RNA vaccines have never been manufactured before, so making millions of doses was bound to take time. Here’s how it’s done.

A few short months ago, there was a fair amount of public skepticism that RNA vaccines would work against COVID-19, given that no such drug had been approved for preventing any other disease.

Now that the injections have been shown to offer near-total protection, millions want to know why they can’t have one immediately.

Blame balky sign-up systems, the need for transport and storage of the drugs at ultracold temperatures, and poor coordination among various levels of government, among other bottlenecks. But ultimately, success depends on the source: a handful of high-tech facilities where stainless-steel bioreactors are steadily synthesizing genetic code to quench the pandemic.

Supply so far has fallen short of pent-up worldwide demand, but industry experts say there were bound to be challenges in making millions of something that had never been made before. And the pace is picking up.

Compared with the lengthy process for producing most traditional vaccines — some flu vaccines are grown in chicken eggs, for example — the RNA approach is expected to be more nimble and efficient in the long run, both for this disease and others. Add the government investment that allowed the makers of the RNA vaccines to start setting up their factories last year before knowing whether the drugs worked, and the result has been dramatic, said Parviz Shamlou, executive director of the Jefferson Institute for Bioprocessing.

“It is remarkable,” said Shamlou, whose industry training institute, located in Lower Gwynedd, is part of Thomas Jefferson University. “It’s never been done in human history.”

For a crash course in the manufacturing technology, we spoke to two scientists whose RNA research at the University of Pennsylvania, starting more than 15 years ago, was instrumental in making the vaccines possible.

They are Katalin Karikó, now a senior vice president at BioNTech SE, the German firm that joined with Pfizer Inc. to produce one of the RNA vaccines, and Drew Weissman, an immunologist at Penn’s Perelman School of Medicine.

The underlying science for synthesizing RNA has been established for decades. From start to finish, small batches can be made in a laboratory in just a week or two.

But it is one thing to make it in a lab, using a robot arm to make less than a quarter-teaspoon of fluid at a time. Producing millions of doses in a high-tech factory?

Karikó likened it to the difference between cooking dinner for two and putting on a banquet for hundreds. The equipment is far larger and more sophisticated. Staff must be trained. Ample quantities of raw materials must be secured from specialty suppliers.

“These things are not sitting on somebody’s shelf, because nobody wanted it before,” she said.

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Quality control is on another level entirely. For a product destined to be injected into the human body, regulators require every step to be checked and double-checked. Each batch of product is tested for purity and consistency. Facilities must have “negative-pressure” systems to ensure that air from one room does not mix with air from another. Protective gowns and shower caps are worn.

And, oh, the record-keeping. Weissman, who is currently advising the government of Thailand on how to set up its own RNA vaccine facility, said the required documentation can seem more taxing than the manufacturing itself.

“The joke is that there are two people watching one person work to keep the records of what they’re doing,” he said.

Remember that most traditional vaccines consist of a weakened or inactivated form of a virus, enabling the recipient’s immune system to develop customized defenses in the event of a live infection.

The RNA vaccines, on the other hand, consist of genetic instructions for human cells to make just a fragment of the coronavirus: the familiar “spike” proteins that protrude from the surface of each virus particle. The key is packaging that recipe in a form that can be delivered inside human cells.

The first step: Writing the code and making lots of copies.

This is done by inserting the spike recipe into bacteria, not viruses, because it is easy to grow lots of them — using large, stainless-steel tanks called bioreactors. Each time the bacteria divide and multiply, there is a new copy of the recipe.

Although the bacteria are useful for making lots of copies, the version of the code inside them is not the right form for use in an RNA vaccine. Instead of RNA, it is “written” using RNA’s cousin, DNA, nestled inside a circular region of genetic material called a plasmid. So in the next step, it must be transcribed.

Chemicals are used to extract the plasmid DNA from all those bacteria. The DNA then serves as a template for making RNA: the version of the code that is needed for the actual vaccine.

Enzymes are used to snip open the bacterial DNA and “linearize” it. Other enzymes are used to assemble the RNA copy, using the DNA template as a guide.

“One letter at a time, it kind of marches down the DNA and it adds a letter to the RNA, corresponding to the one in the DNA,” Weissman said.

Think back to biology class. RNA is comprised of four different chemical bases, or letters: A, G, U, and C. In the case of the spike protein, the RNA code is 5,000 letters long.

The form of RNA used in the vaccines is called messenger RNA. That’s where Moderna, the maker of the other RNA vaccine for COVID, got its stock ticker symbol: MRNA.

Once it is made, the RNA undergoes multiple purification steps and testing. The genetic molecules are then encapsulated into tiny spheres, made of waxy substances called lipids.

This requires a fancy bit of chemistry, with the end result a high-tech vaccine delivery vehicle: billions of microscopic particles with genetic code packaged inside. The outer coating of lipids protects the RNA until it is injected.

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When these waxy spheres encounter the membrane of a human cell (which is also made of lipids), it is swallowed inside. The genetic recipe is delivered. The human cell responds by making the spike protein. And the immune system recognizes it as a foreign presence, and gets to work crafting customized defenses.

But before the needle delivers that dose into a person’s arm, first comes the cold chain.

Many the coordinator of a vaccine clinic has lamented the low temperatures needed to store the RNA vaccines.

The one made by Pfizer and BioNTech must be kept at minus 94 degrees Fahrenheit, though it can be moved to a refrigerator for five days before use. The Moderna vaccine requires freezer storage at minus 4 degrees, though a refrigerator is OK for 30 days before it is administered.

But in a sense, that reflects a positive attribute of these drugs. The reason RNA must be kept in freezers is because otherwise it degrades quickly. That’s what happens once it is injected in the human body. It delivers the recipe and degrades within a week or so, with no lasting effects beyond immunity to the coronavirus.

Nevertheless, that means that until they are administered, the vaccines require special treatment.

First, the lipid spheres are coated in sugars, which allows them to remain stable and distinct from each other when frozen, rather than freezing into one big ball. The resulting mixture is then loaded into vials, which are stored in large industrial freezers, such as those at Pfizer’s “freezer farm” in Kalamazoo, Mich.

When it comes time for delivery, the vials are packed in dry ice and fitted with GPS-enabled thermal sensors, allowing the company to monitor the location and temperature of each batch.

Lots of moving parts. Could it move any faster? Maybe, but it’s not just about one company operating one facility.

From start to finish, each vial of fluid requires the involvement of dozens of partners. Last month, for example, BioNTech announced that fellow drugmaker Sanofi had agreed to fill and package the RNA vaccine at its Frankfurt, Germany, facility starting this summer.

Weissman likened the complexity to what is involved in manufacturing a car or airplane.

“It’s a constantly turning wheel,” he said, “where everybody is dependent on everybody else.”

More vaccines made with other technologies are on the way. But at tens of millions of doses and counting, RNA appears here to stay.