Great post! I wish more scientists would share technical ideas in exactly this way! I've got one suggestion and one bit of blue sky speculation.
Suggestion: I have happy memories of mastering crossflow while helping my postdoctoral mentors develop HPV vaccines a quarter century ago - but in the years since then I've almost entirely shifted to using Optiprep to concentrate and purify viruses. It's great for smaller-scale work. Here's a detailed protocol:
Consider it my bona fides on the suggestion that more scientists should publicly share technical ideas!
Speculation: How much purity do you actually need here? The FDA appropriately sets endotoxin limits for things that will be injected, but pseudomonas sometimes causes skin/lung/gut infections that might theoretically be best addressed by applying the phages topical/aerosol/oral. The FDA doesn't set endotoxin limits for foods, cosmetics, or dietary supplements - so it's not clear to me what the scientific rationale might be for limiting endotoxin in phage preps that will be administered via the same routes as foods, cosmetics, and supplements.
Thanks so much Chris! Totally agree about more technical science posts.. I try to encourage them (and publish them in my other, phage-specific blog) but haven't found too many doing it.
Great to have this method! I hadn't heard of optiprep but looking at your doc, seems it's iodixanol - I now remember someone mentioning they used iodixanol for phage and I've never had a chance to try it!
As for your Q re: how much purity we need - yes technically we don't need to hit the intravenous EU limits for phages that aren't administered IV. We don't always aim to for topical or oral phage treatments (at least the ones I've been involved in). However the phage therapy community generally seems to want to default to getting rid of the EU for all treatments these days, even for research, since there's more and more immunology and PK studies being done - they want to have EUs removed so they can better study phage distribution/response without it being clouded by all the LPS contaminants in the mix. Also I think bringing phage preps down to IV-safe EU levels is just becoming a safe default for compassionate use phage therapy generally - there's often discussion in the clinical meetings I've been in, where it's like 'will this phage we pour into the surgical wound get absorbed into circulation? should we probably use 'IV-grade' for this one? Yeah sounds good). Others are moving toward making phage batches that can be clean and waiting on shelves, and since there's so many more doctors wanting to use intravenous as the administration strategy, it seems it's becoming the reasonable thing to do aim to make the phages 'IV-grade' for any patient.
Iodixanol (Optiprep) was originally developed as an intravenous X-ray contrast agent - and we inject it into mice all the time with no ill effects. The ultracentrifuge isn't all that scalable, but for smaller pilot batches it's truly a one-step process for getting injection-grade material.
My thinking was transformed by the pandemic. In the before-times, I would have reflexively agreed that phage developers should err on the side of cautiously demanding IV-grade purity for all applications. After watching the US FDA drag its feet on Covid vaccine adjuvant purity and updated booster doses, I worry that withholding lifesaving medicines while waiting for ideal purity actually isn't a very cautious approach in the pathogen-addled real world. It's all a matter of striking a balance, but my nightmare is a situation where a topically applied phage stock doesn't quite meet IV-grade purity standards and then the system makes it so the patient gets nothing instead of a somewhat crude stock that realistically might have helped. And by "helped" I literally mean life or death. The pushback on purity is my way of saying the work you're doing is incredibly important!
Actually, I'm being unduly pessimistic about scalability. We used Optiprep to purify the virus-like particles for this pilot monkey-scale vaccine study and we still have quite a lot more material left over.
I can illustrate my thinking with a case study. A few years back, my sister had an ear infection that got so far out of hand it started actively gnawing its way through her mastoid process toward her brain. She said it was by far the most intense pain she ever experienced - and she's given birth to three kids. The bacterium couldn't be cultured and the first round of broad spectrum antibiotics they tried didn't work. Fortunately, the second round of antibiotics worked - so I didn't have to box up my Nanopore and fly across the country to find out which antibiotic resistance genes the damn thing had. If I imagine the alternate scenario where the second antibiotic didn't work and we found out a universally resistant pseudomonas superbug was about to kill my sister, I would have campaigned for access to any phages that might conceivably help. If phage scientists had responded with "sorry, but we don't have pure enough stocks yet," I would have argued that my sister's ear canal was already awash in endotoxins - so what possible harm could it do to try adding a drop of crude phage stock to the existing ocean of bacteria.
Aside from the life-or-death quick-and-dirty topical application scenarios, there's also a broader public policy problem to wrestle with. Ever since the Asilomar Conference, bioethicists have been wringing their hands about how to guard against vague, ill-defined, abstruse hypothetical risks. The hand-wringing artificially stoked inordinate public fear about risks that never materialized. An old made-for-TV movie called "The Henderson Monster" puts the overblown hysteria on unintetionally hilarious display:
And it's not just some quaint bit of history - Greenpeace's monstrous current campaign to blind half a million children by denying them access to golden rice rests on exactly this same type of unsubstantiated "but Frankenstein!" thinking.
In the US, it takes the FDA many months to carefully consider authorizing updated Covid booster doses. This hasn't had the intended effect of promoting public confidence in the idea that the FDA employs careful high standards - it has instead had the disastrous blowback effect of making most of the public think the updated Covid booster doses must be dangerous, ineffective, or unnecessary. In 2023, 47% of Americans got the low-bar flu boost while only 23% got the high-bar Covid boost. Setting standards artificially high without clear rationales based on predictable known risks can be dangerous too.
Totally agree that inaction/slowness as default is actually often dangerous too. As for providing phage for eg. your sister's situation - how do you think about the aspect of liability for the scientists that provide the phage? I think that ends up being a big concern. If they provide the phage and something bad happens, is their career over? For physicians too, I believe they have to risk their career to do it (or at least that would be the perception). I have been meaning to look into Right to Try act etc, and see how things might shift to allow patients to try more experimental medicines more easily. Maybe it is an education hurdle that needs surpassing; eg. educating the physicians and scientists that the likelihood of a crude phage lysate causing a problem is so low that it's unlikely there would be liability concerns? That said, scientists and doctors generally want to see clinical safety studies before they believe that it's safe (safe enough to risk their career), which have only been done for some phages under some conditions.
Couldn't the phage sharing be covered under a standard material transfer agreement? The NIH's standard MTA has a clause saying "not for use in human subjects," but for a phage material transfer the clause could be removed without touching all the other indemnification clauses that protect the scientist. https://www.techtransfer.nih.gov/sites/default/files/documents/pdfs/slaform.pdf
The US Senate is considering an improved right-to-try law called the Promising Pathway Act. I'm hoping they might rename it the Seliger Act
I agree that physicians are generally hesitant about unproven medicines, but when things come down to a choice between realistically-might-help versus certain-death then the first-do-no-help hesitancy starts to be a very bad look for physicians.
His basic idea is to empower individuals to make informed choices about which n-of-1 experments they might want to do on themselves. Through that lens, if my sister in the grim alternate universe where antibiotics didn't work had made an informed choice to gamble by dripping a not-so-pure phage stock into her own ear - and her n-of-1 self-experimentation didn't cure the infection or it caused some unexpected side effects - I wouldn't blame the phage scientist or the physician or my sister. To put it another way, it should really be my sister signing the MTA, not her physician.
It's horrible that individuals are sometimes forced to roll the dice on these terrifying high-stakes gambles. And it's disturbing to contemplate who might get blamed when the gambles don't pay off. But I guess you can call me a knee-jerk liberal when I argue that stripping an individual of the ability to choose what to do with their own body is almost never the best way out of this type of trap.
Ah yes! I totally agree with you on that. At least when I was in Australia, their drug committees (and TGA) were very reasonable about requiring more purity than necessary. We would treat oral or topical infections with freshly made phage lysates on occasion (these were the minority though because doctors wanted to treat IV usually). Now I’m back in the US and I have yet to be on the producer side of a compassionate case - it will be interesting to see what FDA is like by comparison. In urgent cases from what I’ve heard they’re also very amenable. And purification is achievable for an academic lab at this point, as long as they have someone to do the work. So I don’t know if I’d say purification barriers for compassionate cases are directly preventing actual treatments/lives saved right now. It’s more that it’s no one’s job to do it (since it’s not reimbursable until the clinical trials are done), and thus most patients/docs don’t even know about it.
Thanks again for this post and for your spirited replies. The discussion led me to a bureaucratic effort to convince my institution to let me use the new OpenMTA option at Addgene:
In my imagination, you might likewise be able to use OpenMTA to transfer phages. The patient could sign the "Recipient Scientist" line and their physician's institution would sign the "Recipient" line. The approach essentially views the patient as a citizen scientist who's setting out to do a self-experiment. I see it as more respectful approach than the usual framework of only ever viewing the patient as a passive subject.
Great post! I wish more scientists would share technical ideas in exactly this way! I've got one suggestion and one bit of blue sky speculation.
Suggestion: I have happy memories of mastering crossflow while helping my postdoctoral mentors develop HPV vaccines a quarter century ago - but in the years since then I've almost entirely shifted to using Optiprep to concentrate and purify viruses. It's great for smaller-scale work. Here's a detailed protocol:
https://ccrod.cancer.gov/confluence/display/LCOTF/PseudovirusProduction
Consider it my bona fides on the suggestion that more scientists should publicly share technical ideas!
Speculation: How much purity do you actually need here? The FDA appropriately sets endotoxin limits for things that will be injected, but pseudomonas sometimes causes skin/lung/gut infections that might theoretically be best addressed by applying the phages topical/aerosol/oral. The FDA doesn't set endotoxin limits for foods, cosmetics, or dietary supplements - so it's not clear to me what the scientific rationale might be for limiting endotoxin in phage preps that will be administered via the same routes as foods, cosmetics, and supplements.
Thanks so much Chris! Totally agree about more technical science posts.. I try to encourage them (and publish them in my other, phage-specific blog) but haven't found too many doing it.
Great to have this method! I hadn't heard of optiprep but looking at your doc, seems it's iodixanol - I now remember someone mentioning they used iodixanol for phage and I've never had a chance to try it!
As for your Q re: how much purity we need - yes technically we don't need to hit the intravenous EU limits for phages that aren't administered IV. We don't always aim to for topical or oral phage treatments (at least the ones I've been involved in). However the phage therapy community generally seems to want to default to getting rid of the EU for all treatments these days, even for research, since there's more and more immunology and PK studies being done - they want to have EUs removed so they can better study phage distribution/response without it being clouded by all the LPS contaminants in the mix. Also I think bringing phage preps down to IV-safe EU levels is just becoming a safe default for compassionate use phage therapy generally - there's often discussion in the clinical meetings I've been in, where it's like 'will this phage we pour into the surgical wound get absorbed into circulation? should we probably use 'IV-grade' for this one? Yeah sounds good). Others are moving toward making phage batches that can be clean and waiting on shelves, and since there's so many more doctors wanting to use intravenous as the administration strategy, it seems it's becoming the reasonable thing to do aim to make the phages 'IV-grade' for any patient.
Iodixanol (Optiprep) was originally developed as an intravenous X-ray contrast agent - and we inject it into mice all the time with no ill effects. The ultracentrifuge isn't all that scalable, but for smaller pilot batches it's truly a one-step process for getting injection-grade material.
My thinking was transformed by the pandemic. In the before-times, I would have reflexively agreed that phage developers should err on the side of cautiously demanding IV-grade purity for all applications. After watching the US FDA drag its feet on Covid vaccine adjuvant purity and updated booster doses, I worry that withholding lifesaving medicines while waiting for ideal purity actually isn't a very cautious approach in the pathogen-addled real world. It's all a matter of striking a balance, but my nightmare is a situation where a topically applied phage stock doesn't quite meet IV-grade purity standards and then the system makes it so the patient gets nothing instead of a somewhat crude stock that realistically might have helped. And by "helped" I literally mean life or death. The pushback on purity is my way of saying the work you're doing is incredibly important!
Actually, I'm being unduly pessimistic about scalability. We used Optiprep to purify the virus-like particles for this pilot monkey-scale vaccine study and we still have quite a lot more material left over.
https://www.sciencedirect.com/science/article/pii/S0264410X23001287
I can illustrate my thinking with a case study. A few years back, my sister had an ear infection that got so far out of hand it started actively gnawing its way through her mastoid process toward her brain. She said it was by far the most intense pain she ever experienced - and she's given birth to three kids. The bacterium couldn't be cultured and the first round of broad spectrum antibiotics they tried didn't work. Fortunately, the second round of antibiotics worked - so I didn't have to box up my Nanopore and fly across the country to find out which antibiotic resistance genes the damn thing had. If I imagine the alternate scenario where the second antibiotic didn't work and we found out a universally resistant pseudomonas superbug was about to kill my sister, I would have campaigned for access to any phages that might conceivably help. If phage scientists had responded with "sorry, but we don't have pure enough stocks yet," I would have argued that my sister's ear canal was already awash in endotoxins - so what possible harm could it do to try adding a drop of crude phage stock to the existing ocean of bacteria.
Aside from the life-or-death quick-and-dirty topical application scenarios, there's also a broader public policy problem to wrestle with. Ever since the Asilomar Conference, bioethicists have been wringing their hands about how to guard against vague, ill-defined, abstruse hypothetical risks. The hand-wringing artificially stoked inordinate public fear about risks that never materialized. An old made-for-TV movie called "The Henderson Monster" puts the overblown hysteria on unintetionally hilarious display:
https://youtu.be/PUsYNtLSfx8?si=BeiDnqFYUcrkelfj&t=4794
And it's not just some quaint bit of history - Greenpeace's monstrous current campaign to blind half a million children by denying them access to golden rice rests on exactly this same type of unsubstantiated "but Frankenstein!" thinking.
In the US, it takes the FDA many months to carefully consider authorizing updated Covid booster doses. This hasn't had the intended effect of promoting public confidence in the idea that the FDA employs careful high standards - it has instead had the disastrous blowback effect of making most of the public think the updated Covid booster doses must be dangerous, ineffective, or unnecessary. In 2023, 47% of Americans got the low-bar flu boost while only 23% got the high-bar Covid boost. Setting standards artificially high without clear rationales based on predictable known risks can be dangerous too.
Totally agree that inaction/slowness as default is actually often dangerous too. As for providing phage for eg. your sister's situation - how do you think about the aspect of liability for the scientists that provide the phage? I think that ends up being a big concern. If they provide the phage and something bad happens, is their career over? For physicians too, I believe they have to risk their career to do it (or at least that would be the perception). I have been meaning to look into Right to Try act etc, and see how things might shift to allow patients to try more experimental medicines more easily. Maybe it is an education hurdle that needs surpassing; eg. educating the physicians and scientists that the likelihood of a crude phage lysate causing a problem is so low that it's unlikely there would be liability concerns? That said, scientists and doctors generally want to see clinical safety studies before they believe that it's safe (safe enough to risk their career), which have only been done for some phages under some conditions.
Couldn't the phage sharing be covered under a standard material transfer agreement? The NIH's standard MTA has a clause saying "not for use in human subjects," but for a phage material transfer the clause could be removed without touching all the other indemnification clauses that protect the scientist. https://www.techtransfer.nih.gov/sites/default/files/documents/pdfs/slaform.pdf
The US Senate is considering an improved right-to-try law called the Promising Pathway Act. I'm hoping they might rename it the Seliger Act
https://bessstillman.substack.com/p/in-memory-of-jake
I agree that physicians are generally hesitant about unproven medicines, but when things come down to a choice between realistically-might-help versus certain-death then the first-do-no-help hesitancy starts to be a very bad look for physicians.
A cardiovascular researcher named Lutz Kraushaar has some provocative thinking in this space:
https://open.substack.com/pub/drlutz/p/individualised-lifestyle-medicine
His basic idea is to empower individuals to make informed choices about which n-of-1 experments they might want to do on themselves. Through that lens, if my sister in the grim alternate universe where antibiotics didn't work had made an informed choice to gamble by dripping a not-so-pure phage stock into her own ear - and her n-of-1 self-experimentation didn't cure the infection or it caused some unexpected side effects - I wouldn't blame the phage scientist or the physician or my sister. To put it another way, it should really be my sister signing the MTA, not her physician.
It's horrible that individuals are sometimes forced to roll the dice on these terrifying high-stakes gambles. And it's disturbing to contemplate who might get blamed when the gambles don't pay off. But I guess you can call me a knee-jerk liberal when I argue that stripping an individual of the ability to choose what to do with their own body is almost never the best way out of this type of trap.
Ah yes! I totally agree with you on that. At least when I was in Australia, their drug committees (and TGA) were very reasonable about requiring more purity than necessary. We would treat oral or topical infections with freshly made phage lysates on occasion (these were the minority though because doctors wanted to treat IV usually). Now I’m back in the US and I have yet to be on the producer side of a compassionate case - it will be interesting to see what FDA is like by comparison. In urgent cases from what I’ve heard they’re also very amenable. And purification is achievable for an academic lab at this point, as long as they have someone to do the work. So I don’t know if I’d say purification barriers for compassionate cases are directly preventing actual treatments/lives saved right now. It’s more that it’s no one’s job to do it (since it’s not reimbursable until the clinical trials are done), and thus most patients/docs don’t even know about it.
Thanks again for this post and for your spirited replies. The discussion led me to a bureaucratic effort to convince my institution to let me use the new OpenMTA option at Addgene:
https://www.nature.com/articles/nbt.4263
https://biobricks.org/open-material-transfer-agreement/
In my imagination, you might likewise be able to use OpenMTA to transfer phages. The patient could sign the "Recipient Scientist" line and their physician's institution would sign the "Recipient" line. The approach essentially views the patient as a citizen scientist who's setting out to do a self-experiment. I see it as more respectful approach than the usual framework of only ever viewing the patient as a passive subject.