Now you know what upstream processing is, what happens once you’ve expanded the cell culture and product formation? Is that it? The short answer is NO! You can’t just give the cell broth to someone! The cell broth produced after upstream processing contains cells, product impurities (improperly folded product, product that has been modified in some way), media components, cell debris, etc. This stuff is potentially dangerous to the end user, and is also likely to cause the product to degrade quicker, leading to crappy storage.
In order to create a pure, consistent, safe and defined product, we must isolate it from the rest of the cell broth. This is called downstream processing. Downstream processing consists of various unit-operations in which the product is isolated and purified, producing a bulk drug substance (where the majority of the mass is the product). Fill/finishing (where more impurities are removed and excipients are added to improve long-term stability / change the state of the product to allow storage and transport) then produces the drug product from the bulk drug substance.
*In most cases, for FDA / regulatory body approval, the product needs to be at 95% purity, and majority of impurities must be less than 1 part per million in the final drug product.
Downstream processing unit operations can be split into 4 categories based on their purpose:
Recovery: Initial isolation of the product from other components of the cell broth. Unit operation examples: centrifugation, filtration.
Isolation: Further isolation of the product from cell broth components. Unit operation examples: precipitation, capture chromatography, liquid-liquid extraction.
Purification: Removal of impurities/ other remaining non-product components. Unit operation examples: Crystallisation, Polishing chromatography.
Fill/Finish: Removal of remaining impurities + preparing drug product for storage and transport. Examples: Ultrafiltration, nanofiltration, freeze-drying.
After downstream processing and fill/finish, the product can now be used!
If you have any questions, please leave a comment below and I will get back to you ASAP
Cell expression systems are the type of cell line used to express a protein or gene for commercial, research or medical use.
In bioprocessing, cells are used to create the desired product – proteins, antibodies, vaccines, etc. But there are so many different types of cell lines you can choose to use! Eukaryotic or prokaryotic, mammalian or fungi, human or animal.. theres a lot to choose from! Each cell line comes with its own advantages and disadvantages, and will be better for producing some products more than others.
In this post I will cover 3 main choices of cell line – prokaryotic (E. Coli), fungi (yeast) and mammalian, but in reality there are so many cell lines within those groups you can choose from! For example, Chinese Hamster Ovary, or CHO cell lines, are commonly used for producing monoclonal antibodies since they are mammalian cells, which are capable of complex protein folding and modification.
Cell Systems
Prokaryotic: Escherichia Coli (E. Coli)
You’ve probably heard of E. Coli! E. Coli are a type of bacteria, and a very common cell expression system. This is because they are simple and well-characterised, since decades of research has been done using E. Coli as an expression system. Prokaryotic cell systems are simple in that they are unicellular, thus do not function as part of a larger organism. Prokaryotic cells also tend to proliferate at a rapid rate due to this (reproduction is carried out via mitosis). However, due to their simplicity the products they produce tend to also be very simple – protein folding and modification is very limited in these cells.
DNA integration and protein expression tends to be created by introducing plasmid vectors that carry the desired gene to the E. Coli cells. We will cover what plasmids are in more detail in a later post, but they are basically circular DNA that bacteria commonly use to transfer genes between one another. The plasmids can be engineering to carry the gene of interest (GOI) into the E. Coli cell, where it can then be acted upon by the cell’s genetic machinery to produce the desired protein.
E. Coli is used to produce a range of products, such as biofuels, vaccines and insulin! E. Coli can produce product titres (product concentrations) of up to 10 grams/ litre.
Pros:
Simple genetics (less DNA than other cell types)
Easy cultivation
Well-characterised: effective cultivation
Proven to be effective
Rapid growth and proliferation
Characterised media
Simple nutritional requirements
Cons:
No glycosylation of proteins
Inclusion bodies (condensed globs of protein)
Endotoxins
Not as similar to humans as other cell types
Limited protein modification
Fermentative metabolism (can proliferate without oxygen and produce toxic byproducts)
Eukaryotic: Fungi – Yeast
Yeast is another common cell expression system that you may have heard of. Yeast has been used to make many food products, including bread, wine and beer! As a eukaryotic cell, yeast is more similar to human cells than bacteria, and are capable of more protein modification than bacteria. However, yeast cells remain unicellular and can reproduce asexually, which makes it easier to grow them. As one of the first domesticated organisms in human history, yeast is very well-characterised, allowing for rapid and efficient growth.
Yeast cells are manipulated to produce more of the desired product via metabolic engineering. This is when pre-existing metabolic pathways in the cell are manipulated to create higher production of the desired product – for example a gene could be manipulated to produce more of the rate-limiting protein in a pathway that produces the desired product.
Yeast has been manipulated to produce many medicinal products such as insulin, antibody fragments and collagen. Product titres of yeast systems can reach 0.4 – 10 grams/litre.
Pros:
High cell density
Easy to grow
Characterised media
High product titre
Easy genetics
Product is secreted by cell
Simple nutritional requirements
Cons:
Yeast cells tend to over glycosylate the product
Possible toxicity
O2 mass transfer is hard with yeast cultures
Large amount of cell debris created
Complex downstream processing
Mammalian Cells
Mammalian cells are the most similar to human cells, and some cell lines are derived from human cells (look at HeLa cell lines in the Cell culture post)! Mammalian cells are eukaryotic cells, and are generally multicellular. Due to their similarity to human cells, products produced using mammalian cell systems tend to experience less rejection by the human body and are more effective. Complex mammalian proteins can also be made since mammalian cells are able to glycosylate and modify proteins the same way our cells do. However, due to their multicellular state reproduction (mitosis) can be limited – for example nerve cells tend to never undergo mitosis once matured. Also, mammalian cells have much slower growth.
Mammalian cell systems are manipulated into creating the desired protein via genetic engineering. This can cause either transient or stable expression. In transient expression, a vector (i.e plasmid) containing the gene of interest or mRNA of interest is introduced to the cell, where it is then acted upon by the cells genetic machinery to produce the product of interest. However since the gene is not incorporated into the host DNA it may degrade over a few generation doublings. In stable expression, the gene of interest is incorporated into the host cell’s DNA. This means the gene will be passed to future generations of the cell and will not degrade, so all cells produced from that cell will continue to express the desired product.
Mammalian cells are used to produce various products, such as monoclonal antibodies, growth hormones and interferon. Research is also being done to determine how to use mammalian cells to produce organ or tissue replacements. Product titres using mammalian cell systems are from 0.1 – 10 grams/ litre
Pros:
Product quality
Correct folding
Correct modifications
Potentially a simpler downstream process
Wider range of products possible
Cons:
Contamination risk
Slow growth
Not well characterised
Complex nutritional requirements
Long developmental time
Difficult scale-up
Safety concerns (use of viruses as vectors)
Potentially complex downstream process
How do you select what cell line to use?
When selecting a cell line, many factors come into play – price, developmental time, product quality, product yields, safety, purification, scaling of the process. This makes it difficult to select the perfect cell line to use for many products. However, ideally the cell line should meet these criteria:
Acceptable product quality and product quality attributes
Stable expression of the product throughout upstream processing
Desired yield of the product reached
Purification without loss of product quality
And there you have it! If you have any questions, leave a comment below and I will get back to you ASAP!
Finally! We’ve done all the waffling about the topic, now we can get straight to it! It’s hard to explain mass balancing with words, so I think working through an example will be the most effective way of explaining a mass balance.
Note: This exercise was taken from Doran’s book: Bioprocess Engineering Principles. I HIGHLY recommend getting this book, it’s basically the bible of bioprocessing. If you want to buy it, click the photo to be taken to the amazon page:
Bioprocess Engineering Principles by Pauline M. Doran: Click on photo to get amazon link!
The production target is 2kg of acetic acid per hour. However the maximum acetic acid tolerated by cells is 12% mass acetic acid (only 12% of the outlet stream can be acetic acid). Air is pumped into the fermenter at 200gmol per hour. What minimum amount of ethanol is required? What minimum amount of water must be used to dilute the output stream to 12% acetic acid? What is the composition of the outlet gas?
First, lets define the assumptions:
Steady state
No system leakage
100% conversion of ethanol to acetic acid
O2 and ethanol available to all cells
Inlet air and outlet gas are dry
Feed has the density of water: 1kg/m3
Other feed components are negligible
Inlet air has composition of 21% O2 and 79% N2
Define mass balance equations:
We know N2 is not involved in any reactions by looking at the chemical equation. So for the total mass of the process and N2:
Mass in = Mass out
We can see from the chemical equation that ethanol and oxygen are converted to water and acetic acid, so the equation for these will be:
Mass in + Mass generated = Mass out + Mass consumed
Define basis:
Since this is a continuous process, we have to decide a point for where we will do the mass balance. To make things more simple, let’s say that we’ll do this mass balance for an hour of fermentation – then we can define the input and output as a constant amount. Also, we should stick to one unit to make it easier to double check our answers. Let’s choose kilograms since the acetic acid is given in kg.
Make a flow sheet
I don’t know why this is important (besides visualising the process) but you will have to do it in your exams, so here is what the flow sheet for this process will look like:
Process flow sheet for continuous acetic acid fermentation
Calculations!
Now we know that there is 200gmol of inlet air. As mass = MW x moles, we can find out the mass of the inlet air by determining the mass of O2 and N2. Since we assume the composition of air is 21% O2 and 79% N2, we can find the molar mass:
Moles of O2: 21% of 200 = 0.21 x 200 = 42 moles
Moles of N2: 79% of 200 = 0.79 x 2oo = 158 moles
To find the mass in grams:
Mass = MW x moles
O2: 42 x (16 x 2) = 42 x 32 = 1344g = 1.344kg
N2: 98 x (14 x 2) = 158 x 28 = 4424g = 4.424kg
Now that we know the mass of O2 and N2, we can find the total mass of the inlet air:
1.344 + 4.424 = 5.768kg
So the total mass of the inlet air is 5.768kg!
Now, we want 2kg/hr of acetic acid, which is also 12% of the product stream. To find the total weight of the product stream is then just the mass of acetic acid divided by the percentage/100:
Mass of product stream = 2 / (12/100) = 2/ 0.12 = 16.67kg
So now we know the total mass of the product stream. Since we assumed that the rest of the media is negligible, the only other component of the product stream is water. So the mass of water would be:
Total mass – mass of acetic acid = 16.67kg – 2kg = 14.67kg
And voila! Now we know the total mass of 2 streams.
Stoichiometric mass balances
Now lets find the mass of everything in the equations!
We know that there is 2kg of acetic acid. With this and the chemical equation, we can find the masses of every other molecule in the equation.
From the chemical equation we can see that for 1 mole of acetic acid produced, there is 1 mole of ethanol and oxygen used and 1 mole of water produced. Lets convert our 2kg of acetic acid in to moles:
So for every mole of acetic acid produced, 33.3 moles of ethanol and oxygen are used, and 3.33 moles of water is formed. Let’s convert them into mass:
Mass = Moles x MW
Ethanol: 33.3 x 46 = 1582g = 1.582kg
Oxygen: 33.3 x 32 = 1065g = 1.065kg
Water: 33.3 x 18 = 599.4g = 0.599kg
We know how much water there is in the product stream. So, if 0.599kg of water is produced for 2kg of ethanol, then the water that was added in the feed stream must be the mass of water in the product stream – mass of water produced in the process
14.67kg – 0.599kg = 14.07kg water in feed stream
We also now know the amount of ethanol required to make 2kg of acetic acid. Thus to find the total amount of the feed stream we only need to add the mass of acetic acid and the mass of water in the feed stream together:
14.07 + 1.582 = 15.652kg (total mass of feed stream)
Now for the gas! We know that 1.065kg of oxygen is used to make 2kg of acetic acid. Therefore, to find the amount of oxygen leaving the system (that hasn’t been used), we only need to subtract the oxygen used from the amount of oxygen that entered the reactor in the inlet air:
1.344 – 1.065 = 0.279kg O2 in outlet air
Since nitrogen is not involved in the equation, the mass of nitrogen is the same going in and going out. Therefore the total mass of the outlet gas is:
4.424 + 0.279 = 4.703kg mass of outlet gas
And were done! Lets put it in a table to see if we have filled everything in:
IN
IN
OUT
OUT
FEED STREAM
INLET AIR
PRODUCT STREAM
OUTLET GAS
WATER
14.07
X
14.67
X
ETHANOL
1.582
X
0
X
ACETIC ACID
X
X
2
X
OXYGEN
X
1.344
X
0.279
NITROGEN
X
4.424
X
4.424
TOTAL
15.652
5.768
16.67
4.703
Since the total mass balance for the process is mass in = mass out, we can check if we’ve done our mass balance properly:
And thats how you do a mass balance! If you have any questions, please leave a comment and I will get back to you ASAP! Also, I know how difficult it is to understand this, so I will go through more examples in the future since thats the best way to learn mass balances in my opinion. If you have any of your own mass balance problems please let me know so I can go through them!
In bioprocessing it’s usually difficult to calculate things exactly due to the complexity of biological processes. Therefore we tend to make a lot of assumptions.
In your exams or even at work you will need to put all the assumptions you make when doing a mass balance, so I thought I’d write a list of all the common ones that you can use.
Common Assumptions
No system leakage (best assumption!!)
Inlet air and outlet gas are dry (liquid does not leave system)
Cell broth has density of water: 1kg/m3
Complete conversion of input material (nutrients to product)
Composition of inlet air is 21% mol O2, 79% N2
Mass of cells inoculated at start of process is negligible (mainly for batch processes)
CO2 leaves in outlet gas
Other media components can be ignored
O2 and nutrients are available to all cells (vessel broth is homogenous)
There are probably more examples of assumptions you can make, but these are generally applicable to all processes. If you have any questions or want me to add anything, leave a comment and I will get to it ASAP!
So we’ve gone through what mass balancing is and the mass balancing equations. Here is one of the harder parts: calculating the mass balance when there are reactions occurring. This is called stoichiometric mass balancing. This will be basic stoichiometric mass balancing (aka what I learnt in year 1). There is also elemental mass balancing, which is more cell-based and a bit more complex so I’ll go through that in another post.
Stoichiometry (Textbook Definition): The chemical and biochemical reactions involved in the rearrangement of atoms and molecules to form new groups.
Stoichiometry is basically calculating the amount of a reactant or product based on the related chemical equation. To do this you must know the mass of at least one of the molecules, the moles of each molecule and the molecular weight of each molecule.
The best way to teach this is by example, so here is a chemical equation:
Chemical equation for alcoholic fermentation.
This is the chemical equation for alcoholic fermentation. In this equation, 1 mole of glucose (left) is broken down into 2 moles of ethanol (right) and 2 moles of carbon dioxide (right). Now, imagine we have 300 grams of glucose. How much ethanol would we produce?
Here are the 3 main rules:
Matter cannot be created or destroyed. Therefore, the total mass on the left of the equation must be the same as the total mass on the right.
Elements cannot be created or destroyed in a chemical equation. Therefore, both sides of the equation will have the same amount of each element.
Stoichiometric balances work with moles, not mass – you must convert mass into moles when doing stoichiometric balances.
Firstly, since stoichiometry is done in moles, let’s convert our 300g of glucose into moles. From the basic calculations and equations post, we know that moles = mass / molecular weight. To find the molecular weight of glucose, we must add the molecular weight of all the elements in glucose together. If you google the periodic table (or just google the molecular weight of each element) you will find that:
Carbon (C): Molecular weight of 12
Hydrogen (H): Molecular weight of 1
Oxygen (O): Molecular weight of 16
However, since there is more than 1 of each element (as indicated by the subscripts in the equation) we must multiply the molecular weight of each element by how much there is in glucose.
(C x 6) + (H x 12) + (O x 6) = (12 x 6) + (1 x 12) + (16 x 6) = 180
Hence the total molecular weight of glucose is 180. To find the molar mass:
Moles = Mass / MW = 300/180 = 1.67 gmol
Now that we know the molar mass of glucose, we can determine the molar mass of ethanol. If you look at the equation, you can see that there is 2 moles of ethanol produced for every 1 mole of glucose (if there is no number in front of a molecule it means there is only 1 mole). Therefore, the molar mass of ethanol would be 2 times the molar mass of glucose. So:
Molar mass of ethanol = molar mass glucose x 2 = 1.67 x 2 = 3.33 gmol ethanol
From this, we can find the mass of ethanol using the MW of ethanol and the molar mass calculated above:
Mass = MW x moles = 46 x 3.33 = 153g
Where 46 is the molecular weight of ethanol. And voila! There we have the amount of ethanol produced with 300 grams of glucose. If you were to find the mass of carbon dioxide produced (which is roughly 147, you can calculate it yourself if you want practice). you’ll find that:
Both sides of the equation equal 300 grams! This is a good way of checking if you’ve done the mass balance right, since the mass must be the same on both sides of the equation.
Now, lets try a harder one…
Chemical equation for the conversion of glucose to L-glutamic acid.
This LOOKS complicated, but the same method applies. How would we work out how much oxygen is needed to produce 40g of glutamic acid (C5H9NO4)?
Firstly, lets find the molecular weight:
(C x 5) + (H x 9) + (N x 1) + (O x 4) = (12 x 5) + (1 x 9) + (14 x 1) + (16 x 4) = 147
Therefore the molar mass of L-glutamic acid is:
Molar mass = Mass/ MW = 40/147 = 0.272gmol
From the chemical equation, we know that 1.5 moles of oxygen is needed to make 1 mole of L-glutamic acid. Therefore we need 1.5 x more of oxygen:
0.272 x 1.5 = 0.408gmol
To find the mass of oxygen needed:
Mass = MW x moles = 0.408 x (16 x 2) = 0.408 x 32 = 13.06 grams
Therefore to make 40g of L-glutamic acid, we need 13.06 grams of oxygen.
And there you have it! It sounds complicated, but once you understand it its actually pretty simple! However I don’t know if its just simple to me, so if you have any questions about anything please leave a comment below and I will get back to you!
In my previous post I explained (briefly) what mass balancing was. Here I will go over the rules and equations used for mass balancing.
Mass Balancing: General equation
The most important equation that covers all aspects of mass balancing is this:
[Mass in + mass generated] – [Mass out + Mass consumed] = Mass accumulated
Pretty self-explanatory, right? Mass in is the amount of what you put into the system, mass generated is the mass of anything generated inside the system, mass out is the mass of what leaves the system, mass consumed is the mass of what is consumed in the reactor and mass accumulated is the mass of anything that is still inside the system. This equation can apply to the whole unit operation (the mass of everything all together) or to individual components (water, nutrients, product).
However, depending on the type of process or material you have, you can simplify the equation.
Change in Process variables
A fermentation can be split into 2 states:
Steady state: In this state, process variables do not change with time. This means that there is also no change in mass for each component over time. This state generally applies to continuous fermentation, where input, output and the system are the same over time.
Unsteady state: Process variables change over time. This is the case for batch or semi-batch operations, where material is not added or removed from the reactor at a constant rate. Due to this, the mass of different components may differ at different stages of the fermentation.
Depending on the state of the process, the mass balance equation will change!
Mass Balance: Simplified equations
Steady state
Since mass does not change over time, there is no accumulation of mass within the reactor/ system. Therefore, the equation can be simplified to these terms:
Mass in + Mass generated = Mass out + Mass consumed
If there are no reactions occurring in a steady state process, meaning the molecules in that component do not interact with each other (i.e. cell mass), there is no mass generated or consumed. Then, the equation can further be simplified to:
Mass in = Mass out
Both these equations can be applied to either the process as a whole (if the above is applicable) or to individual components.
Unsteady state
In an unsteady state process, we should use the general equation. However, we tend to assume (at least at the bachelor degree level) there is no accumulation by assuming total conversion of input to output. Why? This is simpler, and in a batch or semi-batch process the unconverted nutrients tends to be a very small value. We also only do a mass balance of the whole process, not random time points, since this is more useful and much easier.
Therefore, if we assume no accumulation:
Mass in + Mass generated = Mass out + Mass consumed
No reaction
For some components of the process, there is no reaction occurring between them and other components, resulting in no accumulation, generation or consumption. This applies to elements (hydrogen, oxygen, nitrogen) since they can’t be broken down. It can also apply to other components but always double check that they aren’t reactive! In this case, the amount entering should be the same as the amount leaving, in which case:
Mass in = Mass out
Overall process
Mass cannot be created or destroyed, it can only be transferred. Because of this, for the whole process, regardless of whether its steady, unsteady, batch or continuous, the mass of everything entering the reactor will always be the same as the mass of everything leaving the reactor. This is because reactors are closed systems, and do not allow free exchange of mass in or out of the vessel. Therefore, the mass balance for the whole process will be:
MASS IN = MASS OUT
Those are all the rules! My advice would be to use the general equation for each component, and if there is no accumulation/ consumption / generation, just cross them out of the equation. Usually though, the only equations you need are:
MASS IN = MASS OUT
for components with no reaction, and
MASS IN + MASS GENERATED = MASS OUT + MASS CONSUMED
for components with reactions
If you want to know whether you can use the mass in = mass out equation, you can use this guide:
Does mass
in = mass out?
Material
Without
reaction
With reaction
Total mass
Yes
Yes
Total number of moles
Yes
No
Mass of the total amount of molecules
Yes
No
Number of moles of a molecule
Yes
No
Mass of an element
Yes
Yes
Number of moles of an element
Yes
Yes
This is complicated and hard to explain with words, so I will do some working out of example questions in later posts to help you understand it better. However, if you have any questions about these rules, please leave a comment and I will get back to you ASAP!
Mass balancing is a fundamental part of bioprocessing that is used to determine the amount of a component coming in or out of a system. A process stream is usually made up of multiple components. For example, feed entering the reactor will contain nutrients, water, growth hormones, etc. whilst the stream leaving the reactor will contain product, cells, waste (ammonia, carbon dioxide) and many other components!
We want to know how much of each component is entering, leaving or inside the reactor or system so that we can characterise the process and understand it better. However, with so many reactions occurring in biological processes, we won’t always know how much of each component there is off the top of our head! This is where mass balancing comes in.
Mass balancing is the calculation of what is going in or out of the system based on what information we have. For example, if we know x amount of cells and x amount of feed are going into the reactor, what is the amount of product and waste produced? Remember: we need to know one side of the equation (in or out) to figure out the other!
In broader terms, mass balances refer to the balancing of mass going in and out of a system. A system can be anything that has a clearly defined boundary that separates it from the external environment. For example, a cell has a cell membrane, a water bottle has plastic walls, a bioreactor has metal walls. There are 3 main types of systems:
Open: Allows energy and matter exchange freely with external environment
Closed: Only allows energy exchange freely with external environment
Isolated: Does not allow energy or matter exchange with external environment
In most bioprocesses, the system (bioreactor) is closed – matter cannot be exchanged freely with the surroundings. When matter is exchanged in the reactor, it is because we intentionally add or remove components.
A simple diagram of a system.
Remember: matter cannot disappear! It can only be transferred. Therefore when we add a component to a system we expect to either stay in the system, come out or be transformed into another component.
Now that we know what mass balances are, lets move on to how to do them! If this post wasn’t clear, please leave a comment and I will try to explain it in simpler terms.
This is a quick introduction to sterilisation. We will go through this sterilisation in more detail in its own post, but since it is quite complicated this will just be a brief explanation so you understand the concept.
What is sterilisation?
Sterilisation is the removal or prevention of contamination at any point throughout the process.
Why do we need sterilisation?
Contamination is a big no-no in the industry. If your cell culture is contaminated, you must discard it. This is because contaminants can cause danger to the end user of the product. If your product is contaminated with bacteria and someone ingests/ injects it, they could get seriously sick. Therefore regulatory bodies require that there is no contamination present at any point of the process. Also, contaminants can compete with your cell line for nutrients and oxygen in the broth, which will reduce cell numbers, and could stop production of your product.
Levels of sterilisation required
No sterilisation: Process does not need to be sterile. This is mainly for processes that do not create a product that will be ingested i.e. Waste water treatment.
Protected fermentation: For processes that require some degree of sterility but do not tend to get contaminated if treated properly i.e. disinfection of needles, pasteurisation of milk.
Unprotected fermentation: Require a high degree of sterility due to high likelihood of contamination if exposed to the external environment. This goes for bioprocesses that grow cells in media since media contains many components (nutrients, oxygen, etc.) that are highly favourable by contaminants i.e. bacteria.
Examples of contaminants
Bacteria
Viruses
Mycoplasm
Fungi
How do you avoid contamination?
Bioreactors usually operate as closed systems, meaning they do not exchange any material with the environment around it. Here are some methods of preventing contamination in reactors:
Sterilise the reactor and any ancillary equipment (parts that you attach to the reactor)
Sterilise whatever is going into the reactor (air, buffers, media, nutrients, etc.)
Introduce a pure inoculate (cell culture) and feed. Before introducing the cells into the reactor make sure they are not contaminated (using a viable cell count to detect unusual growth). The feed should also be sterilised before introducing it to the reactor.
Maintain an aseptic operation. Make sure the bioreactor is properly sealed and there are no cracks or holes that expose it to the external environment.
Methods of sterilisation
Inactivation/ killing of viable microbes
This can be done by:
Heat treatment (heating the components to a high temperature to kill contaminants)
Irradiation
Chemical treatment (using strong chemicals i.e. disinfectants to kill contaminants)
Physical Removal of microbes
This can be done using filtration or centrifugation.
If you have any questions, please leave a comment below and I will get back to you ASAP!
In order to successfully expand cells without any trouble, a reactor must meet these requirements:
Operate aseptically: The reactor must be sealed and sterilised so that contamination will not occur during the process. This is important as contaminants not only compete with your cells for nutrients but also can harm your customer if it is in the final product. Contaminated cell cultures must be discarded according to regulatory bodies.
Agitate and aerate effectively: Cells need oxygen to survive, hence effective aeration is needed. Effectively agitated broths produce homogeneous flow: where all the liquid has the same concentrations of oxygen, nutrients, pH buffer, etc. This is important as we want all cells to be exposed to the same conditions so that they grow and proliferate in the same way e.g. if a region of the liquid has too little oxygen all the cells in that region will die.
Control: We must be able to control the conditions in the reactor (temperature, pH, air flow rate) not only so that the cells are growing in conditions we know optimise survival and growth, but to ensure that we have an idea of what conditions the cells are in and therefore predict how are cells will proliferate and grow.
Minimise evaporation: We don’t want the liquid in the reactor evaporating since it contains cells and useful nutrients. Evaporation also means that our predictions of what is happening inside the reactor won’t be accurate.
Allow monitoring/ sampling: So that we can analyse and understand what is happening in the culture.
Minimise labour: More labour = more staff. Staff cost money!
Maximise computer control: Also to reduce cost of staff. Automation can also reduce human error.
Geometric similarity for scale-up: The reactor dimensions (i.e. height of vessel to diameter of vessel ratio, number of impellers, impeller diameter to vessel diameter etc. ) affect the conditions inside the reactor and thus cell growth. Therefore it is much simpler to scale-up cell expansion by maintaining the same reactor dimensions throughout upstream processing.
Flexible operation: In case of reactor failure, change of cell culture, fermentation modes or reactor types (see Fermentation modes and Types of reactors), a process must be flexible and allow easy changes. One example is to increase the number of reactors you have.
If you have any questions, please leave a comment below and I will get back to you as soon as possible!
In upstream processing there are many different types and sizes of bioreactors. The reactors usually differ based on method of mixing. Each reactor type tends to have it’s own advantages and disadvantages, and some will be more suitable for a certain cell type than others. For example, for an extremely shear sensitive cell it may be better to grow it in a bioreactor that is mixed by bubbles instead of impellers. However, for cells that need a lot of oxygen, a stirred tank reactor would be more suitable since they have good air dispersion. These factors also affect bioreactor size, as explained below.
What does reactor size depend on?
Bioreactor size mainly depends on type of product, product potency and product demand. For example, antibiotics are in high demand as the majority of the population need them, and therefore large vessels are needed. For personalised gene therapy, such as CAR-T cell therapy, a large quantity of the product is not required since the cells are grown only for one individual. Vaccines are also in high demand, but since they are much more potent antibiotics, it does not require as big of a vessel.
Common types of reactors
Stirred Tank Reactor
A stirred tank reactor (STR) is probably the most common type of reactor found in industry. Mixing is achieved via stirring using an impeller, and aeration is done via a sparger (a pipe that pumps in air). Baffles are also used in STRs to reduce vortexing (mini whirlpools) of the fluid.
A stirred tank bioreactor. Mixing is done via stirring with an impeller. Baffles on the edge of the reactor prevent vortex formation.
STRs are generally the most efficient at mixing large volumes of liquid and achieving liquid homogeneity (oxygen concentration, nutrient concentration, are more even throughout fluid). Also, STRs are well-characterised, which has made processes using STRs easily automated. This is why it is the most common reactor in the industry.
However, STRs generally have a higher power consumption (amount of power dissipated into the fluid) than other reactor types. Power consumption is related to the shear stress inflicted on the broth, which can cause damage to cells. Therefore STRs tend to be unsuitable for cells that are shear-sensitive, such as mammalian cells. STRs are best suited for cells that are not shear-sensitive (such as bacterial cells).
Additionally, some cells are anchorage dependent (need to be attached to a substrate in order to proliferate), which is common for mammalian cell lines. These kinds of cells cannot be grown in a traditional STR. However, micro-carriers (basically porous balls that cells can anchor to in a fluid) have been increasingly popular, allowing anchorage-dependent cells to be grown in STRs.
Rocked bed bioreactor
A rocked bed bioreactor uses rocking (moving in a seesaw like pattern) to create mixing in the fluid. It generally consists of a pillow-shaped container attached to a rocking surface (look below). Aeration is achieved via surface aeration – air diffuses from the surface of the fluid into the bulk fluid.
Rocked bed reactor. The cell broth is mixed via rocking. Aeration is achieved via surface aeration.
Rocked bed bioreactors are good for cells that are shear sensitive, since power dissipation tends to be lower. However, surface aeration means that there is lower oxygen transfer than in a STR. Also, a rocked bed system is only effective at low volumes due to difficulty of rocking large volumes and inefficient mixing. Online monitoring and control is also difficult since probes will not be immersed in the fluid all the time (as the fluid is rocked back and forth).
Rocked bed bioreactors are commonly used to scale-up from a flask to a bioreactor, as they can raise cell numbers to higher cell numbers than a flask.
Packed bed bioreactor/Perfusion Culture
Packed bed reactors contain a solid substrate within the vessel for cells to attach to. Mixing and aeration are done by continuous flow of liquid through the vessel. The most common packed bed reactor is the Hollow fibre reactor. The hollow fibre reactor has numerous little pipes spanning the length of the vessel, on which cells can grow on the outside. The fibres contain perforations (holes) to allow liquid to flow through and to increase the surface area.
A hollow fibre bioreactor. Mixing and aeration is achieved by a constant flow of liquid through reactor – waste out, oxygenated feed in.
Hollow fibre reactor diagram (as shown from above). Cells grow around fibres, which have pores to transfer waste into the fibre and feed containing nutrients out of the fibre and to the cells.
Packed bed reactors are good for anchorage-dependent cells as they provide a surface for the cells to attach and proliferate on. They also have better homogeneity than static (not mixed) cultures since there is constant fluid flowing through. As a tank is used, scalability and online monitoring and control is better than in a rocked bed or static culture, especially for anchorage-dependent cells.
However, sampling tends to be difficult and the sample is usually destroyed in the process, due to the complex design. This factor also makes cleaning the reactor very complicated. Cell distribution may also be uneven, which can cause cells to grow differently.
Packed bed reactors tend to be used for expansion of anchorage-dependent cells, though they are getting phased out by micro-carriers.
Shaken cultures
Shaken cultures are cell cultures mixed by shaking. This is done by placing a flask on a shaking system. Aeration is done via surface aeration. Shaken cultures are usually kept inside incubators in order to maintain constant temperature and oxygen levels.
Shaken culture. A flask is placed on top of a shaking platform to mix the contents.
Shaken cultures are well understood and characterised, making them easy to use. This also makes them easy to automate. However, they are not scalable, there is no online monitoring or control and cannot be used for large volumes of cell broth.
Shaken cultures are often used early in the process to grow cell numbers before cell expansion to a rocked bed reactor or small STR.
If you have any questions or want me to explain some other types of reactors, please leave a comment below and I will get onto it as soon as possible!
Bioprocessing Explained 