BTEC 6303

What is bioprocess?

any process that makes use of microorganisms or cells in culture, or enzymes to manufacture products

What is a batch system?

nothing gets added except from the gasses. It's a closed system. Starts with a more concentrated culture.

What is the growth curve of batch systems?

inoculum is added while cells are in exponential growth phase

What are stirred tank bioreactors used for?

For the production of antibodies

What happens in lag phase

period of little or no cell division since cells acclimatize to new medium/conditions

What happens in log phase/ exponential growth phase?

cells begin to divide, enter period of growth or logarithmic increase

What happens in the stationary phase?

Growth rate equals death rate, fewer nutrients available and buildup of waste products. Change in pH is a factor in cell death

What happens in the death phase?

logarithmic decline phase starts and continues until the population is diminished to a few cells or dies out completely

What is a fed-batch systems

Grown in batch, then nutrients added incrementally near end of exponential phase

What are the different advantages of fed-batch over batch systems?

\-Minimize substrate inhibition- toxic substrates can be added during exponential growth to minimize the initial lag phase

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\-Less catabolite repression- addition of nutrients in increments avoids repression

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\-Extended production time- keeps the organism semi-starved and rapidly producing more and more of the desired protein product

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\-higher density cell cultivation possible

What happens at the end of fed-batch systems?

After 15 days terminate the reaction, have to stop the process

What is a continuous culture?

\-substrate and other nutrients are added to the system at a fixed, continuous rate

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\-While cells are continuously growing, harvest is occurring

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\-runs for 30-60 days

What are some advantages of continuous culture?

\-highest volumetric productivity

\-Savings in time, labor, and energy

\-Uniform product quality

\-Better automation, monitoring, and process control

\-reduces manual handling and human error

What are some disadvantages of continuous culture?

\-Cell adhesion to reactor walls and sensors

\-Difficult to maintain monoseptic conditions

\-Mutation and instability of some strains

\-Large volume of media - expensive- new- however, yield can offset cost

\-More complex process validation- because of duration of process

\-High upfront cost

What are the culture parameters that can be optimized?

\-media- composition, feeding profile

\-Aeration - stirring speed, gas flow rate, DO

\-Temperature

\-pH

\-Induction strategy

\-Protease inhibitors

\-Selection agent

What happens in downstream processes?

Separate protein product from the rest of the culture

What is protein purification?

the removal of all impurities and contaminants (cells, cell debris, other proteins, endotoxins, viruses, DNA) to make sure the drug is the right identity and is of high purity.

FDA requires biotherapeutics to be what?

requires them to be 99.9% pure

What is product recovery?

It is often work intensive and expensive, and can account for up to 85% of production costs.

During purification, stabilize proteins in solution:

\-Buffered from pH extremes

\-Add protease inhibitors

\-Work at low temperatures

\-Add antimicrobials

What are the steps of downstream bioprocess?

\-Recovery- removal of cells and cell debris prior to chromatography

\-Purification0 yields highly purified product in single step

\-Inactivates endogenous/adventitious viruses

\-Removal of product/process related impurities and viruses

\-Removes endogenous/adventitious viruses.

\-Final, formulated bulk drug substance

What are the 3 steps of product recovery?

1. Centrifugation
2. Filtration
3. Precipitation

What is continuous centrifugation?

\-separates cells/debris, and other insoluble components from medium

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\-most common harvest method for large-scale cell culture broth volumes of 2,000 and 20,000 liters

What is product recovery filtration?

size-based separation method. Different types of filters can remove cells, cell debris, viruses, other proteins, nucleic acids, endotoxins

What is tangential flow filtration?

a type of microfiltration that can be used for less than 2000

What is depth filtration?

the filter can not only reduce the turbidity, but also remove up to 50% of DNA impurities and 15% of HCPs at neutral pH

What is product recovery: Protein precipitation?

\-recombinant protein is usually less than 1% of supernatant

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\-similarities between proteins allow the separation of proteins from non-protein material in the supernatant

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\-salts, alcohols, acids cause proteins to settle out of solution-attracts proteins away from water and concentrates protein

What is size exclusion chromatography?

\-uses gel beads with pores of different sizes

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\-larger proteins move quickly around the beads, while smaller proteins slip through the pores and therefore move more slowly through the beads

What is ion exchange chromatography?

\-relies on the charge of the protein

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\-More negatively charged proteins would bind to the positively charged resin.

What is affinity chromatography?

\-usually the primary capture step

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\-relies on the ability of proteins to bind specifically and reversibly to uniquely shaped compounds called ligands

What are the different examples of affinity chromatography?

\-capture of Mabs using Protein A which binds specifically to Fc region of IgG

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\-Capture of Mab using antigen

Hydrophobic interaction chromatography

sorts proteins on the basis of their repulsion of water

How is virus inactivation achieved by?

\-low pH during or after protein A chromatography

\-heat

\-UV light treatment

\-Nanofiltration (20nm)

\-Solvent/detergent treatment for lipid-enveloped viruses

What occurs after viral removal?

\-After viral removal, concentrated by diafiltration

\-then terminal sterile filtration

\-yield 60-80% of starting amounts

What does tangential flow filtration rely on?

relies on the chemical concept of equilibrium - substances move from area of high concentration to area of low concentration

What does tangential flow filtration used to do?

Used to concentrate, then diafiltration removes cell contents and culture media salts, buffers, or additives, and replace it with cleaner, suitable buffer that will serve to stabilize proteins

What is the formulation of biologics mostly for?

Mostly for subcutaneous or intravenous formulation, aqueous or lyophilized

Formulation of biologics product concerns:

1. aggregation
2. oxidation
3. deamidation

cryoprotectant serve for what?

protects from freezing

Ex. sucrose and trehalose

lyoprotectant serve for what?

protects from lyophilization - drying

What does Polysorbate 80 do?

common for IgG liquid formulation

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\-protects from freeze thaw and agitation-induced aggregate formation

What do preservatives do?

prevent microbial growth, particularly for multi-dose, single container formulations.

Ex. benzyl alcohol, m-cresol, and phenol

What does fill and finish do?

divides bulk formulation into single dosage forms

\-includes choice of container, cap, label, and packaging

Lyophilization (freeze-drying) does what?

\-a vacuum is used to hasten the evaporation of water from the frozen preparation

\-will maintain protein structure, and can be stored at room temperature for long periods of time

High Performance Liquid Chromatography (HPLC)

uses high pressure to force the extract through a column in a shorter time

mass spectrometry

highly sensitive method used to detect the size and identity of most protein fragments

LC-MS for amino acid sequencing?

primary structure or sequence of amino acids determined by LC-MS

NMR (nuclear magnetic resonance)

\-used to determine the complex tertiary and quaternary structures in solution phase

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\-measures current generated by nucleus of atoms subjected to a magnetic field. Behavior depends on molecular environment

X-ray crystallography

\-is used to determine the complex tertiary and quaternary structures of crystals

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\-diffraction mainly based on electron density from electron rich atoms C, N, O