Industrial engineers have to understand current processes used in production systems, alternative engineering methods available in various steps of the process, visualize ideas or concepts that will give better performance - productivity, analyze the benefit and recommend.
To provide IE suggestions for improving covid vaccine manufacturing process, IEs have to first understand the manufacturing processes of covid vaccines.
Information on Manufacturing Processes of Vaccines
"How Pfizer Makes Its Covid-19 Vaccine"
https://www.nytimes.com/interactive/2021/health/pfizer-coronavirus-vaccine.html
It is a complex manufacturing and testing process that takes 60 days.
STEP 1
Pull DNA from Cold Storage
Vials of DNA from the master cell bank is the source of every batch of Pfizer’s Covid-19 vaccine. The vials are kept at –150°C (–238°F) or below, and they contain small rings of DNA called plasmids.
Plasmid is Coronavirus spike protein gene.
Each plasmid contains a coronavirus gene, the genetic instructions for a human cell to build coronavirus proteins and trigger an immune response to the virus.
The plasmids are thawed. A batch of E. coli bacteria are modified to take the plasmids inside their cells in a vial.
Step 2
Grow the Cells
The vial of modified bacteria (E. coli) is swirled into a flask of amber-colored growth medium, a sterile and warm environment that encourages the bacteria to multiply.
STEP 3
Ferment the Mixture
The bacteria are allowed to grow overnight and then moved into a large fermenter that contains up to 300 liters of a nutrient broth.
The bacterial broth spends four days in the fermenter, multiplying every 20 minutes and making trillions of copies of the DNA plasmids.
STEP 4
Harvest and Purify the DNA
When the fermentation is complete, chemicals are added to break open the bacteria and release the plasmids from their enclosing cells.
The mixture is then purified to remove the bacteria and leave only the plasmids.
STEP 5
Test for Quality
The plasmids are tested for purity, and compared against previous samples to confirm that the coronavirus gene sequence has not changed.
To know more about plasmids and their use in vaccines, read
Plasmid-Based DNA Vaccines
By Leonardo A. Gómez and Angel A. Oñate
https://www.intechopen.com/books/plasmid/plasmid-based-dna-vaccines
http://www.addgene.org/collections/covid-19-resources/
STEP 6
Cut the Plasmids
After plasmids pass the quality checks, proteins called enzymes are added to the mixture. The enzymes cut the circular plasmids and separate the coronavirus genes into straight segments, a process called linearization that takes about two days.
STEP 7
Filter the DNA Plasmids
Any remaining bacteria or plasmid fragments are filtered out, leaving one-liter bottles of purified DNA. The DNA sequences are tested again. Each bottle of DNA will produce about 1.5 million doses of the vaccine.
STEP 8
Freeze, Pack and Ship
Each bottle of DNA Plasmids is frozen, bagged, sealed and packed with a small monitor that will record its temperature in transit.
Up to 48 bottles are packed in a container with enough dry ice to keep them frozen at –20°C (–4°F).
These containers are sent to facilities where further processing is to be done. (Andover plant in US and BioNTech facilities in Mainz, Germany).
STEP 9
Transcribe the DNA Plasmids into mRNA
Five bottles of DNA are thawed for a day, then mixed with the building blocks of messenger RNA. Over several hours, enzymes pry open the DNA templates and transcribe them into strands of mRNA. The finished vaccine will carry the mRNA into human cells, which will read the coronavirus gene and begin producing coronavirus proteins.
The mixture is moved into a holding tank, then filtered to remove any unwanted DNA, enzymes or other impurities. Each batch will yield up to 7.5 million doses of the vaccine.
About Transcription
Transcription is the process of copying a segment of DNA into RNA. The segments of DNA transcribed into RNA molecules that can encode proteins are said to produce messenger RNA (mRNA).
Both DNA and RNA are nucleic acids, which use base pairs of nucleotides as a complementary language. During transcription, a DNA sequence is read by an RNA polymerase, which produces a complementary, antiparallel RNA strand called a primary transcript.
Transcription proceeds in the following general steps:
RNA polymerase, together with one or more general transcription factors, binds to promoter DNA.
RNA polymerase generates a transcription bubble, which separates the two strands of the DNA helix. This is done by breaking the hydrogen bonds between complementary DNA nucleotides.
RNA polymerase adds RNA nucleotides (which are complementary to the nucleotides of one DNA strand).
RNA sugar-phosphate backbone forms with assistance from RNA polymerase to form an RNA strand.
Hydrogen bonds of the RNA–DNA helix break, freeing the newly synthesized RNA strand.
If the cell has a nucleus, the RNA may be further processed. This may include polyadenylation, capping, and splicing.
The RNA may remain in the nucleus or exit to the cytoplasm through the nuclear pore complex.
If the stretch of DNA is transcribed into an RNA molecule that encodes a protein, the RNA is termed messenger RNA (mRNA); the mRNA, in turn, serves as a template for the protein's synthesis through translation. Other stretches of DNA may be transcribed into small non-coding RNAs such as microRNA, transfer RNA (tRNA), small nucleolar RNA (snoRNA), small nuclear RNA (snRNA), or enzymatic RNA molecules called ribozymes as well as larger non-coding RNAs such as ribosomal RNA (rRNA), and long non-coding RNA (lncRNA). Overall, RNA helps synthesize, regulate, and process proteins; it therefore plays a fundamental role in performing functions within a cell.
https://en.wikipedia.org/wiki/Transcription_(biology)
STEP 10
Test the mRNA
Inspection scientists repeatedly test the filtered mRNA to verify its purity and confirm the genetic sequence is correct.
The output of the batch will be 10 bags of coronavirus mRNA. Each bag holds 16 liters and represents the raw material for about 750,000 doses of the vaccine.
STEP 11
Freeze, Pack and Ship
The bags of mRNA are frozen to –20°C (–4°F) and shipped to packing facilities (Pfizer facility in Kalamazoo, Mich.,).
The Andover plant can produce two batches of mRNA a week, each about 10 bags (1.5 million doses). The facility made its first test batch last July, and recently doubled its mRNA capacity by adding a second suite.
STEP 12
Prepare the mRNA
The the bags of mRNA are kept frozen until needed and then thawed enough to produce 3.6 million doses of the vaccine, or 600,000 vials.
The thawed mRNA is mixed with water in preparation for making the vaccine.
STEP 13
Prepare the Lipids
In a separate process, the oily lipids that will protect the mRNA and help it enter human cells are produced. The lipids are measured out and mixed with ethanol. The ethanol will eventually be removed from the finished vaccine.
STEP 14
Assemble the mRNA Vaccine
A rack of 16 pumps precisely controls the flow of the mRNA and lipid solutions, then mixes them together to create lipid nanoparticles. When the lipids come into contact with the naked strands of mRNA, electric charge pulls them together in a nanosecond. The mRNA is enveloped in several layers of lipids, forming an oily, protective vaccine particle.
Synchronizing eight pairs of pumps is not an ideal solution, but Pfizer engineers chose to scale up existing technology instead of trying to build a larger, unproven type of precision mixing device.
The newly made vaccine is filtered to remove the ethanol, concentrated and filtered again to remove any impurities, and finally sterilized.
Read section Lipid Based Delivery in
STEP 15
Prepare the Vials
Empty vials are washed and heat sterilized. A set of 13 cameras performs a high-speed visual inspection, taking more than 100 photographs of each vial. Any vials with cracks, chips or other imperfections are removed from the line. A separate machine puts each vial under vacuum to confirm it doesn’t leak.
STEP 16
Rush to Fill the Vials
The flow of vials is narrowed to a single-file line. Machines inject 0.45 ml of a concentrated vaccine solution into each vial, enough for six doses after dilution. The vials are sealed with foil and capped with purple lids, at a pace of up to 575 vials per minute.
The mRNA will deteriorate if left unfrozen for too long. In 46 hours, the process to get the liquid vaccine into vials has to be completed and then vials are to be put into deep freeze.
STEP 17
Package, Freeze and Test
The filled vials are inspected again, and then labeled and packed into “pizza boxes,” small plastic trays that hold 195 vials each. The trays are bundled in stacks of five and loaded into industrial freezers. Each freezer holds 300 trays.
It takes a couple of days for the vaccine to reach the –70°C (–94°F) required for long-term storage, and each freezer is tested to ensure that every shelf can maintain that ultracold temperature.
Once frozen, the vials of vaccine are held for four weeks of testing.
Pfizer currently operates on a 60-day timeline from start to finish, and more than half of that time is dedicated to testing.
STEP 18
Pack and Ship the Finished Vaccine
After testing, the vaccine is ready to ship. Workers pull trays from the freezers and pack them in shipping boxes with temperature and location sensors. The minimum order is one tray of 195 vials, and a box holds up to five trays. Each box contains 45 pounds of dry ice. Pfizer is also evaluating different formulations of the vaccine, including freeze-dried and ready-to-use versions that would not require ultracold storage.
Commercial production of the Pfizer vaccine began in September 2020. As of April 22, the plant had delivered more than 150 million vaccine doses to the United States. Pfizer expects to deliver 220 million doses by the end of May, and 300 million by mid-July.
How Pfizer Makes Covid-19 Vaccine - More Details on Steps
https://www.nytimes.com/interactive/2021/health/pfizer-coronavirus-vaccine.html
It is a complex manufacturing and testing process that takes 60 days.
STEP 1
Pull DNA from Cold Storage
Vials of DNA from the master cell bank is the source of every batch of Pfizer’s Covid-19 vaccine. The vials are kept at –150°C (–238°F) or below, and they contain small rings of DNA called plasmids.
Plasmid is Coronavirus spike protein gene.
Each plasmid contains a coronavirus gene, the genetic instructions for a human cell to build coronavirus proteins and trigger an immune response to the virus.
The plasmids are thawed. A batch of E. coli bacteria are modified to take the plasmids inside their cells in a vial.
How to Store DNA
1.Storage in vitreous state
2. Medium length storage
3. Repeatedly freezing and thawing DNA - studies show that repeated freeze and thaw cycles with up to 19 cycles have no detected DNA degradation.
https://www.news-medical.net/life-sciences/How-to-Store-DNA.aspx
Evaluation of DNA Plasmid Storage Conditions
Manabu Murakami*
Department of Pharmacology, Hirosaki University, Graduate School of Medicine, Aomori, Japan
The Open Biotechnology Journal, 2013, 7, 10-14
https://benthamopen.com/DOWNLOAD-PDF/TOBIOTJ-7-10/
You have just received your plasmids from Addgene. Now what?
https://www.addgene.org/recipient-instructions/myplasmid/
Plasmid DNA Purification - Macherey-Nagel
https://www.takarabio.com/documents/User%20Manual/PT4022/PT4022-1.pdf
Step 2
Grow the Cells
The vial of modified bacteria (E. coli) is swirled into a flask of amber-colored growth medium, a sterile and warm environment that encourages the bacteria to multiply.
STEP 3
Ferment the Mixture
The bacteria are allowed to grow overnight and then moved into a large fermenter that contains up to 300 liters of a nutrient broth.
The bacterial broth spends four days in the fermenter, multiplying every 20 minutes and making trillions of copies of the DNA plasmids.
STEP 4
Harvest and Purify the DNA
When the fermentation is complete, chemicals are added to break open the bacteria and release the plasmids from their enclosing cells.
The mixture is then purified to remove the bacteria and leave only the plasmids.
Plasmid DNA Purification - Macherey-Nagel
https://www.takarabio.com/documents/User%20Manual/PT4022/PT4022-1.pdf
STEP 5
Test for Quality
The plasmids are tested for purity, and compared against previous samples to confirm that the coronavirus gene sequence has not changed.
To know more about plasmids and their use in vaccines, read
Plasmid-Based DNA Vaccines
By Leonardo A. Gómez and Angel A. Oñate
https://www.intechopen.com/books/plasmid/plasmid-based-dna-vaccines
http://www.addgene.org/collections/covid-19-resources/
STEP 6
Cut the Plasmids
After plasmids pass the quality checks, proteins called enzymes are added to the mixture. The enzymes cut the circular plasmids and separate the coronavirus genes into straight segments, a process called linearization that takes about two days.
STEP 7
Filter the DNA Plasmids
Any remaining bacteria or plasmid fragments are filtered out, leaving one-liter bottles of purified DNA. The DNA sequences are tested again. Each bottle of DNA will produce about 1.5 million doses of the vaccine.
STEP 8
Freeze, Pack and Ship
Each bottle of DNA Plasmids is frozen, bagged, sealed and packed with a small monitor that will record its temperature in transit.
Up to 48 bottles are packed in a container with enough dry ice to keep them frozen at –20°C (–4°F).
These containers are sent to facilities where further processing is to be done. (Andover plant in US and BioNTech facilities in Mainz, Germany).
STEP 9
Transcribe the DNA Plasmids into mRNA
Five bottles of DNA are thawed for a day, then mixed with the building blocks of messenger RNA. Over several hours, enzymes pry open the DNA templates and transcribe them into strands of mRNA. The finished vaccine will carry the mRNA into human cells, which will read the coronavirus gene and begin producing coronavirus proteins.
The mixture is moved into a holding tank, then filtered to remove any unwanted DNA, enzymes or other impurities. Each batch will yield up to 7.5 million doses of the vaccine.
About Transcription
Transcription is the process of copying a segment of DNA into RNA. The segments of DNA transcribed into RNA molecules that can encode proteins are said to produce messenger RNA (mRNA).
Both DNA and RNA are nucleic acids, which use base pairs of nucleotides as a complementary language. During transcription, a DNA sequence is read by an RNA polymerase, which produces a complementary, antiparallel RNA strand called a primary transcript.
Transcription proceeds in the following general steps:
RNA polymerase, together with one or more general transcription factors, binds to promoter DNA.
RNA polymerase generates a transcription bubble, which separates the two strands of the DNA helix. This is done by breaking the hydrogen bonds between complementary DNA nucleotides.
RNA polymerase adds RNA nucleotides (which are complementary to the nucleotides of one DNA strand).
RNA sugar-phosphate backbone forms with assistance from RNA polymerase to form an RNA strand.
Hydrogen bonds of the RNA–DNA helix break, freeing the newly synthesized RNA strand.
If the cell has a nucleus, the RNA may be further processed. This may include polyadenylation, capping, and splicing.
The RNA may remain in the nucleus or exit to the cytoplasm through the nuclear pore complex.
If the stretch of DNA is transcribed into an RNA molecule that encodes a protein, the RNA is termed messenger RNA (mRNA); the mRNA, in turn, serves as a template for the protein's synthesis through translation. Other stretches of DNA may be transcribed into small non-coding RNAs such as microRNA, transfer RNA (tRNA), small nucleolar RNA (snoRNA), small nuclear RNA (snRNA), or enzymatic RNA molecules called ribozymes as well as larger non-coding RNAs such as ribosomal RNA (rRNA), and long non-coding RNA (lncRNA). Overall, RNA helps synthesize, regulate, and process proteins; it therefore plays a fundamental role in performing functions within a cell.
https://en.wikipedia.org/wiki/Transcription_(biology)
STEP 10
Test the mRNA
Inspection scientists repeatedly test the filtered mRNA to verify its purity and confirm the genetic sequence is correct.
The output of the batch will be 10 bags of coronavirus mRNA. Each bag holds 16 liters and represents the raw material for about 750,000 doses of the vaccine.
STEP 11
Freeze, Pack and Ship
The bags of mRNA are frozen to –20°C (–4°F) and shipped to packing facilities (Pfizer facility in Kalamazoo, Mich.,).
The Andover plant can produce two batches of mRNA a week, each about 10 bags (1.5 million doses). The facility made its first test batch last July, and recently doubled its mRNA capacity by adding a second suite.
STEP 12
Prepare the mRNA
The the bags of mRNA are kept frozen until needed and then thawed enough to produce 3.6 million doses of the vaccine, or 600,000 vials.
The thawed mRNA is mixed with water in preparation for making the vaccine.
STEP 13
Prepare the Lipids
In a separate process, the oily lipids that will protect the mRNA and help it enter human cells are produced. The lipids are measured out and mixed with ethanol. The ethanol will eventually be removed from the finished vaccine.
STEP 14
Assemble the mRNA Vaccine
A rack of 16 pumps precisely controls the flow of the mRNA and lipid solutions, then mixes them together to create lipid nanoparticles. When the lipids come into contact with the naked strands of mRNA, electric charge pulls them together in a nanosecond. The mRNA is enveloped in several layers of lipids, forming an oily, protective vaccine particle.
Synchronizing eight pairs of pumps is not an ideal solution, but Pfizer engineers chose to scale up existing technology instead of trying to build a larger, unproven type of precision mixing device.
The newly made vaccine is filtered to remove the ethanol, concentrated and filtered again to remove any impurities, and finally sterilized.
Read section Lipid Based Delivery in
STEP 15
Prepare the Vials
Empty vials are washed and heat sterilized. A set of 13 cameras performs a high-speed visual inspection, taking more than 100 photographs of each vial. Any vials with cracks, chips or other imperfections are removed from the line. A separate machine puts each vial under vacuum to confirm it doesn’t leak.
STEP 16
Rush to Fill the Vials
The flow of vials is narrowed to a single-file line. Machines inject 0.45 ml of a concentrated vaccine solution into each vial, enough for six doses after dilution. The vials are sealed with foil and capped with purple lids, at a pace of up to 575 vials per minute.
The mRNA will deteriorate if left unfrozen for too long. In 46 hours, the process to get the liquid vaccine into vials has to be completed and then vials are to be put into deep freeze.
STEP 17
Package, Freeze and Test
The filled vials are inspected again, and then labeled and packed into “pizza boxes,” small plastic trays that hold 195 vials each. The trays are bundled in stacks of five and loaded into industrial freezers. Each freezer holds 300 trays.
It takes a couple of days for the vaccine to reach the –70°C (–94°F) required for long-term storage, and each freezer is tested to ensure that every shelf can maintain that ultracold temperature.
Once frozen, the vials of vaccine are held for four weeks of testing.
Pfizer currently operates on a 60-day timeline from start to finish, and more than half of that time is dedicated to testing.
STEP 18
Pack and Ship the Finished Vaccine
After testing, the vaccine is ready to ship. Workers pull trays from the freezers and pack them in shipping boxes with temperature and location sensors. The minimum order is one tray of 195 vials, and a box holds up to five trays. Each box contains 45 pounds of dry ice. Pfizer is also evaluating different formulations of the vaccine, including freeze-dried and ready-to-use versions that would not require ultracold storage.
Commercial production of the Pfizer vaccine began in September 2020. As of April 22, the plant had delivered more than 150 million vaccine doses to the United States. Pfizer expects to deliver 220 million doses by the end of May, and 300 million by mid-July.
Book - Vaccine Development and Manufacturing
Emily P. Wen, Ronald Ellis, Narahari S. Pujar
John Wiley & Sons, 06-Oct-2014 - Technology & Engineering - 456 pages
Vaccine Manufacturing and Production is an invaluable reference on how to produce a vaccine - from beginning to end - addressing all classes of vaccines from a processing, production, and regulatory viewpoint. It will provide comprehensive information on the various fields involved in the production of vaccines, from fermentation, purification, formulation, to regulatory filing and facility designs. In recent years, there have been tremendous advances in all aspects of vaccine manufacturing. Improved technology and growth media have been developed for the production of cell culture with high cell density or fermentation. Vaccine Manufacturing and Production will serve as a reference on all aspects of vaccine production by providing an in-depth description of the available technologies for making different types of vaccines and the current thinking in facility designs and supply issues. This book will provide insight to the issues scientists face when producing a vaccine, the steps that are involved, and will serve as a reference tool regarding state-of-the-art vaccine manufacturing technologies and facility set-up.
Highlights include:
Comprehensive coverage of vaccine production : from a process point of view- fermentation to purification to formulation developments; from a production point of view - from facility design to manufacturing; and from a regulatory point of view - requirements from government agencies
Describes the challenges and issues involved in vaccine production and manufacturing of the different classes of vaccines.
Preview the book
https://books.google.co.in/books/about/Vaccine_Development_and_Manufacturing.html?id=51O_BAAAQBAJ
More articles -
on Manufacturing Processes of Vaccines
AstraZeneca
Gavi
Moderna
Pfizer
Cost Estimates of Vaccines
Industrial Engineers - Engineers Specializing in Cost Reduction
Please give your suggestions as comments.
Industrial Engineering Suggestions and Ideas for Cost Reduction of Covid Vaccine Manufacturing
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GreenLight’s mRNA Manufacturing Process - Cost Reduction Potential
Optimization of an mRNA vaccine assisted with cyclodextrin-polyethyleneimine conjugates
Optimization of Lipid Nanoparticles for Intramuscular Administration of mRNA Vaccines
Using Enzymatic DNA technology
Plasmid manufacture is the bottleneck of the genetic medicine revolution
Jonny Ohlson
Drug Discov Today. 2020 Nov; 25(11): 1891–1893.
Plasmid DNA Production: Productivity with Modular Single-Use Minibioreactors
Those time savings allow for a significant acceleration in clients’ clinical development programs. It speeds programs to initial clinical studies and helps during later stage development when the time available to perform process development activities is restricted by the potential of complicating clinical development programs.
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