Transcript of: Vaccine Development – Pitfalls and Strategies

Presented by Dr. Jacobson on 23 June 2020  Watch the Webinar 

So, thank you so much for being here, Dr. Jacobson. I’m going to turn the presentation over to you now.

Very good. Thanks so much, Karen. I’m excited to be here. Thank you so much University Lab Partners for giving me this opportunity. So, today I’m going to talk a little bit about the pitfalls, strategies and opportunities in vaccine manufacturing. I’ve entitled it walking softly through the minefield, since there are a lot of pitfalls to avoid along this path towards opportunities.

At the beginning of the talk, let me first just ask a question. I wanted to get an idea just for the audience what its makeup is. Samuel, if you could put up our first poll, I’d like you to have an idea from we’re primarily made up of bio entrepreneurs, people with just general interests in the field, active vaccine researcher/scientist, or even anti-vaccine activists, I know that has become very big lately. So, if you could do that and submit, I’d be very interested. This will kind of help me to fine tune the talk as best as I can on the fly and be able to maybe address some of your concerns or just tailor the talk in response to the actual makeup of our audience.

And, Samuel, I want to ask one more question, one more poll to the audience before we get started also. I wanted to get an idea for how many folks, how long people think is a reasonable timeframe to develop a vaccine to take it from start from its very inception and actually take it into the clinic. If you can put that slide up, Samuel, that’d be great. Do you think it’s reasonable for six months? Do you think it’s a year? Do you think it’s two years? Do you think it’s five years or even 10 years or more?

There are a lot of different opinions out there. A lot of folks… I have a good friend, he thinks 18 months is absolutely absurd that it takes… should be much shorter than that. So, I’d be very interested to know what the audience thinks. Interesting. So most folks have about two years. So, let’s see what’s sort of like in popular media, what public figures, what politicians tend to think on the subject.

Before I get too involved, I just want to read a disclaimer. The work included in this webinar is based on peer-reviewed publications, SEC filings, interviews, corporate press releases, news articles, and other forms of publicly-available information. Any investment decisions should not be based solely on what is presented during the course of this webinar.

This webinar may contain forward-looking statements that involve known and unknown risks and uncertainties, and none of the statements made are on behalf of a publicly-traded or private entity. The views expressed in this webinar do not necessarily reflect the views of ScienceDocs or its management. The content of this webinar is the exclusive property of ScienceDocs and is not to be shared or disseminated without the express-written consent of ScienceDocs.

So, now that we’ve got that basic piece of housework underway, let’s have a look. So you’re hearing a lot of different things in the media and a lot of different things from high out, and these are very, very different perspectives. On the left, that man needs no introduction. That’s our president. And look what he said. I’ve heard very quick numbers, a matter of months, and I’ve heard pretty much a year would be an outside number. So I think that’s not bad at all, not a bad range. But if you’re talking about three to four months, in a couple of cases, and a year in other cases, wouldn’t you say, doctor, would that be about right?

So as of June 5th, 2020, is there are 2 million corona vaccine doses that are “ready to go.” Once scientists figure whether it’s safe and effective. Then we’ve got Dr. Fauci, right? So he’s saying if COVID is like other coronaviruses, it isn’t likely to be a long duration of immunity. He’s talking about the actual infection itself. And he says the US should have a couple of hundred million doses of COVID-19 vaccine by the start of 2021. And he said that start of 2021 and I’m sorry. That’s a typo. It’s on June 3rd 2020, he said that. And then there’s a link so you can go see that yourself.

The reality is that, or a different opinion I should say, this comes from Sanofi Pasteur during my time as a contractor there in their seminar and understanding our science is that most vaccines never make it to the market. Some make it but they don’t reach the original product goals. A few make it and they perform as designed. And this can take 10 years or more to complete. So that’s quite a contrast to what the president and what Dr. Fauci are saying.

So, let’s try to get a handle on these different timelines and understand why it might take so long and why thinking something you’re going to have a couple million doses just sort of “ready to go,” is really not realistic at all. So, first, let me talk about the challenges to vaccine development. Why it can have such a long timeline? And the path to developing a successful vaccine, it’s a heavy load to lift. I got this little picture from one of the world’s strongest men competitions in case anybody’s curious. So, what’s going on here? What are the obstacles that we have to surpass?

Most vaccines require boosters, so that means you can’t just get a single shot, one and done, that just doesn’t really exist. There are missed opportunities to vaccinate. Sometimes certain people will oppose vaccinations for various reasons. Other times they don’t get it early enough. Mass production, huge. I mean, you can’t get something to the patients if you can’t make it. It’s that simple. Everything has to be made and it has to be made at large scale. And it has to be made safely. It has to be made reproducibly. And it has to be made under GMP conventions.

Not so easy to do. It’s expensive. Some folks have completely left the vaccine game because it is expensive and the rewards aren’t so great. Another challenge, there’s no centralized database of vaccination status. So it’s very hard to tell who’s been vaccinated, who’s not, how to kind of keep track and manage that. I’ve got a question on the subject for folks a little bit later in the talk. Possibly adverse reactions, those exist. But, fortunately, they’re usually on the rare side. And then less than 100% efficacy.

So, even if you get a vaccine, you’re not 100% protected. You can’t just go, “Ooh, I’m safe. I’m safe for the season.” Let’s consider flu. It’s a well-vetted vaccine, been around for a long time. But you’re only batting 50 to 60% during… it really depends on the season, but it’s nowhere as close to 100%. It works, it’s better than nothing. If you do get the flu and you had the vaccine, you’re way less likely to suffer a serious complication. So, in that respect, it’s good but it’s certainly not at 100%.

And then, the vaccine… this is all borne out. And on my final bullet point here, the vaccines licensed for use in the United States. So, I would encourage all the attendees, I believe this is a live link, it’s not just cut it out, put it in your browser. Go and have a look. For me, this list was shockingly short. I thought it would be quite a bit more products that are licensed for use in the United States but it’s really very, very few. And I should have mention that today’s talk, I’m going to concentrate on vaccines directed toward viruses. I’m going to leave the ones towards bacterial infections alone since the talk will just get too broad.

Just sort of a little qualifier but it really is striking to see just how few of these we have that are approved. And I should add, probably there’s another obstacle there is we live today in a very litigious society. For something like measles, right, a very well-vetted vaccine, is an attenuated virus. To actually prove that it doesn’t cause any sort of disease… if somebody actually started out de novo trying to work with the measles vaccine because of the implications, because of legal liabilities, probably it wouldn’t even be attempted.

So you got some real powerful forces that are opposing vaccine development that you have to be aware of. That being said, it’s certainly a worthwhile endeavor because when it works, it’s a very, very powerful tool to fight disease. So, getting back to our timeframe, understanding why it takes so long if you choose to go this route, you have to understand there’s six phases of vaccine development. Let me just tell you a little bit how it’s sliced, right? You start at the very beginning. That’s the preclinical before you go to clinic.

You’re going to work with animals in vitro systems. You’re going to look at toxicity, immunogenicity, the potential maybe the combined different antigens or viruses for a combination of vaccine, stability data and potency, correlates of protection, right? You’ll look and see antibody titers in animals if they actually correlate protection when exposed or challenged by the virus. You’ll begin developing your manufacturing strategy, right? And then that will all culminate in the filing of an Investigational New Drug Filing, right? An IND, right?

So then once you’re on your IND, you’re going to make the jump to Phase I, right? And that’s typically quite small, right? For one of these studies, you’ll have 10 to 40 human volunteers, again, we’re talking about a vaccine trial. It will go weeks to months, you’re primarily looking at safety, right? And you’re also looking at your examined dose size and the route of administration. You’re going to look a little bit at mode of action and immune response. And also you’ll get to understand the characterization of your vaccine a little better.

If Phase I proceeded properly, everything’s safe, then you’re able to go to Phase II, and that’s a little bit bigger in scale, all right? It increases in scale and increases in complexity. Now, you go from 10 to 40 human volunteers, 10 to 40 healthy human volunteers usually, to 50 to 500 volunteers. This will range from months to years. And you’ll look at rates and severity of common adverse events. You look at the immune response of target groups.

If that goes well, valuing to Phase III, right? That can go to two to five years. So now you’ve got several thousand, right, anywhere from 20,000 to 50,000 volunteers. I mean, it’s pretty… now you get a really large scale. Placebo controlled, you look at safety, efficacy, establishment of final manufacturing procedures. Typically, you’re going to have validation lots or consistency lots. You’re going to make three lots around your manufacturing scale, you make them consecutively, and you’re going to show that this is a reproducible process, that all of your critical quality attributes, your acceptance criteria are consistently met.

And if that goes well, then you get to a BLA filing and that might take one to two years in that process, right, that’s why I made a separate number, number five, the BLA approval, the back and forth, the filing. And then even then, once you get approval, FDA approval, you’re still not done because now you have to have post market surveillance. You’re monitoring after licensure, safety, efficacy, new indications. So it’s sort of a never ending process. So you can see just how long this takes. And that’s just to get you to your first vaccine. And you’ll see in my final slide, I’ll talk a little bit how the first vaccines typically not the best, it’s an iterative process.

So even once you get something that’s approved in monitoring at the post market surveillance level, you still may improve upon it years. So, it can take years and not decades. So, it’s really getting people’s hopes up, raising hopes unnecessarily, giving people false expectations. They think that they can just have something that’s any good and most importantly is safe in just a matter of months.

All right. So, I mentioned the previous thing about combination vaccines. I figured I’d like to talk to them a little bit, we’ve got a bunch of bio entrepreneurs in the audience. So, this is sort of a useful tool, a useful thing to develop. You sort of get more bang for your buck if you can do it. What’s the most typical combination vaccine? Probably the MMR vaccine, the measles, mumps, rubella, typically that young kids get, that’s probably the best example I can think of off the top of my head.

So, like I said, you’re getting several for the price of one, right? You’re preventing multiple diseases or you prevent one disease caused by different strains or serotypes in the same organism. You offer the benefit, right, fewer injections. But one of the challenges now is stability which I’ll talk about later in the talk as well, is it becomes a little bit more complex. So, each component in your mixture has got to be stable. And each component of the mixture, you’ve got to understand its potency. So you’re going to have to understand the potency of each virus or each antigen by in and of itself and then in tandem with the other mates in this combination, this combinatorial regime.

So, again, you’re going to still have to do your validation lots, the component lots, right, if that will represent three, preferably consecutive, production lots. The adverse events, right, of your combination vaccine. They got to be less than or equal to the adverse events seen of each individual component. The immune response at protective levels, right? It’s got to be, again, in tandem with these other components in the mixture, they have to be at least at the same level as they were individually. So each bulk component is got to be tested for identity, sterility, and purity.

So, a little more complicated. But if it works, you have a very powerful tool in your arsenal. And I should say, you can read more about this if you’re so inclined, right? There’s a guidance, FDA guidance, the Guidance for Industry for the Evaluation of Combination Vaccines for Preventable Diseases: Production, Testing, and Clinical Studies. Okay. So, that takes us to our next slide.

So, you got to understand there are multiple types of viral vaccines. And this is the breakdown. This is the sort of how I see the field. I’ve tried to make it as straightforward as possible. And I’ll talk a little bit about the different classifications, the different categories, and an example from each class. So, you’ve got the live, attenuated ones. So this is a virus, right? It’s live. It’s typically attenuated. So, unlike the wild-type virus, it doesn’t really cause a disease at all. If it does, it’s a very, very mild disease. What do we got there, right? We got YF-VAX for yellow fever. You got VARIVAX. Those are two members of this class.

Another one is like you’ve got a vaccine, right? You’ve got a virus, but now it’s either been killed, right? The virus is intact but it’s been inactivated. And you typically might use a reagent like formaldehyde to kill the virus. It’s no longer infectious. If you expose it to cells or an animal or human, it won’t cause an infection. It’s dead, but it is antigenic. So it’s killed, inactivated. Sometimes when you say sub-virion, it’s actually the split. So, not only if you kill the virus, you treat it with some sort of detergent and it breaks up into little pieces. The flu vaccine from Sanofi Pasteur, that’s a wonderful example of a killed vaccine that’s also split, right? And that’s Fluzone, right? And then IPOL is a vaccine for polio.

Then another classification would be the recombinant vaccine. So now you just take, with the magic of genetic engineering, you can get the cDNA of the component of the virus, express it in a recombinant system and then use that artificially expressed protein, that fragment of a virus, and immunize people. The amount of immune response against that fragment and that will be enough to actually fight against exposure to the intact live virus. One good example is SHINGRIX vaccine against shingles, RECOMBIVAX HB. Actually, that’s not really a viral vaccine so I cheated a little bit but you get the picture.

Then it gets a little bit sort of more of efficiency as we kind of go down the spectrum, right? We go from live to now virus-like particles. So these aren’t really viruses at all. What they are is a tremendously cool technique. When you take the different structural proteins of a virus and in a recombinant system, you’re able to express those, have been pieced together so it’s almost like, it can be like a hollow shell of the virus. So it’s not infectious, right?

You don’t have to worry about anybody getting infected, just not a problem at all. And there’s some commonly available vaccines, GARDASIL, CERVARIX, right, those are all virus-like particles, FDA approved. I put up there another one, in case anybody’s curious, Hecolin, that’s not approved in the United States. I put a little footnote there to that effect. But it is approved for use in China and for the prevention of hepatitis E.

Then you have genetically engineered vaccines. So this is where you’d have a virus, right, an unrelated virus, and you take the viral protein from your virus of interest and you’d expressed it in the context of a different virus. So a virus that’s really not terribly infectious, that really won’t cause a lot of harm to a person. But now it harbors just approaching from the virus that you’re trying to vaccinate against. So, that’s a different approach, also very interesting.

Now, as we go down on the spectrum here, we’re getting to more and more chemical approaches. DNA, RNA. To tell you the truth, there’s a lot in the news about this, but there are none that are currently licensed. DNA takes a bunch of different forms. Most typically they’re plasmid, plasmid vaccinations, right? We’ve got a circular DNA containing your gene of interest, RNA also, that it’s quite a bit in the news. We reject the RNA that gets translated into protein and hopefully you mounted immune response against that.

And then finally down at the bottom list is sort of our most synthetic. And that’s the peptide, right? It’s a short stretch of proteins, chemically synthesized. Fields have been around for a long time but none are currently licensed. So, of the list here, I don’t have to tell you that I think a peptide-based vaccine probably is the least likely to come to fruition. And I base that on, there’s 30 years of folks out there doing research on this and truly, as yet to come to fruition. I’ll talk a little bit more about it.

But the only real utility I could see for a peptide vaccine is not so much mounting an immune response, but maybe if it acts as a decoy and it now prevents a virus from docking or disrupts the viral replicative protein, that might be useful during the course of infection, but I don’t think as a prophylactic agent. I could be wrong and I certainly hope that I am because if I am wrong, that means people at the end will benefit. But history is weighing very heavily against this molecular candidate.

Okay. Again, now let’s sort of got going a little bit on peptides. I don’t want to spend too much time on it. I’ll give you a brief overview since it’s out there. People are working on these things. What actually is going on? Well, they’re typically very poor… They have very poor immunogenicity, right? Usually, it would take a defined region that’s associated protection of the viral protein. When you deliver it, right, you got to worry now the peptide is highly susceptible to proteolytic cleavage.

So, you’ve got to get something that’s stable. You probably have some sort of chemical group on it. It’s going to have to be immunized in the context of an adjuvant drill so never mount an immune response to it. A lot of times you’re trying to… humoral immunity means the level of B-cell. You’re trying to get your body to generate antibodies against the foreign agent. Well, they really aren’t just short stretches of proteins, these are proteins with an intricate fold, a three-dimensional conformation, very hard to duplicate that with a peptide.

With regard to cell-based immunity, peptides have a tendency to actually be tolerizing, right? They’re not uploaded properly in the antigen-presenting cells. It they don’t go through the antigen presentation pathway, they conduct outside of the cell to the MHC and actually induce tolerance, which is really the opposite of what you’re trying to do, right, so. Okay. So, yeah.

So then it’s, yeah, then like I said, just of sort of weak, weak immune responses, in general. So, like I said, you’ve got to have either your adjuvants, some other sort of protein that comes with it like KLH or Complete Freund Adjuvant. You see those a lot in mice from urine systems, but it’s really not something that’s likely to ever get FDA approval and be used in the context of a huge trial on humans or that can be made in a large scale and done on people.

So, enough said about peptide vaccines. We’re kind of going the opposite way which I initially presented the list. But now you got the DNA/RNA vaccines, right? So, and I kind of put these guys together for both of them, you can see. The use of vaccines is attractive from the standpoint of manipulation, manufacture, and standardization, right? You got a genetic code, right? You can change a single base there, right, single base. And that could change an amino acid sequence. So, they’re tunable so to speak, right?

Non-infectious, you don’t have to worry about anybody getting infected, you don’t have to worry about a live vaccine that might revert into an infectious disease and get somebody sick. When I put these facts up here, these are all things from peer-reviewed publications. Both have been documented to stimulate innate immune responses, right? The innate immune system is not talked about as much as the adaptive immune system, but it’s certainly equally important. Also, both of the RNA and DNA modalities, they can induce T and B cell immune responses, that’s all been documented, right?

There are some concerns, right? If they’re taken up by an antigen presenting cell, right? There’s something called the original antigenic shift, it’s basically just… there are different terms for this. We also talked about antibody-dependent enhancement. But in a nutshell, it’s just sort of a lack of diversion, diversification of an immune response where we kind of get locked in, for one response, for one class of viruses. Well then what happens with the different serotype, a different variant of the virus arises or different members of the same family that the immune system is kind of locked in and might not be able to adapt properly to that.

And you can actually end up with a worse infection than if you’re never vaccinated in the first place. It’s something that you have to be aware of. Both of these, they can be made fairly quickly. Again, the biggest challenge is probably going to be the scale. What’s nice about this is if you have the length of a full length of a protein when it gets made in the human cell, it’s going to be properly folded, right? And then if it’s a human whole-cell, it’s also going to have the proper post translational modification pattern like constellation?

Some of the caveats, DNA, right? There’s a potential to integrate in the human genome, right? That’s something that’s really… it’s out there, it’s never been fully addressed. Maybe with some of these recombination systems where it can be kind of that we’re developing through gene therapy that it can be trained to integrate into regions of the genome where it won’t cause harm. I think that will be the biggest advance to come in the field but it isn’t quite there yet. I mean, they talk about CRISPR technology. In the clinic, they haven’t had the best immunogenicity.

And then for RNA, right, there’re bigtime concerns with instability. And then, again, this isn’t humans and in a lower immunogenicity. Some of the other studies that are peer-reviewed that I talked about in the earlier bullet points, those are in animal systems, and it gets a little bit different. Once you go from a mouse to human, it can be quite a bit different.

Now, let’s talk about scalability. Just to give you an idea for how much can you really make of these RNAs or DNAs, there’s a lot in the news about that, wanted to investigate it a little bit. I went to the press release of Nitto Denko Avecia, right? They’re talking about being able to make DNA at a 1.6 molar scale which is… it’s pretty good to get, and that’s the largest… you can go to that web link and see. You can go to that web link and see. And so in theory, if you had a 50 mer and you wanted to make it a 100 microgram dose, that would translate to a 264 million doses. But that’s a big theoretical leap.

That means, just because it’s made, you still got to purify it, multiple steps in the purification, so I would think you would need to get close to that build. And that’s still just a very, very small molecule. So, I’m trying to give you a feel for the challenges that are out there to make something and has something that’s widely available to many, many people. So, I’ll continue on a little bit more about RNA vaccine, some more bullet points on what’s going on there.

So, in addition to something that’s being either it’s chemically synthesized or it’s biochemically synthesized, you have a nice little feature here where it can be self-amplifying, right? So contains the genes, the RNA replication machinery, right, where the structural proteins, they replicate along with the gene of interest. So, this may be difficult to do with the GMP level. There’s some great papers that are there, peer-reviewed publications, studies that show that it kind of almost mimics an infection in a way.

So I think the closer you are to mimic any infection using the immune systems, exploiting the full capabilities of the immune system, the better you’re going to be. So, this is something that bears watching and could be the way in the future. Right now, I think it’s something that probably couldn’t be done so easily at the GMP level but time we’ll see. But with all RNAs, you’ve got some issues with stability, right? You can’t really make something, have it distributed and have it in stable, right? It’s not compatible with developing successful vaccine. You have to have some sort of shelf life. And it’s just something for just months, it’s not going to cut it.

So these are some reports that are out there. So, freeze-dried mRNA with trehalose or naked mRNA is stable for about 10 months at four degrees Celsius. That’s not great, but at least something. If you encapsulate mRNA with cationic liposomes or cell penetrating peptides, right, they can protect it from RNA. But I don’t know what happens if you have some sort of excursion in the temperature, how good that’s going to be. And then there’s another, all reviewed in the article below so you can read a little bit more about it. A lyophilized mRNA vaccine was stable for five to 25 degrees for 36 months and 40 degrees for six months.

So, you see that’s getting a little bit better and something that’s more usable in a commercial context. So, it’s really what appear that you need to either have the RNA or the DNA either encapsulated or lyophilized, right? Just use it naked or in solution, it will just never hold up. All right. So, whenever people talk about if any oligonucleotide or even peptide also is made synthetically, right, you’ve got to look at the link, the link between length, coupling efficiency, and yield, right?

So, if you need a 50 mer, you really got 50 different reactions going, right, 50 sequential reactions linking one base to the next, right? And the higher that efficiency of coupling, the better yield you’re going to get, right? And you can see, if you look at the coupling efficiency, just a few percent, right, it’s a very good record 97%. I mean, just when you start getting a much larger oligonucleotide, it really drops precipitously. So, that’s something to keep in mind. And there’s no straight coupling efficiency across the board. Each all of the nucleotide or each peptide, there’re different studies in and it of themselves. So, that’s another challenge for manufacturing.

All right. Virus-like particles, you hear a lot about this. These are commercially available. They have been licensed. So, it’s a good technology if it works, right? Again, subassembly is viral structural proteins, really it’s great. They’re not infectious, right? They don’t contain any viral genetic material. They can have a very high antigen load. They resemble the parent virus taken up my cells, right? And they’re processed by your immune system very similarly to the way that the parent virus from which they were derived is processed. Like I said, they’re employed in licensed vaccines. They can generate cell-mediated and humoral immunity. The VLP, you can bulk express it in a bioreactor culture, right?

Again, like I said, anything you want to commercialize, you can’t do that unless you can really make it and make it at large scale. Again, I’ll talk about the challenges also in case you’re looking at a different technology, you want to understand it or different company, what they’re up to. There are some challenges again with stability, right? So, you’re challenged a little bit like that, for example, right? Commercial HPV vaccine Gardasil, right, has to be refrigerated at two to eight degrees, protected from light, and you can’t freeze it.

There’s also analytical challenges in their characterization. If they get stressed, that can change the particle size distribution, also can affect stability. So, they’re kind of little bit on the fragile side but really none was near what you’ll have to worry about with an RNA, right? We’re getting more to like the live vaccines, right? So this is an actual virus that’s live, can cause an infection, right. So, most of the viruses out there, most viral disease, right, is a… or immunity to most viral diseases are best achieved by live attenuated viruses.

Just think about historically what we’ve seen. We’ve seen polio, right? Some of the first early polio vaccines, smallpox vaccines, a lot of viral infections, right? The pathogenesis is complex, right? It’s dependent on an amplification in more than one target organ, right? You think of live viruses such as measles and yellow fever viruses. They don’t just replicate in like a muscle cell. It’s something that their natural life course across the context of the entire organism.

So, again, there’s a concern of, if you ever use a live viral vaccine that’s attenuated, there always is the possibility of reversion to virulence. That was a concern with the early live polio vaccines. Now, we’re kind of moving into this replication deficient virus mode where those are much less likely to have that happen, right, since by design they don’t replicate. So, that’s a very good approach where they infect the cell that is [inaudible 00:35:04] and it’s just one round of infection. So, you’re getting the best of both worlds, right? You’re getting an exposure to a viral infection, you’re mounting the proper immune response, but you’re not risk getting a full blown infection and you don’t risk reversion, right?

And then, this also interact with the innate immune system, I mentioned that before, right. They’re recognized as potential pathogens but a different molecular pattern, recognition pathway, right? And data from animal studies has been pretty consistent and it indicated that not only are these live experimental, in the case of flu, right, most effective, but the directed… if you’d administered them via the actual way that people typically get infected that you’ll better protection than generated by parenterally-administered inactivated vaccines.

So, I was just going to show you a little bit. Whenever you develop a vaccine, you’ve got to balance immunogenicity versus reactogenicity, right? And I couldn’t find one from an actual study that there was an actual virus that was administered. But I did find one that was actually a peptide vaccine, and I only include this and it shows you what can happen if you get a real sort of rough reaction? So, anything that gets to the clinic, really, you can’t have a lot of swelling, soreness, fevers, fatigue.

Kind of interesting if you have a pet that you look and see after it gets vaccinated. Typically, they’re not doing so well in the next day or two, but you’ll never see that for a naturally approved human vaccine. And I guess, Samuel, takes us to our next question. I believe I had a question about would you be willing to tolerate a very reactogenic vaccine or how many people would be willing to take that vaccine that’s very reactogenic but at the same time it’s effective? I just wanted to feel how much are folks willing to be subjected to?

Would you be willing to tolerate having like a golf ball sized? Well, if you know you’re going to get protection. Or would you just alternatively say, “Gee, that’s something I’d really rather forgo and just kind of take my chances because it’s just not worth the pain and discomfort.” So most people would not want the golf ball size well. You can see, people are much more discriminating than their pets. They would never accept something like this.

All right. So, again, recombinant vaccines, right? So, again, these are things that you can make some sort of incubator, right? I’ll talk a little bit about the different cells that are used. Some folks have asked me in the past what’s the most common cell type. There are zillions. There are tons and tons of different cells that are out there in cell lines. The CHO cells, Chinese hamster ovary cells, they’re sort of the workhorse for the therapeutic proteins. And about 70% of today’s therapeutic proteins are made in CHO cells, right? And I put the reference there so you can look this up and see what information you glean from it.

So, the major step, right, you have steam processing, you got to have a frozen vial that comes from a master cell bank. These are common paradigms across all sorts of cell culture based vaccines. Yeah, you got a producer clone, right, and ends with the harvest of the culture broth, right? And then, you’ve got to have critical parameters. You don’t just put it in there and it just magically grows. You got to get the right cell density, you have to have the duration of production phase where you get the maximum amount of accumulation recombinant protein.

You got to find out what’s the best density, cultural conditions. You got to have to define culture media, for how long the process has to go for, probably the… I’ll just switch back to this one. Yeah, this again, for the different cells here, this just shows like the past 10 years and total on all the research. So, you can see there are different cells. The HEK cells, BHK, NSO. But you see what’s consistent over the… again, here’s the reference where it came from. Over the course of the last 10 years and in total things in PubMed, that the percentage of CHO cells is pretty constant so that’s kind of like your workhorse and that’s something that’s sort of the most common cell type.

And then just to give you an idea, I mean, of how big these things can get, you can have up to like a 20 kiloliter bioreactor. I know a lot of folks have sort of asked me, what did you make and what’s the size and then you can get… this takes for a while to do. It’s like 29 days and you get two tons of cells, up to two tons of cells to be generated. So that’s pretty good. And then for your yields and stuff, you think about dosing and getting something to many, many people, you get production titers close to anywhere from one to 10 grams per liter in the different batches depending whether it’s sort of a batch mode or a fed-batch mode when you’re continuously replenishing reagents.

And whenever you’re working with a sort of recombinant protein like that, you’ve got to try to take measures to mitigate it against the loss of biological activity. Something that might diminish or it might cause an unwanted immunological reactions or other side effects. So you’re going to have to do a lot of steps for quality by design, analyzing protein quality. All right.

This takes me to my next slide, my next little pathway [inaudible 00:40:53]. I really have to think about these things. And, again, I’m trying to present something, is what’s the fastest but [inaudible 00:41:02] times not by no means compromising safety to kind of get a vaccine that’s useful, and how we can maybe trim the 10 years down as best we can without outdoing something that’s completely reckless, right? I put a little flow diagram together. People can kind of ask themselves these questions. First, if a virus can be grown in cell culture, right, then that’s great, then you might be able to do multiple passages or figure out a way to get an attenuated virus.

And that’s probably way of getting a lot of virus and a lot of vaccine. If it’s reactogenic, then you’ll have to think about somehow inactivating or splitting it. If not, then you can sort of certainly go the route of the VLPs, right? And then you kind of sort of work your way down. If no, right, then, you’re going to ask if maybe just neutralizing antibodies would just by themselves would suffice, then maybe you can get by with a recombinant protein. And that sort of put a question mark here for the oligonucleotide, that’s not… since there’s really nothing that’s been commercially licensed, it’s still really not clear exactly where it sits along the scheme.

Then there’s some other technologies that really are not on the slide that I just really didn’t mention. There’s been different techniques or different ways that in aquaculture, in agriculture, some very successful ways to develop vaccines, and that might be something that we should emulate in people as well. There’re different vaccines for fish to get different diseases where they take that orally. There’s even a coronavirus, I’m sorry, yeah, the coronavirus type vaccine in chickens, believe it or not, and that’s for an infectious bronchitis, that’s a coronavirus. And that’s actually been tested to work and protect chickens where they give it to them orally.

So, these are things that maybe at some point they’ll find their way into this flow diagram as well or should be thought about. All right. So, one really important thing when developing a vaccine is to have a correlate of protection, right? You’ve got to try to figure out early on if generating immune responses actually going to translate into protecting a person against the disease when they’re exposed to the live wild type virus, right? So, you got to be careful. Some of these animals really don’t mimic humans very well. A lot of stuffs done in mice but a mouse immune system is quite a bit different.

In fact, to give you a reference here, you can look that up. Don’t take me for my word for it. You can see for yourself. And if one’s going to use a mouse, it’s probably the better to use one that’s actually humanized. We have a human immunological synapse, something that better recapitulate disease, but you have to be wary of these mouse models. Obviously, mouse is the cheapest system available, cheapest mammalian system so that it tends to be used quite a bit, but other things are much better. Like for flu, probably a ferret is a better animal system to work with. Certainly primates as well might be or that much closer to people.

So, you got to be aware of some of these things. You have to have proper potency assays, right, that they can be cell based mechanism of action, immunochemical. You really want an immune response that’s responsible for and statistically interrelated with protection, right? It makes no sense if you get a great immune response but it’s really not going to protect you. I mean, you’ll get maybe something in early clinical stages but you really won’t get something that goes much further. That’s something to be aware of.

All right. Sorry, you got to have now… we’re talking about actually manufacturing something. So, it’s one thing to have an idea, right? It’s completely different to have a manufacturing process. One thing I never really appreciated when I was in academia is just how difficult it is to have a good manufacturing process, right? It’s a science in and of itself, right? The initial identification of the epitopes, the response you get in a preclinical animal system, those are all very, very upstream, right? But you still really haven’t answered the real crack, the tough nut, like how are you going to make this, right?

I mean, there’s a million things that are going on, right? And how are you going to make it reproducibly? You can’t just make something that works some of the time or once in a while. It’s going to work every time. It’s going to have the same level of potency each time. Otherwise, you’ll just be rolling the dice with each one. So, one thing that’s real critical here, right, something called the design of experiments, right, DOE, right? So, a lot of times, if you don’t have a DOE when you look at different factors in your formulation, in your process, you’ll just be doing things by trial and error, right? And I would say anything you get involved with, any process, make sure somebody has done a DOE experiment, right?

There’s a great paper here or chapter in a book, right, you can look it up right? You can save up to 50% of time by performing a proper DOE. And whatever you do in terms of your formulation, the DOE should also kind of look how it interacts with the process, right? You don’t do those independently, right? You got to develop your process as you’re getting your formulation, right? You wouldn’t want to do some sort of initial study, have great potency in mice and then when you finally make it, you find it’s really not nearly as potent as it once was. So, that’s something very much to be cognizant of, right?

Another thing is you’re trying to go for a live vaccine, right. I talked a little about the center flow diagram. Some of these viruses just can’t be grown in cell culture, right? That would be something like HBV, right, human noroviruses. Or they might not really produce yields of progeny that are high enough for use in a vaccine, right? HIV, right? HCV, right, other herpes viruses, right? HIV, HCV, those are really kind of been notorious. We work many decades on those and we still have no vaccine. So, like I said, it’s definitely a worthy endeavor, but it’s something that really, it can be very challenging.

And anything you make, you’ve got to also know the manufacturing strategy, you’ve got to have mechanism intact to minimize any post translational modifications, right? You don’t want microheterogeneity, right? They could have an impact, right, on solubility, potency, biological activity, all these things you have to take in mind. And then we actually make it. In terms of speed, you want to stay away from something like a traditional stainless steel tank. Single-use bioreactors are the way to go if you’re making your product in a bioreactor, right? You don’t have to validate those, they’re reliable, right? They’re clean, presterilized, so you don’t have to deal with all the requirements for installation qualification and personnel training. So, those are some points to keep in mind.

All right. So, another thing to kind of speed, stuff up, right? So, the critical path, right, for IND filing, right? It’s the availability of your tox lot for toxicological studies. So, you want to get that going as soon as possible, right? It takes about six months. So, you don’t want to have that at the very end, right, because they’ll just add like another six months. It’s got to be done in parallel, really not in sequence or not sequentially. But you could do it like that but it’s going to take longer, your investors are going to get mad, you might run out of money. All these are steps to trim the path.

You’re not cutting any corners, you’re just doing things efficiently, right? For process development, you want to get that done as early as possible. Your formulation development, method development, those are all concurrent, right? Again, you’re doing this in parallel, right? And if you’re making clones, you get these things done before screening, screening the actual clone. Sometimes for your tax slot, right, if you’re working with different clones, you can use a pool of clones, right, that will be acceptable before the highest yield clone. So this way, you can get your stuff in tax as soon as possible, right?

And then you want to have your GMP production start prior to the full completion of toxicological study. You can kind of get a little bit of a jumpstart, right? And as I said, the toxicological study can be on a pool of material, right? So you don’t have to necessarily wait to your very best that clone comes available. So, all right. I talked about manufacturing, right, some of the strategies there, right? What are some of the other levers we can pull?

So, there’s certainly regulatory mechanisms. Now, I listed four different pathways. There’s four different mechanisms. They’re really six, right? And I’ll talk about this a little bit. Because you’re hearing a lot in the news and I’d be remiss if I didn’t mention those, right? So, just a couple things, right. When they talk about approval or designation, they usually accompany these terms, right? The approval mechanism, right, approval is just simply a mechanism to market authorization, right? And the designation, right, it’s granted you’re drug based on meeting certain criteria and providing certain benefits. So, that’s a distinction between approval and designation.

And, again, here are the four main expedited pathways. But there are a couple other ones I didn’t quite talk about, right? There’s another one called Emergency Use Authorization. You hear a lot about that. That kind of scares me because there I think you might be thinking about taking away some of your safety mechanisms. Let’s say I want to drive from New York to Boston, right? And I wanted to get the faster. There are ways I can do it. Let’s just say, I could go through stoplights, stop signs, take the brakes off my car, that would get me there but it will be a perilous ride.

And basically, the fast track designation, it gives the FDA authority, right, the regulations on it now where they have the authority to actually… do they allow an unapproved medical product, right, or unapproved uses of approved medical products, right, that can be used in emergency to diagnose, treat, prevent serious or life threatening diseases or conditions, right. And this is generally for CVRx, right? Chemical, biological, radiological, nuclear. So, yeah, so it’s like in CBRN, that’s kind of what it’s used for.

What scares me about it is something could get into use much faster than it should be. I don’t see it as a real typical thing, I talk about it because of the current times in which we live in that, in conjunction, there’s another program that’s out there or like I’ve said there’s a Supreme Court case many years ago. Actually, it’s called Jacobson versus Massachusetts. No connection to me, it’s 1905 way before my time. Supposed with a name like Jacobson, he was a great guy. But in all seriousness, that basically gives the state the authority to give you a vaccination at their discretion, that was done in response to smallpox.

So, that’s a fast way of getting things into use but I don’t think it’s something that’s certainly not a normal development pathway. So I’ll say a few words about this. We’ve got the accelerated approval, right? That’s the accelerated approval on number three, right. That let’s drugs for serious conditions, right, that fulfill an unmet medical need, right, to be approved based on a surrogate endpoint, right? I’ll say a few words about priority review, right? The designation means the FDA goal is to take action on an application within six months. So usually it’s about 10 months.

So that’s a way to kind of get that fast track designation, right? You’re going to get a priority review, you’re going to get your vaccine develop much faster, fast track, right? It’s also for an unmet need that gets developed [inaudible 00:54:28]. And then, you got the Breakthrough Therapy Designation. So, those are all the four mechanisms. The FDA is very involved in those. You’ve got to request those pretty early. And then I’ll say just a few words before I move on as we get a little close on time. That you also have a voucher program, right, for tropical diseases, neglected diseases, orphans, orphan diseases that affect less than 200,000 people.

If you’re an entrepreneur and you get one of those vouchers, right, your product, you request that during development, you’re getting a priority review for any other product that you want to develop in the future. So the beauty of that is you can actually sell these. Well, if you’ve got a product, it can develop faster but you can also transfer that to another entity. And typically those are the pretty high market value. We’re talking 200 to 300 million dollars. So, if you got a small company, that’s certainly a strategy to consider getting some sort of voucher, priority review voucher, that can be sold and certainly be quite a boon to your financial resources.

So, I’m going to go through the list pretty quickly. You guys can look at that. And see we’re getting close in the hour there. Stability is a big commitment that has to be met, right? I put up here, there’s the stability for Phase I, the requirements for Phase II, for Phase III, everything that’s expected. So I just wanted to give the bio entrepreneurs in the audience and also folks that have an interest in vaccines, what’s really required to make a good vaccine with regard to stability, right?

And then I’ll finally conclude with my few words and opportunities. So, irrespective of what you think about the folks, the fellow who originated this quote, when you sort of read this, as we know, there are no knowns, no knowns, there are things we know we know. We also know that there are known unknowns and that is to say we know there are some things that we do not know. But there are also unknown unknowns and those are the ones we don’t know.

So, kind of painful to read this, but after about the 10th time, you realize there’s a certain amount of wisdom there. So, what do we got here? We’re concluding on the opportunities, we’ve got the no knowns, right? Those are the viruses that just reemerging, right? No unknowns, things that are emerging, right? And, again, I’m going to say right here, most of these actually are zoonotic, they come from animal species. So, it’s something that’s a pattern that’s going to probably reproduce itself. I would see our opportunities are the no knowns, the known unknowns, right? Unknown in terms of how infectious they’re going to be. And then, something that’s completely unknown, we really can’t do much about.

And, really, after the final few slides, I’m just going to devote to the viruses, I call these the known unknowns. The ones that really don’t have a vaccine, we know about them that they don’t have vaccine. And that’s sort of a potential opportunity. I put a little iceberg here because in terms of tip of the iceberg. I mean, it’s not what I present towards the end. It’s really just not even a snowflake on the iceberg. I put a quote up here from a book from The Coming Plague, right?

And in a nutshell, what it’s saying is, the more the world develops, the more we interact with different folks, the more we’re putting ourself at risk to encounter one of these zoonotic organisms. So, even those is written in 1994, actually got this book coincidentally in December of 2019, I thought it would be interesting reading it. I never knew it would be so appropriate. But you see, this is going to be a recurrent paradigm, right? I know there’s talk about the current situation that it’s a bioweapon or escape from a lab, I kind of hope that… I wish that it were so that would mean it’s a one and done issue and it could be addressed.

But if it’s something that came from nature, you can see that this is something that’s going to happen over and over again. And the situation we find ourselves in, it’s really a light tap as opposed to what could be a significant punch. If you look at this, we’ll conclude by it. I’ll show you the viruses that are out there that we don’t have a licensed vaccine for. It’s a scary list when you look at some of these, when you look at the mortality rate is, when you look at the symptoms. If these ever became wildly infectious, it would be very, very problematic, much worse than the current situation that we’re now in.

And if you scroll down the list here, I’ve referenced the different studies on the clingov trial sites, you can kind of get an idea what’s out there and where we stand. It’s sort of a frightening list. And it kind of terrifies me that someone like the cutest organisms really are the most problematic. And I put up here a squirrel monkey and a spider monkey. These guys harbor viruses like you wouldn’t believe. You’ll never go to a zoo and you’ll never see an Old World monkey mixed with a New World monkey. That’s been tried but these guys have so many viruses which they’re immune to or what they really have.

These herpes viruses, they spread very readily to the Old World monkeys and they give them like a cancer or leukemia and most of them have died within several months. So, heaven forbid, that’s something that due to gene swapping could ever find its way and potentially as possible into the human population. So, this kind of gives you a glimpse. You got this long list here. Here’s something like known unknowns or known knowns that we can maybe it’s worth going from one of these vouchers or orphan diseases and developing something against this. So, having something in our arsenal if the unthinkable actually happens.

And with that, I’m going to get my concluding slide and see close to time here. And I don’t mean this disparagingly in any way to anybody’s efforts, just all I mean to say is that it’s a very iterative process, vaccine development, right? The first vaccines probably not going to be your best. It’s just really the first horse out of the starting gate. And I borrowed this quote from a gentleman who is a very well respected, well established gene therapy researcher. I heard him say at a bio conference, just because a horse is first out of the gate does not mean that it’s secretariat.