprising how many scientists working with Benzonase nuclease, and then they try to adapt their process to that nuclease. Instead of picking a nuclease that is optimally performing in the conditions that is relevant for the process. it brings a whole new set of tools and simplifies things. And since they're more efficient at these salt levels, you can also use less nuclease and thereby save costs.


Thank you, Jørn. This has been a very informative discussion and thanks to all of you for watching, and we hope that this has helped you to understand how you can, solve the paradox encountered when clearing DNA from viral vector manufacturing. Have a great day.

Thank you. It was a pleasure.

And are there any examples or case studies that you can share where salt active nucleases have successfully been used in Biomanufacturing?

There are some case studies, but I think that is a topic for the next discussion.

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Why Salt Active Nucleases Are Vital to Biopharmaceutical Manufacturing

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