Bioprocessing

The production of genetic mutations via a physical change in DNA is known as [blank_start]mutagenesis[blank_end]

Microorganisms can be used as host for production of recombinant products such as proteins, enzymes, etc. Microorganisms utilised in this way are known as [blank_start]expression[blank_end] hosts

Advantages and Disadvantages of various Expression Hosts: E.coli: Recombinant protein glycosylated? [blank_start]No[blank_end] Recombinant protein exported? [blank_start]No[blank_end] Number of potential vectors. [blank_start]High[blank_end] Expression yields. [blank_start]Low[blank_end] Saccharomyces Cerevisiae: Recombinant protein glycosylated? [blank_start]Yes[blank_end] Recombinant protein exported? [blank_start]Yes[blank_end] Number of potential vectors. [blank_start]Low[blank_end] Expression yields. [blank_start]Moderate[blank_end] Pichia Pastoris: Recombinant protein glycosylated? [blank_start]Yes[blank_end] Recombinant protein exported? [blank_start]Yes[blank_end] Number of potential vectors. [blank_start]Low[blank_end] Expression yields. [blank_start]Moderate[blank_end] Bacillus: Recombinant protein glycosylated? [blank_start]No[blank_end] Recombinant protein exported? [blank_start]Yes[blank_end] Number of potential vectors. [blank_start]Low[blank_end] Expression yields. [blank_start]High[blank_end]

One of the main advantages of E.coli is that it is a gram negative bacteria and thus produces many [blank_start]endotoxins[blank_end] which are undesirable in the resulting product.

A system according to which successive batches of product (e.g. a vaccine) are prepared using the same master compound is called a [blank_start]seed lot[blank_end] system.

The first generation of amplified cells from one of the cells from a master seed are typically known as a [blank_start]working[blank_end] seed. From this, a [blank_start]production[blank_end] seed is taken and it is from this seed that the product is yielded.

Typically, a product should be produced by a seed no further than [blank_start]5[blank_end] generations from the master seed.

A master seed lot must be kept sterile at all times.

A common form of storage of a master seed is in liquid nitrogen at or below around 70 degrees celsius.

A [blank_start]colony forming[blank_end] unit is a unit used to estimate the number of viable bacteria or fungal cells in a sample.

Different types of media are used to isolate or identify particular organisms: [blank_start]Selective[blank_end] media allow certain types of organisms to grow, and inhibit the growth of other organisms [blank_start]Differential[blank_end] media are used to differentiate between closely related organisms or groups of organisms.

[blank_start]Transport[blank_end] media are essentially solutions of buffers with carbohydrates designed to preserve the viability of bacteria during transfer between locations without allowing their multiplication. The primary objective of the use of the this medium is to maintain the specimen as near its original state as possible.

[blank_start]Lyophilization[blank_end] or freeze drying is a process in which water is removed from a product after it is frozen and placed under a vacuum, allowing the ice to change directly from solid to vapor without passing through a liquid phase.

Which of the following methods of preserving cultures allows the cultures to be stored indefinitely?

Difference between sterilisation and disinfection: [blank_start]Sterilisation[blank_end] is the process of freeing an article or a surface from all living microorganisms including viruses and bacterial spores [blank_start]Disinfection[blank_end] is the process of freeing an article or a surface from all or some of the living microorganisms but not necessarily the bacterial spores.

Generally, the preferred method of sterilisation is via [blank_start]heat[blank_end]

Generally, animal derived materials should not be used for culture due to the risk of the [blank_start]Prion[blank_end] protein being present. This type of protein can trigger normal proteins in the brain to fold abnormally thus causing severe illness in humans.

The presence of Prion proteins in animal material can cause Transmissible Spongiform Encephalopathies in humans, including the human variant of mad cow disease called...

The [blank_start]Crabtree[blank_end] effect is a regulatory system whereby respiration is repressed by fermentation, except in low sugar conditions. This means that certain organisms, mainly yeasts such as Saccharomyces Cerevisiae, undergo fermentation over respiration when in aerobic conditions as long as they are in a high sugar environment.

Thus, due to the crabtree effect, most fermenters in industry run [blank_start]aerobically[blank_end]

The process by which fermentors have built in tools which allow them to clean themselves following fermentation thus removing the operating costs/potential contamination caused by human cleaning is called [blank_start]Cleaning In Place[blank_end]

Sterilisation in place is also sometimes necessary in stainless steel fermenters in order to ensure that the vessel is microbiologically clean. This process typically utilises [blank_start]high temperature steam[blank_end]

One of the main challenges faced by [blank_start]single use fermentation[blank_end] is the prospect of leachables and extractables migrating into the product during fermentation.

Comparison between Stainless Steel and Single Use Fermentation: Conducts fermentation faster = [blank_start]Single Use Fermentation[blank_end] Produces more product annually = [blank_start]Single Use Fermentation[blank_end] Lower cost for single fermentation = [blank_start]Stainless Steel Fermentation[blank_end] Lower lead time to build fermentation plant = [blank_start]Single Use Fermentation[blank_end] Significant reduction of cross contamination = [blank_start]Single Use Fermentation[blank_end]

The upstream processing is the bioprocessing phase in which cell lines are generated. The downstream processing, on the other hand, is the phase that deals with extraction or separation of desired products from the biomass.

When removing insolubles from a sample, what is the most common mode of doing so?

When isolating the desired product from the fermentation end product, which compound is typically the chief impurity? [blank_start]Water[blank_end]

After insolubles are removed and the product has been isolated, a process whereby contaminants that resemble the product very closely in physical and chemical properties are separated must occur. This process is called Product [blank_start]Purification[blank_end]

In terms of centrifugation, bacteria need a [blank_start]high[blank_end] speed whereas yeast cells and filamentous fungi (molds) need a [blank_start]lower[blank_end] speed

Chromatographic Separation Techniques: [blank_start]Ion exchange chromatography[blank_end] is based on electrostatic interactions between charged protein groups, and solid support material (matrix). [blank_start]Affinity Chromatography[blank_end] is typically used to separate out enzymes, hormones, antibodies, etc and involves binding of a protein to a matrix via a protein-specific ligand. [blank_start]Gel Permeation Chromatography[blank_end] separates molecules based on their molecular size using a porous matrix - the path of elution is proportionate to the molecular size.

Freeze drying, also known as Lyophilization, is the process of removing water from a substance by sublimation, causing it to rapidly change from a [blank_start]solid[blank_end] to a [blank_start]gas[blank_end], completely bypassing the [blank_start]liquid[blank_end] stage