Class 20 Scaleup Book Chapter 10 It seems insignificant but there is a general chemical rule that says each 10x scale up of the batch size takes as much time as the original research and costs 10x as much. Thus 1 year project to make 10gm of material will take 5 years to get to ton scale and the original $50K will become $5Billion. That is probably an overestimate and you could get away with a piddling $50million.

Section 10.2 largely talks about reactors and I will let you read the descriptive part. Some special points: with big reactors we have to pay a lot of attention to keeping the whole volume well-stirred with no backwaters where organisms might become modified due to different oxygen or nutrient levels. foaming is often a major problem because biopolymers will drop the surface tension of the water. Fluorinated compounds and silicones can be added to stop foaming but may cause other problems.

Sensors (Section 10.3) It is easy to measure temperature and pH in a reactor but such basic measurements do not give a lot of information about the chemistry taking place.

There is a lot of interest in developing sensors that provide more chemical information. While they can work well in the lab they tend to be much less reliable in complex mixtures where there may be interfering compounds or the sensor may become fouled ( coated with slime).

Gas analyzers should at least be relatively reliable. The best answer would probably be a small cheap mass spec. Maybe airport detection systems will turn out to be useful.

HPLC is very powerful for liquids, but slow and hard to automate.

Once you have a mass of data on a fermentation, you have to figure out what to do with it. That can be quite difficult.

Sterlization Section 10.4 Many fermentation must be sterile, with no organisms except those that should be there. In this context “kills 99.9% of all bacteria” is only a start. Whether or not a homogenous population of bacteria is extinguished becomes a probability as discussed in the book. There is a further problem that the population may not be homogeneous, so a sub-population will be resistant to the treatment and survive, recall 'Conan the bacterium' from a previous lecture. It is becoming clear that this is an issue with cancer treatments, tumors are not homogeneous and small numbers of survivor cells with a different genetic makeup will grow once the main tumor is killed by a drug.

There follow Chris Brigham's notes on sterilization

What is sterilization? The Centers for Disease Control and Prevention (CDC) define sterilization as process that destroys, eliminates or inactivates (kills) all forms of microbial life including bacterial endospores. Not to be confused with disinfection, which kills most forms of microbial life (except bacterial spores).

Methods of sterilization Chemical treatment Exposure to radiation – UV – Gamma – X-ray Sonication Filtration* Heating* *Used in large-scale processes

Liquid growth medium is commonly sterilized in the vessel where it is to be used (i.e., the fermenter). If steam is used: Allowance must be made for dilution of medium by condensate Typically adds 10-20% extra volume High quality steam should be used No introduction of metals or other toxic substances to culture Variation of temperature with time during sterilization Reduction in number of viable cells during batch sterilization

Rate of heat sterilization. Fist order cell death kinetics, in a batch vessel where the only process affecting the number of viable cells is death: N is the number of viable cells, t is time, and k d is the specific death constant. Direct integration of the above equation is only valid when temperature is constant: OR Where t hd is the holding time, N 1 is the number of viable cells initially, and N 2 is the number of viable cells at the end of holding

Generalized time-temperature profiles for heating and cooling during sterilization.

General equations for temperature as a function of time during the heating and cooling periods of batch sterilization.

Variables involved in the heating/cooling equations:

Continuous sterilization Continuous steam injection with flash cooling Heat transfer using heat exchangers High-temperature, short time continuous sterilization can maximize cell destruction while minimizing damage to the fermentation media.

Temperature v. time, continuous sterilization SteamHeat exchangers Steam – dilution of medium by condensate; potential of foaming (problematic for flash cooler) Heat exchangers – more expensive to construct; susceptible to fouling (reduces heat transfer efficiency) Disadvantages of continuous sterilization methods:

Fluid flow into a bioreactor Ideally, all fluids entering a bioreactor should spend the same amount of time in sterilization No mixing should occur in tubes Risk of contamination goes up Fluid entering the sterile environment of the bioreactor should neither experience mixing or variation in velocity

Plug flow is ideal for fluid entering a bioreactor In reality, fluid in pipes experiences a range of different velocities

Filter sterilization of liquids Media with heat-labile components – Ex: enzymes or serum Polymer membranes – Pore size = 0.2 to 0.45 µm – Membranes must be sterilized before use High flow rates require large surface areas Filtration generally not as reliable as heat sterilization – Virus and mycoplasma could potentially pass through pores – Media typically pre-incubated prior to inoculation

Sterilization of air Sterile air must be provided to bioreactors for aerobic processes The number of microbes in air is ~ cells/m 3 Filtration is generally the process for sterilizing air – Heat sterilization of air is impractical Depth filters are widely used – Made of fibrous materials like glass wool – Don’t perform well with large fluctuations of velocity or with wet air input Steam-sterilizable cartridge filters are replacing depth filters Filters also used to sterilize effluent gases – Prevent release of microbes into the environment

Finally look at the two files from Celligen and Eppendorf (biobundle). These are ads. for disposable reactors. The idea is that going to disposable plastic reactors greatly reduces the problems on contamination between batches. The end