This is a summary of course 8 theory lessons on downstream processing. After learning this summary, I got a 7.2 for knowledge test 2 myself (without rematch)!
This is a summary of the course 8 theory lessons on downstream processing. After learning this summary, I myself obtained a 7.2 for knowled...
DSP 1
Lesson 1: WHICH STEPS ARE STILL REQUIRED?
• Product present in a large volume
• Product intracellular
• Product in a mixture with other compounds
• Product in a mixture with cell debris
• Product not suitable for sale
WHAT CAN WE DO DURING DSP?
Clarification:
get rid of large
molecules
Formulation: to make the product ready for
sale
Successful and efficient protein purification,
selecting the most appropriate techniques,
optimizing their performance to suit the
requirements and combining them in a logical
way to maximize yield and minimize the
number of steps.
The recovery of a highly pure bio-(chemical) product (target bio-molecule) from a complex mixture
on an economical responsible manner.
How to monitor purification / quality?
- BCA assay, SDS page and purification table
What is important during DSP?
Which basic aspects of the starting material / the end-products / the process may lead to differences
in the way DSP is carried out?
Considerations for DSP
• What is your host organism?
• How was the target molecule produced?
• Intend of the product
• Starting material and handling
• Purity of the material at the start and required in final product
• Need for complete/partial removal of impurities
• Scale of purification/ scale-up possible
• Economical constraints (resources, equipment, finance, time)
Define your objectives for the DSP! Purity, quantity, activity, economics, timeframe
Purity:
• Nature of the source material
• Intended use of the product (also affects quantity)
,• Special safety issues; which impurities should be removed or can be tolerated
• Need for speed may override purity (economical motifs)
• Homogeneity might be an issue, the same molecule might be present in different forms due to
proteolytic cleavage, pre-terminated translation or PTMs
• Minor amounts of impurities may have biological implications
Activity
• Often a necessity in protein purification
- Enzymes, antibodies
• Proteins should remain in their native conformation
- No exposure to harsh purification methods and specific conditions
Which ones? How do they affect protein conformation?
• Some chemical methods do not require active protein
• Other methods require long term stability (e.g. crystallography)
• Purification strategies should be matched to the required end condition or storage condition
• Knowledge of enzyme kinetics necessary
Quantity
• Depending on the purpose different amounts of product are desired
• Affects the scale of purification
• Scale of purification further affected by sample volume and contaminants
Homogeneity
• Next to high purity, product should be highly homogeneous (only enzymes no other molecules)
• Heterogeneity introduced in different ways: (multiple molecules, like enzyme + cofactor)
- preterminated translation (mix of different size proteins)
- protease activity (during expression or purification)
- oligomers, aggregates and complexes
- Post-translational modifications
• Important to have a clear view which compound to purify
• Techniques to be applied might be different
THE HOST ORGANISM
• Contaminating molecules present?
• Where is the product produced/released?
• PTMs present?
• Proteolytic activity
, KNOWLEDGE OF THE TARGET AND IMPURITIES
• Basic information (MW, formula, structure, pI-value, solubility, PTM, conformation, stability,
activity, functional groups)
• Differences compared to impurities
• Determine stability window (for example enzyme is only stable for … hours in this buffer)
• Which impurities should be removed?
• Avoid inactivation
• Important for the selection of techniques and assays
STARTING MATERIAL
• Depending on the expression host and localization of the product different starting conditions
• May alter the sequence or conditions for purification
PURIFICATION TABLE
• Designed to follow the progress of purification
• Evaluate outcome of each purification step
• Recovery should be as high as possible and purification factor should increase
You calculate the amount total protein (mg) by (volume (mL) * protein conc. (mg/mL)
Supernatant USP: 100% starting material
Purification factor
Over time the volume will be less due to it being more concentrated (in our case filtration vivaflow).
Specific activity: total activity / total amount of proteins
Recovery (%): (new total activity / old total activity x 100%)
Purification factor: (specific activity new / specific activity old)
These 2 give an indication how the DSP is going
Old activity/ specific activity is the first (supernatant) ALWAYS -> in graph 107
ANALYTICAL ASSAYS
• Determine quantity, activity, recovery, critical impurities
• Follow progress, effectiveness
• Assays for both target molecule and impurities that need removal
• Should fit the properties of the target molecule
• If unavailable new techniques should be developed
Examples: SDS-PAGE, BCA, bradford, spectrophotometer, activity assays, Mass Spectrometry
PURIFICATION TECHNIQUES
• Should fit the properties of the target molecule
• Use different techniques using different target properties
beneficial for purity of the final product
- e.g. different forms of chromatography
• Implies knowledge of target molecule and possible contaminants!
The benefits of buying summaries with Stuvia:
Guaranteed quality through customer reviews
Stuvia customers have reviewed more than 700,000 summaries. This how you know that you are buying the best documents.
Quick and easy check-out
You can quickly pay through credit card or Stuvia-credit for the summaries. There is no membership needed.
Focus on what matters
Your fellow students write the study notes themselves, which is why the documents are always reliable and up-to-date. This ensures you quickly get to the core!
Frequently asked questions
What do I get when I buy this document?
You get a PDF, available immediately after your purchase. The purchased document is accessible anytime, anywhere and indefinitely through your profile.
Satisfaction guarantee: how does it work?
Our satisfaction guarantee ensures that you always find a study document that suits you well. You fill out a form, and our customer service team takes care of the rest.
Who am I buying these notes from?
Stuvia is a marketplace, so you are not buying this document from us, but from seller tessdekleijn. Stuvia facilitates payment to the seller.
Will I be stuck with a subscription?
No, you only buy these notes for $8.58. You're not tied to anything after your purchase.
