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NNadir

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Speculations on Covid-19 Vaccine Production Bottlenecks.

The paper I'll discuss in this post is this one: Optimization of Lipid Nanoparticle Formulations for mRNA Delivery in Vivo with Fractional Factorial and Definitive Screening Designs (Kevin J. Kauffman, J. Robert Dorkin, Jung H. Yang, Michael W. Heartlein, Frank DeRosa, Faryal F. Mir, Owen S. Fenton, and Daniel G. Anderson
Nano Letters 2015 15 (11), 7300-7306)

Recently on this website, I described how I cheered up my doctor by relaying to him "war stories" from the scale up of HIV protease inhibitors in the 1990s, of which I am a "veteran" of sorts. It's here: I hope I cheered my doctor up.

This morning I attended a (Zoom) lecture by an astrophysicist at Princeton University Professor Cristiano Galbiaiti who works on neutrino and dark matter detectors which rely on highly purified gases, who used his expertise and knowledge of them to rapidly design a new ventilator that was cheap, easy to manufacture, and proved to be highly effective for the treatment of Covid-19. It obtained the fastest approval, from conception to patient use, of a new medical device by the USFDA, 45 days.

It is an example of how "impractical science," the discovery of neutrinos in space, allows for "practical" innovation, much as the 1960's space program allowed for the development of small portable computers, on which many of us now rely.

If you member, in the early days of Covid, there was a huge shortage of ventilators, and the Canadian government, which funded the design and manufacture of the ventilators, ordered many tens of thousands of these new ventilators, which were delivered. The end result is interesting: There is now an oversupply of them, and the partners working on this project are now working to get Western governments to donate ventilators to Africa, where, predictably, ventilators are still in short supply.

I have to see my doctor again this week, and I decided to look into the manufacturing process of RNA vaccines, so I could further cheer my doctor up, with some more detailed insights into the manufacture of these vaccines, about which I have a sense of regulatory requirements, but not the actual details.

I am not currently in anyway involved in the manufacture of Covid vaccines, but I have had professional conversations about nucleic acid formulations, and have had other conversations relating to, and sometimes supporting, related "lipid nanoparticle" work, notably liposomes. Anything I say in this post will, however, be largely guesswork and should be taken with a grain of salt.

The HIV protease inhibitors are drugs that are considered "peptidomimetics" and they depended on access to certain amino acids as starting materials, in particular cysteine and phenylalanine (depending on the protease inhibitor in question). Since many chemical synthesis steps were involved in converting them into "isosteres" of peptides, these starting materials were unregulated commodity items required only to meet certain specifications subject to somewhat limited specifications.

What was involved was large chemical reactors, performing organic synthesis on a multi-ton scale, in some cases utilizing fairly dangerous reagents. (Phosgene was one.) Ultimately these were utilized to make the "API" - the active pharmaceutical ingredient, the protease inhibitor.

The situation with respect to the API of nucleic acid drugs is somewhat different, I expect.

Nucleic acids, such as DNA and RNA, are self replicating molecules of course, and so in theory, their production can (and does) proceed exponentially. This is the technology behind forensic and commercial (as in "23 and me" and related companies) "PCR" (Polymerase Chain Reaction) technology. It is the design of the nucleic acid that matters.

A nice overview of the design and some information about production of the API can be found in an open source article that is somewhat, but not, I think, overly, technical: The promise of mRNA vaccines: a biotech and industrial perspective. A cool feature of the design of these vaccines, with which I was unfamiliar, is the realization that they not only encode for the immunogen protein of the SARS-CoV-2 virus, the famous "spike" protein, but they also encode for some proteins designed to aid with the transcription of the RNA to generate the proteins.

I really can't say very much intelligent about the scale up of the API and potential bottlenecks, since these are involved more with biological systems than synthetic systems and I personally have far less exposure (mainly osmotic exposure) to these than I do to synthetic systems. I would imagine that the chief bottleneck might be having useful enzymes for transcription from the DNA parent the RNA, as well as nucleic acids themselves, although, as opposed to amino acids, which in living systems have 20 basic components (actually a few more because of "post translational modifications" ) there are only 8 nucleic acids in living cells, making separations more straight forward. I would imagine (but do not know) that these are synthetically available without isolation from natural sources (as are some amino acids) from ribose, deoxyribose (almost certainly obtained by fermentation) and the nucleobases which are readily available commodities lacking stereocenters. I know for a fact, that enzymes, including certainly the enzymes responsible for nucleic acid transcription are readily available from biological fermentation facilities. It is possible there has been some strain on these supplies, but basically a vast infrastructure exists.

So my guess is that the API's are not much of a bottleneck at all, beyond, it would seem, some industrial scale chromatography in at least some steps; I would imagine, perhaps, affinity chromatography, which is much, much cleaner than other types of chromatography, perhaps even at the level of solid phase affinity extraction. (These are all speculations; I have no information.)

My guess is that the real issue, the biggest bottleneck, is the formulating agents.

Nucleic acids are charged species, hydrophilic species, that is they are soluble in water and other polar solvents. All cell membranes, by contrast, are lipids, aka "fats." The basic rule that "like dissolves like" applies, which is why cells like blood cells are able to stay intact; their membranes are insoluble in water. Therefore the trick is to get the charged nucleic acids through the membrane, and into the cell. This requires amphiphilic lipids, which have hydrophobic properties on one end of the molecule, and hydrophilic properties on another end. The most famous of these are soaps, with which everyone is familiar.

In fact, the SARS-CoV-2 virus is coated with a lipid, and this is why soap, regular hand soap, is so effective at inactivating it, since it strips away the lipid layer on which the virus depends.

Fats are readily available from multiple sources, so much so that they have been utilized as fuels, biodiesel, in automobiles and trucks by the so called "renewable energy" industry, which has succeeded in destroying large swathes of Asian rain forest for palm oil plantations in part to make diesel fuel for German cars and trucks.

I want to be clear on something in this context, since I am about to discuss lipids, the amount of palm oil required to make the world supply of Covid vaccines (if palm oil is a basic source at all, which it may not be) will surely be trivial when compared to produce other commodities, including biodiesel and the palm oil utilized in foods. There is really no legitimate rationale for destroying the South Asian rain forests for commercial interests. The very special lipids that are likely utilized in Covid vaccines might have just as easily utilized corn oil as palm oil, since it is likely that the starting materials, saturated and unsaturated straight chain fatty acids are present in both sources, and perhaps other temperate zone agricultural products such as canola oil or soybean oil.

Anyway.

The lipids in question are amphiphilic, but they are very special lipids of a type, which are widely found in living systems, but which are also synthetically complex except by enzymatic routes.

To give a feel for the complexity of lipids beyond the straight chain fatty acids, here is a chart showing types of a class of lipids that are found prominently in skin, the ceramides:



Some of the lipids described in the paper cited at the beginning of this post are isosteric (similarly shaped) to sphingosines.

The key point in this development is the concept of "ionizable lipids," which address the transport of a charged species, RNA, across the lipid membranes of cells.

(At this point, it behooves me to mention a post in this forum by our leader, pointing to the career of a famous lipid chemist in the early days of lipid chemistry: Watch for NOVA's rerun of "Forgotten Genius" in your area ... or watch it online ...

From the introduction to the paper cited at the outset:

Nucleic acids have tremendous therapeutic potential to modulate protein expression in vivo but must be delivered safely and effectively. Because the delivery of naked nucleic acids results in poor cellular internalization, rapid degradation, and fast renal clearance,(1, 2) lipid nanoparticles (LNPs) have been developed to encapsulate and deliver nucleic acids to the liver. Most notably, the field has seen orders-of-magnitude potency advances in the delivery of 21–23 nucleotide-long double stranded small interfering RNAs (siRNAs) due in part to the creation of new synthetic ionizable lipids and lipid-like materials.(2) Whereas some of these novel lipids were synthesized with rational design approaches by systematically varying the lipid head and tail structures (e.g., DLin-KC2-DMA, DLin-MC3-DMA, L319), (3-5) other materials were discovered by creating large combinatorial libraries of lipid-like materials (e.g., C12-200, cKK-E12, 503O13).(6-8) When formulated into LNPs, these amine-containing ionizable lipids and lipid-like materials electrostatically complex with the negatively charged siRNA and can both facilitate cellular uptake and endosomal escape of the siRNA to the cytoplasm.(6, 9) In particular, the ionizable lipid-like material C12-200 has been widely used to make siRNA-LNP formulations for various therapeutic applications in vivo to silence protein expression.(10-12)

In addition to the ionizable material, three other excipients are also commonly used to formulate LNPs: (1) a phospholipid, which provides structure to the LNP bilayer and also may aid in endosomal escape; (2, 13) (2) cholesterol, which enhances LNP stability and promotes membrane fusion; (14, 15) and (3) lipid-anchored polyethylene glycol (PEG), which reduces LNP aggregation and “shields” the LNP from nonspecific endocytosis by immune cells.(16) The particular composition of the LNP can also have profound effects on the potency of the formulation in vivo. Several previous efforts to study the effect of formulation parameters on siRNA-LNP potency utilized the one-variable-at-a-time method,(17, 18) in which formulation parameters were individually varied to maximize LNP potency; this approach, however, does not allow for examination of potentially important second-order interactions between parameters. Inspired by statistical methodologies commonly used in the engineering and combinatorial chemistry literature,(19, 20) we chose to utilize Design of Experiment (DOE) to better optimize LNP formulations for nucleic acid delivery. Using DOE, the number of individual experiments required to establish statistically significant trends in a large multidimensional design space are considerably reduced, which is particularly relevant for the economical screening of LNP formulations: in vitro screens are often poor predictors of in vivo efficacy with siRNA-LNPs,(21) and it would be both cost- and material-prohibitive to test large libraries of LNP formulations in vivo.

To demonstrate the application of DOE to LNP formulation optimization in vivo, we formulated LNPs with a different type of nucleic acid than siRNA. Recently, messenger mRNA (mRNA) has been investigated for therapeutic protein production in vivo, including applications in cancer immunotherapy, infectious disease vaccines, and protein replacement therapy.(22, 23) Unlike plasmid DNA, mRNA need only access the cytoplasm rather than the nucleus to enable protein translation and has no risk of inducing mutation through integration into the genome.(24) Because there are inherent chemical and structural differences between mRNA and siRNA in terms of length, stability, and charge density of the nucleic acid,(25) we hypothesized that LNP delivery formulations for mRNA may require significant variation from those developed for siRNA delivery...


The authors developed a combinatorial approach using chemical libraries (compounds modified and mixed in a controllable but different way) to screen lipid formulations.

There are many hundreds of papers on the topic of lipid particles utilized in nucleic acid drug development, but for this brief post, we'll just look at the pictures produced here, and return to figure one for further discussion:



The caption:

Figure 1. Formulation of lipid nanoparticles. Lipid nanoparticles (LNPs) are synthesized by the mixing of two phases: (1) a four-component ethanol phase containing ionizable lipid, helper phospholipid, cholesterol, and lipid-anchored PEG; (2) an acidic aqueous phase containing mRNA.




The caption:

Figure 2. Efficacy results of LNPs in Libraries A, B, and C. (a) Serum EPO concentration 6 h post-intravenous injection of 15 μg total mRNA for each formulation in Libraries A and B, including the original formulation (data presented as mean + SD, n = 3). (b) A statistically significant trend of increasing serum EPO concentration was observed with increasing C12-200:mRNA weight ratio and with DOPE phospholipid for Library B formulations, independent of the other formulation parameters. Furthermore, a statistically significant second-order effect was observed between DOPE and increasing weight ratio, as indicated by the larger relative slope of the DOPE best-fit line compared to the DSPC best-fit line. (1 data point = 1 mouse) (c) Serum EPO concentration 6 h post-intravenous injection of 15 μg total mRNA for formulation B-26 and Library C, which had similar formulation parameters as B-26 with differing C12-200:mRNA weight ratios. (Data presented as mean + SD, n = 3.)


EPO here, is one of the early protein drugs on the market, one that got Lance Armstrong and other athletes busted, the anti-anemia drug erythropoietin, which jacks up hemoglobulin in the blood. The idea here is to insert RNA into cells in such a way that rather than being produced in a reactor, it is produced within the blood itself. (I would imagine that tracing this would make anti-doping efforts somewhat more challenging.



The caption:

Figure 3. Efficacy and biodistribution of original and C-35 formulation with Luc mRNA. (a) Efficacy of original and C-35 LNP formulations synthesized with mRNA coding for luciferase in three organs of interest as measured by total flux from luminescence 6 h after intravenous injection of 15 μg total mRNA. (Data presented as mean + SD, n = 3). (b) Representative biodistribution image of luciferase expression for original and C-35 LNP in seven organs as measured with an IVIS imaging system 6 h after intravenous injection of 15 μg of total mRNA.




The caption:

Figure 4. Efficacy of original and C-35 formulation with siRNA. Efficacy of original versus optimized C-35 formulation made with C12-200 and siRNA coding against Factor VII (FVII) protein as measured by serum FVII levels 72 h post-intravenous injection of various doses of total siRNA. FVII levels were normalized with respect to PBS-injected control mice. (Data presented as mean + SD, n = 3.)


The experimental portion of the paper, by the way, describes the synthesis of the mRNA itself that was used in this study; it is very "PCR" like, an exponential growth system. Certainly some clean up is involved, but I suspect this is not a bottle neck.

mRNA Synthesis

mRNA was synthesized by in vitro transcription from a plasmid DNA template encoding the gene, which was followed by the addition of a 5′ cap structure (Cap 1) using a vaccinia virus-based guanylyl transferase system. A poly(A) tail of approximately 300 nucleotides was incorporated via enzymatic addition employing poly-A polymerase. Fixed 5′ and 3′ untranslated regions were constructed to flank the coding sequences of the mRNA.


Now I'ld like to return to figure 1, which I repeat for conveninence, and make some comments.



Without having sourced it myself, I would imagine that cholesterol is easy to get from a variety of sources.

What are interesting are the other three molecules (which may represent classes of molecules in real cases or similar cases).

These I would imagine (and in one case, I know) are chemically synthesized in chemical reactors.

First the "ionizable lipid." There is an element of pseudosymmetry in this molecule, inasmuch as all the long carbon chains are C12, and are almost certainly obtained from lauric acid, the C12 fatty acid, which I know from experience is available in high quality from a number of sources. The core of the molecule is piperazine, the cyclic molecule in the center with two nitrogen atoms in the ring on opposite sides, also a commercially available reagent. Nevertheless, there is symmetry breaking inasmuch as the spacers in the two nitrogens on the piperazine ring have different spacers. This means to make the molecule, one must protect one (and only one) of the nitrogens, which is not entirely straight forward, or otherwise synthesize the ring in such a way that one nitrogen is protected, eliminating the advantage of using very cheap and readily available piperazine. My organic synthesis muscles are no longer strong enough to offer a route off the top of my head to the OH group that is one carbon removed from the amino nitrogens in those side chains, but I'm quite sure if one looks, one can find such a route.

Nevertheless, although very large reactors for making this "ionizable lipid" are certainly available under regulated ("GMP" ) conditions, this would be a bottleneck, if this particular (or a similar) "ionizable lipid" were utilized in formulating either the Moderna or the Pfizer vaccines.

The DSPC is a derivative of phosphocholine, which is found in many foods, notably eggs, and is fact found all over living organisms. In theory it can be made from one of the cheapest industrial chemicals there is, glycerol, a side product of the soap and biodiesel industries which is so cheap that people often dump it rather than sell it if they are far from a facility that uses it. The DSPC is a chiral molecule exhibiting "handedness" (being non-superimposable, like right and left hands). I'm sure that there are lots of enzymatic routes to making phosphocholine, but still, after one has it, one has to acylate the two oxygens with steric acid, also a commodity produced from things like palm oil, corn oil, canola oil...etc...etc. Still one needs a reactor to do this.

Finally there is there is "lipid anchored" "PEG," polyethylene glycol, which is also a derivative of phosphocholine, in fact, it is a derivative of DSPC. It is the PEG side chain that matters. The thing that disturbs me a little bit is the designation, at least in this paper, of a fixed length. PEG in most drugs - it is commonly used in many protein drugs to prevent their rapid breakdown in the body - is a polymer, and generally has a molecular weight distribution centered around a specific molecular weight, and also containing some molecules of higher and some of lower molecular weight. It is generally made by the polymerization of ethylene oxide (oxirane). I would hope that the formulations of the lipids in the vaccines do not actually in real life involve this restriction, since this would be problematic and represent a real bottle neck.

But even if it was...

One of the largest selling drugs in the world for many, many, many years was atorvastatin (Lipitor). It was discovered and initially developed at a company that no longer exists, Parke Davis. Here is the structure of atorvastin:



I was told a story about atorvastatin that I heard; it may or not be true. This drug was developed in the 1980's when industrial chiral synthesis was still somewhat problematic - it was a problem generally solved by the time HIV protease inhibitors came around - but early on it wasn't. I was told that Park Davis felt that atorvastatin would never be a big seller, because it was too expensive to make on an industrial scale, because of the seven carbon side chain with the two hydroxygroups on it, both of which are present as chiral centers. In the early days of scale up of the drug, the cost of the side chain was said to be over $1000/kg in 1980s dollars. I know people who were selling it for $800/kg in the 1990s.

Every chiral organic chemist in the world, both industrial and academic, looked at that side chain and figured that they could make a killing by making it cheaper; everybody in the world was going to get rich making it for $500/kg. Companies were founded on this expectation.

So many people exercised their genius to make the molecule cheaper that it ultimately became a commodity, and everybody was trying to undercut everyone else, because everyone's cheaper route made for an oversupply. I heard, that the cost of the side chain, before the expiry of the Park Davis successor company(ies) (ultimately Pfizer) the side chain was priced well under $100/kg, I heard $50, out of India and China.

So what's the story, morning glory?

Yeah, right now, there are bottlenecks, but they aren't going to last very long. Within a year, maybe sooner, these vaccines are going to be commodities, because, right now, there is clearly intellectual and almost certainly physical infrastructure to do it.

And that's a good thing. In the end, it will come down to logistics and little else.

Cheer up. We have a smart and energetic President who actually gives more than a rat's ass, vastly more than a rat's ass, as opposed to his orange predecessor, and we will beat this thing.

Some suggested further reading:

Martin A Maier, Muthusamy Jayaraman, Shigeo Matsuda, Ju Liu, Scott Barros, William Querbes, Ying K Tam, Steven M Ansell, Varun Kumar, June Qin, Xuemei Zhang, Qianfan Wang, Sue Panesar, Renta Hutabarat, Mary Carioto, Julia Hettinger, Pachamuthu Kandasamy, David Butler, Kallanthottathil G Rajeev, Bo Pang, Klaus Charisse, Kevin Fitzgerald, Barbara L Mui, Xinyao Du, Pieter Cullis, Thomas D Madden, Michael J Hope, Muthiah Manoharan, Akin Akinc, Biodegradable Lipids Enabling Rapidly Eliminated Lipid Nanoparticles for Systemic Delivery of RNAi Therapeutics, Molecular Therapy, Volume 21, Issue 8, 2013,

Dynamic PolyConjugates for targeted in vivo delivery of siRNA to hepatocytes
David B. Rozema, David L. Lewis, Darren H. Wakefield, So C. Wong, Jason J. Klein, Paula L. Roesch, Stephanie L. Bertin, Tom W. Reppen, Qili Chu, Andrei V. Blokhin, James E. Hagstrom, Jon A. Wolff, Proceedings of the National Academy of Sciences Aug 2007, 104 (32) 12982-12987; DOI: 10.1073/pnas.0703778104

Semple, S., Akinc, A., Chen, J. et al. Rational design of cationic lipids for siRNA delivery. Nat Biotechnol 28, 172–176 (2010). https://doi.org/10.1038/nbt.1602

Many, many, many more papers may be found by calling these papers up in Google Scholar, and clicking on the citations list.

I hope you will have a safe and enjoyable Sunday.

Has anyone here ever eaten a fruit called Durian?

I'm catching up on my reading, and I came across a paper titled thus: Food Waste Durian Rind-Derived Cellulose Organohydrogels: Toward Anti-Freezing and Antimicrobial Wound Dressing (Xi Cui, Jaslyn Lee, Kuan Rei Ng, and Wei Ning Chen, ACS Sustainable Chemistry & Engineering 2021 9 (3), 1304-1312).

It contains this text:

Durian is a fruit which is famous all over the world, especially in Southeast Asia as its name is “king of the fruits”. Durian is popular for its special flavor and high-quality nutrients, which could afford health benefits for the human body. However, less than a half part of the entire durian is edible, while the other parts (rind and seeds) are treated as food waste in the durian industry.(18) Durian residues (rind and seeds) are usually used in landfills or burned, which pose a serious problem to the environment and, in the meantime, are also a waste of the natural resource. Durian rind is composed of 31.6% cellulose, 15.5% hemicellulose, and 10.9% lignin in dried durian rind.(19) The content of cellulose in durian rind is larger than that in okara,(20) which makes durian rind a sustainable cellulose resource for hydrogel fabrication, and this can also help reduce the environmental pollution.


Famous all over the world?

Serious problem to the environment?

Never heard of Durian...

Anyone?

PPPL Science on Saturday: From studying the Sun, to searching for dark matter, to fighting Covid.

PPPL's science on Saturday lecture tomorrow morning by Princeton University Professor Professor Cristiano Galbiaiti giving a talk entitled: From studying the Sun, to searching for dark matter, to fighting COVID-19

Sign up here:

Science on Saturday, on Zoom

Almost always the talks are fascinating. Although, the Princeton Plasma Physics Lab is an National Physics Lab, the talks, while often involving physicists (as tomorrow's will), also include, biologists, geologists, chemists, social scientists, oceanographers, climate scientists, etc.

They are hosted by the charming and fun Dr. Andrew Zwicker, head of science education at the lab, who also moonlights as the Democratic NJ Assemblyman in the NJ Legislature.

In previous years, the talks were held at PPPL with a very nice social hour before hand featuring coffee, donuts (great donuts!) bagels and interesting conversation.

As with Covid, the talks have moved to Zoom, they are now accessible around the world. They are held at 9:30 am EST, with a Q&A session after the talks ending usually by 11:30. The talks themselves are about an hour generally.

Check it out!

A fabulous soporific horror book.

I'm a chronic insomniac, but I just found a great tool for going to sleep which isn't pharmaceutical.

I remember when I was 18, having my first college adventure, the arguments between the Trotskyites and the Leninists in the student center, with a few Maoists thrown in the mixture just for fun.

It was New York; what can I say?

I had no idea whatsoever what it was all about, having been raised to think a commie was a commie, and being something of a Rube, it all seemed so excitingly sophisticated. I never did learn to tell the difference.

Eventually arguing over Trotsky and Lenin fell out fashion, thankfully, but in the back of my mind, as an old man, I found myself wondering, what the hell all that passionate stupidity was all about. I mean, I knew it was stupid somehow, but never knew why.

Lately I've been reading Dmitri Volkogonov's Stalin, Triumph and Tragedy. Volkogonov was a Soviet Colonel General who was charged with psychological warfare, and who, given access to the Soviet archives before the fall of the Soviet Union, became a historian. When he died, donated his papers to the US Library of Congress.

It's fascinating, because it gives a behind-the-scenes account of the rise of Stalin who, by aligning himself with Lenin's dead body, defeated Trotsky via the duplicitous manipulations of the weather vane duo Zinoviev and Kamanev who, as sophisticates, felt they could manipulate and control the rise of either Stalin or Trotsky. It was a bad bet. Zinoviev and Kamanev were executed by Stalin in the 1936 Purges.

However, one cannot read Soviet history, especially as it involves Soviet Marxist theology/"theory" without eventually thinking of conversing with Brezhnev about wonderful Soviet tires made of concrete and licorice. Three of four pages, tops, and your eyes are heavy. By the fifth page, you're asleep. Better than Ambien, safer too.

The rise of Stalin is, of course, a horror story, and one feels trepidation because you kind of know the ending, but it is fascinating because Stalin rose by seeming reasonable. I'm coming up on the great Ukrainian famine. It's scary, if one can stay awake through it.

I'm just getting to the point where we learn of "Socialism in one country," of which I've heard in reference to Stalin, but I never actually understood the background and how it differed from Trotskyism.

I wish I was 18 again, and could join in the fun in the student center.

I wouldn't care, but I do need to sleep in order to work well.

I hope I cheered my doctor up.

I had to go see my doctor for a problem not related to Covid-19. As it happened, my son had seen him earlier, whereupon my son reported that he was basically freaking out about the vaccine/mutation situation.

Apparently he was thinking it was never going to end, and was extremely stressed about the vaccine supply apparently because the State of New Jersey had a disconnect with the status of his vaccine supplies and the people who could get it and now he was forced to field a lot of calls from anxious and sometimes angry people. He was worried about the mutants.

So after the exam was done, I asked him, cheerfully, if he was having "fun" with Covid. He looked really frazzled and said, "No! No!"

Happily - unhappily because it was a disease - I am a veteran of the industrial scale up of HIV protease inhibitors, and was intimately aware of what people go through under such a circumstance, a circumstance wherein a logistical screw up, or perhaps, a regulatory or process screw up means, literally, that people will die. So you do everything you can to do everything right.

It's serious, a huge responsibility, but in those times, one in which pharmaceutical teams, I'm happy to say, rose to the occasion - you saw the best in people mostly, and very little of the worst. I was very, very, very proud of the opportunity to have been involved.

"Look," I told my doctor, "for about a year, I was dealing with people begging me for supplies and then, all of a sudden, the whole thing dried up, because so many players rushed in that eventually there was an over supply."

As for mutations, I said, I know, I know, I know, retroviruses suck because they mutate so rapidly. But I told him, and of course he knows this, that this is reason we now give HIV protease inhibitor cocktails, because any virus with a single nucleotide polymorphism, or even a polynucleotide polymorphism is going to be hit by one or the other.

We saw these SNP's and PNP's all the time, but lives are still, almost 30 years later, being saved with protease inhibitors.

I told him about the glycosylation patterns on the coronavirus, contrasting them with HIV, and letting him know that this was a different, easier, ball game and finally about how these nucleotide drugs are easy to re-engineer to a moving target.

"Look," I said, "it's going to be, at the worst case, ultimately, like when you ask me to get a flu shot every year. You're going to ask me did I get my annual Covid shot. The infrastructure will be there, and sales guys will be calling on you to tell you why their vaccine is better than the other guy's, begging you to use theirs."

Expecting a doctor to really know about the industrial operations of the pharmaceutical industry is rather like expecting your car mechanic to know about how an auto parts plant works. Your mechanic doesn't need to know about the metallurgy of a water pump to fix your car. He just needs to know if the part fits and if it works.

I felt my doctor felt better. I hope so. He's under a huge amount of strain, and I could see it.

I feel better because adults are in charge of the country again, which makes everything better automatically. It doesn't mean we're "there yet," but at least we know where we're going.

Life is interesting, and then you die.

Why cats go crazy for catnip.

The following is a news item in the premier scientific journal Science. It should be open sourced, and is here: Why cats are crazy for catnip (Sofia Moutinho, Science, January 20, 2021).

Some excerpts:

Cat owners flood the internet with videos of their kitties euphorically rolling and flipping out over catnip-filled bags and toys. But exactly how catnip—and a substitute, known as silver vine—produces this feline high has long been a mystery. Now, a study suggests the key intoxicating chemicals in the plants activate cats’ opioid systems much like heroin and morphine do in people. Moreover, the study concludes that rubbing the plants protects the felines against mosquito bites.

“This study essentially has revealed a new potential mosquito repellent” by examining the “pharmaceutical knowledge” of cats, says Emory University biologist Jacobus de Roode, who did not participate in the study.

Catnip (Nepeta cataria) and silver vine (Actinidia polygama) both contain chemical compounds called iridoids that protect the plants against aphids and are known to be the key to the euphoria produced in cats. To determine the physiological effect of these compounds, Iwate University biologist Masao Miyazaki spent 5 years running different experiments using the plants and their chemicals.

First, his team extracted chemicals present in both catnip and silver vine leaves and identified the most potent component that produces the feline high: a minty silver vine chemical called nepetalactol that had not been shown to affect cats until this study. (The substance is similar to nepetalactone, the key iridoid in catnip.) Then, they put 10 leaves’ worth of nepetalactol into paper pouches and presented them, together with pouches containing only a saline substance, to 25 domestic cats to gauge their response. Most of the animals only showed interest in the pouches with nepetalactol...

...Next, the researchers measured beta-endorphins—one of the hormones that naturally relieves pain and induces pleasure by activating the body’s opioid system—in the bloodstreams of five cats 5 minutes before and after exposure. The researchers found that levels of this “happiness hormone” became significantly elevated after exposure to nepetalactol compared with controls. Five cats that had their opioid systems blocked did not rub on the nepetalactol-infused pouch...


It appears, however, that nepetalactol also repels mosquitos:

But the researchers wanted to know whether there was a reason for the cats to go wild, beyond pure pleasure. That is when one of the scientists heard about the insect-repelling properties of nepetalactone, which about 2 decades ago was shown to be as good as the famed mosquito-stopper DEET. The researchers hypothesized that when felines in the wild rub on catnip or silver vine, they’re essentially applying an insect repellant.


If you have a cat, and have seen him or her around catnip, you knew, at least intuitively, that it acts on opioid receptors. I didn't know about mosquitos however. I might try some myself.

The full scientific paper to which this article refers is here, and is, I believe, open sourced: The characteristic response of domestic cats to plant iridoids allows them to gain chemical defense against mosquitoes (REIKO UENOYAMA, TAMAKO MIYAZAKI, JANE L. HURST, ROBERT J. BEYNON, MASAATSU ADACHI, TAKANOBU MUROOKA, IBUKI ONODA, YU MIYAZAWA, RIEKO KATAYAMA, TETSURO YAMASHITA, SHUJI KANEKO, TOSHIO NISHIKAWA, MASAO MIYAZAKI, SCIENCE ADVANCES 20 JAN 2021 : EABD9135)

Nepetalactol:

The cabinet page at Whitehouse.gov

When I need to be happy and confident in the future, I'll look at it.

This is America:

The Cabinet, Whitehouse.gov

Recovery of Trivalent Lanthanides and Transplutonium Actinides with Resin Supported Diglycomides.

The paper I'll discuss in this post is this one: Scaling Trivalent Actinide and Lanthanide Recovery by Diglycolamide Resin from Savannah River Site’s Mark-18A Targets (Kevin P. McCann, Mark A. Jones, Edward A. Kyser, Tara E. Smith, and Nicholas J. Bridges Industrial & Engineering Chemistry Research 2021 60 (1), 507-513).

The elements in the two rows below the "main" periodic table are collectively called the "f elements." The row beginning with the chemical symbol Ln (lanthanum) are called "the lanthanides" - and somewhat more commonly in an annoying and misleading term, "rare earths" - and the row beginning with the chemical symbol Ac (actinium) are called the "actinides." They are placed below the main elements of the periodic table only to make the table fit nicely in the width of a sheet of paper. Properly drawn they represent another "step" in the step shapes in the main part of the table, properly the table should have 32 columns, not 18, although the congener relationship in purely chemical terms between, say protactinium and praseodymium is weak and not all that worthy of consideration.

The lanthanides, with some exceptions, generally exhibit the +3 oxidation state, the "trivalent" state, because the "f orbitals" which are being filled across the row do not participate to any appreciable extent in chemical bonding because of shielding effects.

This is not true for the lower actinides before americium, only actinium itself exhibits only the trivalent state. For a very long time, until the 1940's, when interest in actinide chemistry exploded - no pun intended - thorium was thought of as a congener of hafnium and zirconium, because like them, its most common oxidation state is +4, and protactinium was considered a congener niobium and tantalum because its common oxidation state is +5, and uranium, a congener of molybdenum and tungsten because of its common oxidation state of +6.

Actinium, thorium, protactinium, and uranium all occur naturally in weighable amounts, thorium and uranium on a billion ton scale - their decay is largely responsible for the internal heat of the Earth - actinium and protactinium occur in trace amounts, in concentrations so low that they are best accessed not by isolation from the ores in which they occur, but by the use of nuclear reactions, neutron or proton bombardment.

In 1940's, as he worked on the discovery of new synthetic elements in the periodic table, especially neptunium, plutonium, americium and curium, Glenn Seaborg had the insight that the chemistry of these elements could be discerned by recognizing that they were, in fact, "f elements" as opposed to "d elements" like hafnium, tantalum, and tungsten.

The actinides become "lanthanide-like" at americium. Although americium can be oxidized to higher oxidation states, it's major oxidation state is +3; this is also the case with curium, berkelium and californium.

When I was a kid, the first mass spec with which I was used by one of the companies for which I worked had a californium ionization source; I'm an old guy. (Modern mass specs have other types of ionization inductions, notably electrospray ionization (ESI) for which John Fenn won the Nobel Prize.) The californium where I worked (in California) was the 252 isotope, which has a half-life of about 2.64 years, decaying both by spontaneous fission and by alpha decay to curium-248. The spontaneous fission of californium-252 made it a useful source of neutrons, and it was widely used in chemical analysis using neutron activation analysis, which has been mostly displace by high sensitivity ICP/MS instruments that for most elements can record parts per trillion.

Perhaps a current motivation, particularly in the days of antibody payloads is in neutron boron therapy, where small portable Cf-252 sources might displace the need for expensive accelerators, particular in rural or remote regions: Boron neutron capture therapy: Current status and future perspectives (Dymova, M.A., Taskaev, S.Y., Richter, V.A. and Kuligina, E.V. (2020), Cancer Commun., 40: i-i.)

In the 1960s and 1970's an effort was made to produce large (large being milligram quantities) of californium-252 as a neutron source and as an ionization source. (Mass specs in space robots generally use curium sources because of their longer half-lives.)
Another important isotope is plutonium-244, which has a half-life of 80 million years, and which is used as an internal standard in actinide analysis, and as a target for super-heavy element analysis.

The United States is running out of plutonium-244, and the paper listed above is about recovering it, as well as the transplutonium elements therein. The introduction to the paper covers things on which I touched above:

During the late 1960s to the late 1970s, 86 targets containing 8 kg of high-assay 242Pu (Mark-18A targets) were irradiated under high thermal neutron flux (up to 10^(15)) neutrons per cm2 per second) in K-Reactor at the Savannah River Site (SRS). The original intent of irradiating Mark-18A targets was for large-scale production (milligrams) of 252Cf from 242Pu via a series of neutron capture and beta-decay reactions.(1) Twenty-one of the targets were processed in 1971–1973 at Oak Ridge National Laboratory’s (ORNL) Radiochemical Engineering Development Center to recover 252Cf for market development.(2,3) Additionally, the United States’ current supply of 244Pu, approximately 7 g, was recovered from the 21 targets. The United States’ supply of 244Pu is depleting due to its use as a standard reference material for nuclear forensics and in research applications such as superheavy element production.(4,5) The targets also contained hundreds of grams of heavy curium, 246–248Cm. Heavy curium serves as an ideal feedstock for 242Cf, 249Bk, and 252Es production at the ORNL High Flux Isotope Reactor (HFIR). Californium-252 has several industrial uses including a neutron source for various industries, neutron activation analysis, radiotherapy, fundamental research into actinide elements (along with Bk and Es), and heavy element synthesis.(6−9) Due to the increased demands for 244Pu and heavy curium, the Department of Energy (DOE) tasked the Savannah River National Laboratory (SRNL) to lead the design and implementation of a separation flowsheet to recover the rare plutonium and heavy curium materials from the remaining 65 Mark-18A targets.

To recover the valuable materials, the Mark-18A material recovery flowsheet separates the target’s aluminum cladding by caustic dissolution, leaving most of the fission products and the actinides as a solid material. The undissolved material is filtered and then subsequently dissolved in >7 M HNO3 at elevated temperatures. The resulting high nitrate solution, containing dissolved plutonium, actinides, and most of the fission products, will be sent through an anion exchange column using Reillex HPQ resin, similar to its use in the SRS’s HB-Line Facility.(10−13) Reillex HPQ achieves Pu/Am decontamination factors on the order of 14,000.(11,12) The high decontamination factors are a result of Reillex HPQ’s high selectivity for the Pu(NO3)6 2– anion at >7 M HNO3 and little to no affinity for lanthanides (Ln), americium, curium, and fission products. As a result, in the Mark-18A material recovery flowsheet, the Ln, Am, Cm, and remaining fission products remain in the raffinate and require additional processing to recover heavy Cm from the >7 M HNO3 raffinate...


The Reillex HPQ resin is an anion exchange resin, a polyvinyl-N-methylpyridinium resin, that intereacts with the anionic plutonium (IV) hexanitrate anion (-2). In Reillex 402, the methyl group is replaced by a proton, in other N-pyridinium alkyl primary ammonium groups of various chain lengths to give a dicationic species.

The structure of these complexes are nicely shown in this cartoon:



Molecularly Engineered Resins for Plutonium Recovery

(S. Fredric Marsh, D. Kirk Veirs, Gordon D. Jarvinen, Mary E. Barr, and Eddie W. Moody, Los Alamos Science 26 (2000) 454-463)

The Los Alamos people note that some of the designed resins may also extract transplutonium nitrate complexes, but it would appearl that straight up Reillex HPQ (the methyl pyridinium is selective toward tetravalent plutonium nitrate complexes) and thus can be utilized in the current setting, where the target are trivalent species, specifically transplutonium actinides and lanthanides.

To save the world, the required inventory of plutonium - even in the "breed and burn" scenario where only a critical mass (realistically a little more) is required for start up - is on the order of hundreds of metric tons, and it is unrealistic that Reillex HPQ would be of much use on that scale.

These charged resins thrill, perhaps in the form of ionic liquids, the imagination about the possibility of electroprocessing.

I recall that when I was writing over in the E&E forum a lot, an anti-nuke of the particularly dull sort announced that nuclear energy was "too dangerous" because a tunnel at the Hanford site which had been abandoned collapsed. Pretty typically this person who is happily on my ignore list was far less interested in the 7 million people who died last year from air pollution while dumb guys get wedgies in their underwear about tunnels built in the 1950's which may contain (gasp) radioactivity. On investigation, it turns out that the tunnel contained some old abandoned chemical reactor vessels for plutonium purification ob rail cars, with trace plutonium on their surfaces. The number of lives lost associated with the collapsed rail tunnel was zero.

Reillex HPQ would be good to decontaminate the decontamination wash solutions to clean off the chemical reactors such as may exist from reprocessing efforts, but not for large scale processing. Let's be clear though, on a scale of risk, when compared with the very real and rising catastrophe of dangerous fossil fuels, unless one is a complete idiot - and sadly complete idiots exist - plutonium stains on a 50 year old chemical vessel is a non starter.

But in the case of the Mark-18A targets, these contain curium on a scale of a few hundred grams, and thus the resins have much to recommend them. Since the quantities are relatively small, but extremely valuable, column scale separations are entirely acceptable.

The authors note that during the last campaign to milk the Mark-18A targets for their valuable components, which took place in the early 1970s, a Dowex anion exchange resin was utilized. A difficulty with this resin, although it was clearly workable, was the release of sulfate from the sulfonylphenyl groups on which this relatively primitive anion exchange resin was based. Sulfates tended to contaminate the eluted products and raffinates, requiring additional clean up steps. Moreover, combustion of the used resins was problematic.

The more modern resin under discussion here contains only carbon oxygen and thus can be readily destroyed in an appropriate oxidation setting designed to contain residues.

The historical separation process is described as follows:

An Am, Cm, and Cf separation method was developed at the SRS around 1971 using a pressurized ion exchange process that could separate Am, Cm, and Cf.(14−16) The column separation utilized a styrene and divinyl benzene copolymer resin (Dowex 50W) containing sulfonic acid functional groups to recover and separate the transplutonium elements over four successive columns.(16,17) The four 4 ft tall columns decreased from 4 in. diameter down to 1 in.(16) Elements were loaded onto the column from a nitric acid solution and were eluted by a 0.05 M diethylenetriamine pentaacetic acid solution. Fermium came off first and then Es, Cf, and Bk followed by Cm and Am. The elution bands were monitored by an in-line BF3 tube to detect neutrons from 252Cf and 244Cm and lithium-drifted germanium detector and low-energy gamma spectrometer for 244Cm and 243Am.(14,18)


To prevent leaks, the authors were looking for a way to avoid the use of pressure, hence the evaluation of a new approach to the separations. All of the fermium and einsteinium in the targets has now decayed to lower actinides, of course, so they are no longer relevant to the case.

They write:

A well-known trivalent lanthanide and actinide extractant that performs in high-molar nitric acid is the N,N,N′,N′-tetraoctyldiglycolamide (TODGA) ligand.(22−25) In commercial production, TODGA is physisorbed onto a polymethyl methacrylate (PMMA) backbone resin and sold under the trade name DGA Resin (Eichrom Technologies Inc.). The resin is often used to concentrate Am and Cm for bioassay analysis using prepackaged 1–2 mL columns. The bioassay samples typically contain very few elements competing for binding sites on DGA Resin, unlike the significant amounts of both actinides and fission products that will be present in the dissolved Mark-18A targets.(24) As a result, fission products will be in competition for binding sites with the desired actinide material. The purpose of this study was to determine the retention of fission products on DGA Resin to optimize the mass loading of Am and Cm found in the Mark-18A targets for gram-scale recovery. Furthermore, the capability of in-line UV/vis spectroscopy to monitor breakthrough in the raffinate was also tested. The results will be used to design and implement a unit operation to recover Am and Cm from Mark-18A targets at the SRNL.


Although I do not have access to the exact structure of the commercial DGA resin, the structure of these resins is probably something along these lines:



(cf Mohapatra et al., RSC Adv., 2014,4, 10412-10419)

The authors conducted two tests, one using non radioactive fission product simulant with neodymium standing in for the actinides, and a second, also loaded with a fission product simulant spiked with the actinide americium.

Two Mark-18A target simulants were generated by dissolving representative elements, primarily as metal nitrate compounds in 7 M HNO3. The metal concentrations for Mark-18A Simulant 1 (non-radioactive test) and Simulant 2 (radioactive test) are listed in Table S1. The primary elements in the Mark-18A simulants were Mg, Al, Fe, Zn, Sr, Zr, Mo, Ru, Pd, Sn, Cs, La, Ce, Pr, Nd, Sm, Eu, Gd, and Re. Zinc was used as a surrogate for Cd, Re was used as a Tc surrogate, and extra Nd was added as a surrogate for Am and Sm for Cm. To study bounding conditions, Simulant 1 represented a concentrated simulant based off initial Mark-18A target acid dissolution volumes. Simulant 2 represented a more dilute solution due to processing the dissolved targets through other unit operations downstream of target dissolution.


The flow chart calls for the dissolution of the aluminum clad targets with caustic (which dissolves aluminum), leaving behind a solid residue of oxides which are then dissolved in 7M nitric acid. It is the nitrate complex which is separated as anionic species by the resin.


Some pictures from the text:




The caption:

Figure 1. Kinetic results showing adsorption of listed metals from Mark-18A Simulant 1 to DGA Resin at varied contact time lengths.




The caption:

Figure 2. Results of the 8 mL column run at 3 BV/h. Two bed volumes of the simulant through the column (16 mL) followed by three bed volumes of 7 M HNO3 wash (24 mL). Data presented as a ratio of metal concentration in the raffinate to starting concentration in the feed simulant.




The caption:

Figure 3. Percent retention of select elements by DGA Resin at varied flow rates. Values are reported with an overall uncertainty of 20%.





The caption:

Figure 4. Element breakthrough of Mark-18A Simulant 1 through the 52 mL DGA Resin column at a 3 BV/h flow rate. Values are reported with an overall uncertainty of 20%.





The caption:

Figure 5. Element breakthrough of Mark-18A Simulant 2 (241Am tracer) through the 77 mL DGA Resin column. Values are reported with an overall uncertainty of 20%.


A new wrinkle in this method as opposed to the method utilized in the 1970s also concerns the detection. For the purposes of these experiments, the complexes were monitored by their UV absorption spectra.



The caption:

Figure 6. Visible absorbance spectra of the raffinate from Mark-18A Simulant 2 after processing through the 62.5 mL DGA Resin column.





The caption:

Figure 7. Baseline-corrected Nd(NO3)3 579 nm peak measured at various raffinate volumes after processing through the 62.5 mL DGA Resin column.


The authors conclude as follows:

The proposed recovery method for the trivalent actinides and lanthanides by DGA Resin from the Mark-18A simulant was validated. Batch contact experiments showed that the trivalent lanthanides and zirconium have a high affinity for the resin in 7 M HNO3. Elements such as Mg, Al, Zn, Cs, Ba, and Sn are not adsorbed onto the DGA Resin. Some transition metals such as Fe, Mo, Ru, Pd, and Re show low to moderate affinity but will be easily displaced by the more favored trivalent lanthanides and actinides. Breakthrough curves in column experiments illustrated that chromatographic separations occur, as indicated by high concentration gradients in the raffinate for retained elements. Lanthanides break through the column in the order of increasing atomic number...

...Americium tracer experiments validated that Am breakthrough closely follows Nd breakthrough. An in-line UV/vis spectrophotometer was able to track the ingrowth of Nd in the raffinate solution. In full-scale processes, detection of Nd in the in-line UV/vis system will indicate Am breakthrough and serve as a warning that Cm breakthrough is forthcoming. Future work will focus on column design for the full-scale process in SRNL hot cells. Additionally, research is underway to apply the CHON principle and incinerate the loaded DGA Resin to reduce volume while directly converting the loaded metal to a stable oxide form for shipment.


Cool paper I think, with some application, for cleaning chemical reaction vessels used in the essential - if we are to save the world - separation of the higher actinides.

I trust you are enjoying, as much as I am, the first weekend in the already magnificent Presidency of Joe Biden, and are doing so safely.

Conspiracy (2001), Kritzinger's Warning.

The Wannseekonference took place on January 20, 1942. The conference lasted about 2 hours, and was conducted as a lunch meeting. It was held to plan the murder of the Jews of Nazi occupied Europe, and discussed plans for the murder of Jews in countries that had not yet been conquered by Germany, Great Britain, for instance.

The participants were highly educated and efficient men.

The conference was organized by Reinhard Heydrich, a talented musician, skilled athlete who was educated at a German Naval Institute. He later became the "protector" of Moravia and Bohemia, where he was assassinated by British trained Czech partisans several months after the Wannsee conference. Heydrich is considered by historians to have been one of the purest embodiments of pure Nazi evil.

Notes from the meeting were discovered in 1947 from the files of Martin Luther, a minor Nazi official, probably the lowest ranking figure at the meeting, who had, in fact, ended up in a concentration camp himself before the war was over.

Several films, including documentaries have been made about the Wannsee conference, one in German in 1984, another in English in 2001, called "Conspiracy." It stars Kenneth Branagh in a chilling portrayal of Heydrich.

If one has ever been to a high level executive meeting in any capacity, one can recognize the tenor of the meeting as portrayed in "Conspiracy." The goal is to "get things done" while schmoozing in a genial way, juggling for position, clarifying issues, coming to a final agreement and understanding.

In this sense it is frightening, and I advise young people "on their way up" in their careers to study it in this light, to see how easy it can be to slip into a kind of officiousness that is divorced in every way from morality, how easy it is to divorce one's self from ethics by "going along," "not making waves." There is surely an element of that in all kinds of business meetings, including those where the business is criminal.

A scene in the film came to mind, because I have felt myself being consumed with rage and anger and, yes, frankly hatred over the last four years.

The scene comes at the end of the film, after all of the participants in the conference have left, after agreeing in an affable business-like sense to murder all of Europe's Jews in a two hour meeting. Heydrich (Branagh), Adolf Eichmann (played by Stanley Tucci), and Heinrich Mueller, head of the Gestapo, sit together to have a drink and commiserate about the outcome of the meeting. They discuss a story told by Friedrich Wilhelm Kritzinger, a State Secretary in the chancellery, who is portrayed in the film as having had reservations about the murder of the Jews, although it is not clear in the historical record that this actually was the case for the real Kritzinger.

The scene has stuck in my mind for many years as a kind of warning should one let oneself be consumed and defined by hatred.



No one here, I'm convinced, is consumed by race based hatred like Heydrich's Eichmann's and Mueller's hatred of the Jews, but I know for myself, I have repeatedly felt consumed by perhaps a more justified hatred over the last 4 years, which, irrespective of its origins and its justifications, is hatred all the same.

Speaking only for myself, in these days of liberation and relief, I want to let go of it; as hard as it is, I want to let go of the hatred. It does nothing good for me.

Still high...

Amanda Gorman's poem deepens my conviction that this coming generation is going to a great generation like none we've seen, and Yo Yo Ma, the beautiful comforting voice, the happiness, the Amazing Grace.

And then there's that old white guy, sprinting around the White House filled with fire, as if with a magic wand, expelling demons.

That old white guy makes me feel so much better about being an old white guy, which has been something of an embarrassment of late.

Suddenly I feel, at least, the joy of youth.

Yo Yo Ma, how beautiful, how unbelievably beautiful:



This old man is glad to have lived to see this!!!!!
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