Fleet Bioprocessing Flashcards
What is Bioconjugation?
Bioconjugation is a chemical technique used to couple two molecules together via a stable linkage, where at least one is a biomolecule.
A Biomolecule is labelled to produce a conjugate.
A biomolecule could be…
Protein > A Protein biomolecule is made up of amino acids, arranged in a particular sequence (primary structure). Structure defines function.
Enzyme
Nucleic acid
Carbohydrate
Lipid
Bacterium
Examples of biomolecules used at Fleet
Antibodies (IgG ~150kDa, IgM ~900kDa, etc)
Peroxidase from Horseradish (HRP ~47kDa)
Alkaline Phosphatase (AP ~140kDa)
Avidin ~67kDa / Streptavidin ~55kDa
The Dalton(Da) is equivalent to theatomic mass unit (g mol-1). Daltons are commonly used to describe biomolecule/protein MW.
Examples of labels used at Fleet
Biotin
Fluorophores (small molecule, FTC.ED, AF)
Enzymes (HRP, AP)
Small molecule drugs / toxins
Haptens
Examples of conjugations at Fleet
Biotin-BSA
Ab-Biotin
Streptavidin-HRP
Ab- Alexa Fluor 488 (different numbers mean different absorbances = different colours)
HRP-DES-Ab
Conjugations via Lysine
Carboxyl to Amine conjugation using EDC/NHS mechanism:
NHS Ester reagent will react with primary amine on protein to = stable conjugate (amide bond).
Lysine = electrically charged side chain.
NH3+ = hydrophilic, buffer 3 good.
Conjugation reaction = condensation.
Lysine residues on primary amine of protein will react to make stable amide bond.
Examples of protein conjugations via lysine at Fleet:
BSA – Biotin (Bovine serum albumin is a serum albumin protein derived from cows)
Antibody – Fluorophore
Adv: Single step, wide pH range, stable amide bond.
Disadv: Labels need to be functionalised as NHS ester, Labels often have poor water solubility, Reaction progression not easy to measure, Poor site-specificity (ie. lysine could be added on different sites for each product).
Conjugations via Cysteine
Sulfhydryl-maleimide coupling
Maleimide Reagent reacts with Sulfhydryl on protein to = stable conjugate (THIOETHER BOND).
Addition reaction = Simple.
Thioether bond (stable) = pH 7-9, good site specificity. Ellman’s assay to monitor progress > it measures Sulphur.
Two main approaches for Thiolation:
1. Conjugation of reduced protein via cysteine (cystine) residues.
- Conjugation of thiolated protein via modification of lysine residues. (SATA).
Sulfhydryl-maleimide is an example of a bio-orthogonal reaction.
Adv: wide pH range, Cysteines (cystines) are ubiquitous amongst most proteins and can be accessed through reduction/thiolation, good site specificity, Easy colorimetry to monitor reaction progress.
Disadv: Linker/label needs to be functionalised as maleimide, etc, Thioether ‘exchange’ is possible if GSH is present, Accessing protein cysteines can cause structural problems.
Examples of protein conjugations via cysteine used at Fleet:
Antibody – HRP
Antibody – AP
Factors Impacting Conjugation
Properties of the Protein
Protein and linker/label concentrations & stoichiometries.
Reaction time and temperature.
Buffer matrix.
pH.
Designing a Conjugation Experiment Considerations
Protein and linker/label concentrations.
Protein and linker/label solubilities.
Buffer matrices and linker solvents.
Reaction stoichiometries.
Reaction monitoring.
Purification.
Characterisation.
Designing a Conjugation Experiment: Buffer matrices and linker solvents
E.g. Buffer 3 (Phosphate pH 7.5) is our default buffer for conjugation of protein via lysines. However, some proteins may require higher ionic strength (e.g. IgM, KLH) and others are not compatible (e.g. Alkaline Phosphatase activity is inhibited by phosphate).
Buffer 32 (PBS, EDTA, pH 7.0) is our standard Sulfhydryl-maleimide coupling buffer, but TBSE pH 7.5 (buffer 99) is also a favourite.
Buffer 33 (PBS, pH 6.7) is our standard presentation buffer.
Linkers (e.g. SATA, SMCC, PMPI, EMCH, Biotin-XX-NHS, etc) are organic soluble; our default solvent is DMSO (water miscible, non-toxic, good protein-compatibility).
Linker concentration should be high to minimise DMSO content in the reaction (10% is widely tolerated), but not too high so addition volumes are not practical (e.g. >5 µl).
Designing a Conjugation Experiment: Reaction stoichiometries
This is usually driven by the desired incorporation of label, and the protein concentration.
The protein concentration and properties of the linker/label will determine the incorporation efficiency. We perform panels of conjugates to optimise for incorporation and learn what stoichiometries are required.
Protein concentration should be ≥1 mg/ml for higher incorporation efficiencies.
Designing a Conjugation Experiment: Reaction monitoring
Is performed whilst the reaction is on-going (e.g. Ellmans colorimetry to monitor sulfhydryl-maleimide coupling, or analytical SEC to monitor fragmentation of an Ab).
Purification
Purification is determined by what the reagents are and how they are to be separated. Examples of purification include:
Low resolution Size-Exclusion Chromatography (GFC)
High resolution Size-Exclusion Chromatography (GFC)
Affinity Chromatography (AC)
Ion-Exchange Chromatography (IEX)
AKTA Purifier (Chromatography)
Allthese purification approaches can be carried out using an AKTA FPLC system.
Analogous to HPLC butdesigned tooperateat lower back-pressures suitable for protein purification and characterisation.
Characterisation
Once we’ve made and purified a conjugate, some characterisation is required to quantify and qualify the product.
UV-vis spectrophotometry; concentration of protein and absorbing labels (if applicable).
Colorimetry; quantitation of biomolecule or specific functional group (BCA, LAL, HABA, TNBS, Ellmans, SAMSA)
SDS-PAGE; MW and purity with respect to standards, average DAR.
WB; qualification of protein activity/identification of target protein in a mixture.
UV-vis spectrophotometry and Colorimetry
Concentration of a substance follows Beer Law (A = elc).
Absorbances at different wavelengths > can convert to concentrations.
“Extinction” or “absorptivity” (e) of a substance is how strongly it absorbs at a specific wavelength. It is determined empirically to relate Absorption (A) at thatsame wavelength(measured as optical density) to concentration (c).
Absorbance proportional to concentration. Fleet measure at the max absorbance (peak) = greatest sensitivity.
A280 (proteins),A260/A280 (nucleic acids),A350-750 (chromophoric compounds).
UV-vis spectrophotometry and Colorimetry
A340 for TNBS (Amine/lysine quantitation).
A412 for Ellman’s (Thiol/cysteine quantitation).
A495 for SAMSA (maleimide quantitation).
A500 for HABA-Avidin (Biotin quantitation).
A570 for BCA (Protein quantitation).
Applications of Bioconjugation
Assay reagents.
Assay = Test of an analyte to get a dose response.
Immunology
Diagnostics (pregnancy test, thyroid function).
Veterinary
Therapeutics
Applications - Immunology
Raising antibodies requires an immunogen and host animal.
Small molecules (“haptens”) by themselves cannot elicit an immune response. To make small molecules immunogenic they are conjugated to a carrier protein (DYE1).
DYE1 Project > Making compound immunogenic so the host animal will generate anti-molecule against that compound = generates immune response!
More specific = more targeted.
COOH group = can use EDC/NHS chemistry for conjugation (lysine).
Applications - Theraputics (Antibody-Drug Conjugates)
Bioconjugation of a drug (payload) coupled to an antibody.
Conjugation of the drug attenuates it’s cytotoxic effect.
Ab functions as the targeting moiety, to deliver the drug to the desired target site, where the drug is ‘released’ and exhibits it’s cytotoxic effect.
Hinge region on Ab = can target as nicely exposed.
Ab is specific for target antigen > raised in an animal or phage display.
Important considerations for ADCs:
Controlling drug-antibody ratio (DAR) and conjugate heterogeneity (site-specific conjugation strategies are used for these reasons).
Design of the Ab-drug linker (important for getting the right release profile, etc).
Minimising the occurrence of aggregation.
Example at Fleet - Her2:
Her2 > breast cancer > lose hair.
Off target recognition > cytotoxic drug for targeting cancer cells.
Then will be cleaved / hydrolysed off over time.
Low aggregate Ab.
HABA-Avidin
HABA > aromatic, delocalised.
Biotin will displace HABA.
Ab500.
BCA Assay
Redox reaction.
Cu2+ reacts with Ab backbone.
Cu2+ (green) > Copper-BCA complex (purple, proportional to concentration of protein).
Best thing you can do is have protein with known concentration as a standard! (correction factors).
Gyros / Gyrolab
Gyros leverages innovative CD-based technology to enhance immunoassay performance.
Gyrolab automates immunoassays using proprietary CD-based technology.
These compact discs (CDs) are engineered with nanoliter microfluidic channels.
By removing the potential for human error, Gyrolab delivers ultra-robust immunoassay results at nanoliter scale.
Compared to conventional ELISA, Gyrolab produces high-quality immunoassay data in less than half the time, using smaller sample volumes.
Gyrolab immunoassays minimize sample and reagent volumes, reduce analysis time, and increase data quality across various applications.
Alexa Fluor Dyes
Conjugation to Biomolecules:
Alexa Fluor dyes can be directly conjugated to primary antibodies or secondary antibodies. This conjugation allows them to specifically bind to target molecules (such as proteins) within cells or tissues.
When these labeled antibodies encounter their targets, they emit fluorescence.
Excitation and Emission Spectra:
The Alexa Fluor series covers a wide range of the visible spectrum and even extends into the infrared.
Upon excitation (usually by a laser or specific wavelength of light), the Alexa Fluor dye molecules absorb energy.
As a result, they transition to a higher energy state.
When they return to their ground state, they emit fluorescence at a specific wavelength (the emission spectrum).
Signal Amplification:
Alexa Fluor dyes are often used in signal amplification strategies = enhances sensitivity and allows for better visualization.
Streptavidin-conjugated Alexa Fluor dyes are commonly employed for immunofluorescence imaging.
Fleet Example: Gyros FRD11 Job.
