Fleet Bioprocessing

Question 1

What is Bioconjugation?

Answer 1

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.

Question 2

A biomolecule could be…

Answer 2

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

Question 3

Examples of biomolecules used at Fleet

Answer 3

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.

Question 4

Examples of labels used at Fleet

Answer 4

Biotin

Fluorophores (small molecule, FTC.ED, AF)

Enzymes (HRP, AP)

Small molecule drugs / toxins

Haptens

Question 5

Examples of conjugations at Fleet

Answer 5

Biotin-BSA

Ab-Biotin

Streptavidin-HRP

Ab- Alexa Fluor 488 (different numbers mean different absorbances = different colours)

HRP-DES-Ab

Question 6

Conjugations via Lysine

Answer 6

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).

Question 7

Conjugations via Cysteine

Answer 7

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.

2. 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

Question 8

Factors Impacting Conjugation

Answer 8

Properties of the Protein

Protein and linker/label concentrations & stoichiometries.

Reaction time and temperature.

Buffer matrix.

pH.

Question 9

Designing a Conjugation Experiment Considerations

Answer 9

Protein and linker/label concentrations.

Protein and linker/label solubilities.

Buffer matrices and linker solvents.

Reaction stoichiometries.

Reaction monitoring.

Purification.

Characterisation.

Question 10

Designing a Conjugation Experiment: Buffer matrices and linker solvents

Answer 10

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).

Question 11

Designing a Conjugation Experiment: Reaction stoichiometries

Answer 11

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.

Question 12

Designing a Conjugation Experiment: Reaction monitoring

Answer 12

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).

Question 13

Purification

Answer 13

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)

Question 14

AKTA Purifier (Chromatography)

Answer 14

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.

Question 15

Characterisation

Answer 15

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.

Question 16

UV-vis spectrophotometry and Colorimetry

Answer 16

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).

Question 17

UV-vis spectrophotometry and Colorimetry

Answer 17

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).

Question 18

Applications of Bioconjugation

Answer 18

Assay reagents.
Assay = Test of an analyte to get a dose response.

Immunology

Diagnostics (pregnancy test, thyroid function).

Veterinary

Therapeutics

Question 19

Applications - Immunology

Answer 19

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).

Question 20

Applications - Theraputics (Antibody-Drug Conjugates)

Answer 20

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.

Question 21

HABA-Avidin

Answer 21

HABA > aromatic, delocalised.

Biotin will displace HABA.

Ab500.

Question 22

BCA Assay

Answer 22

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).

Question 23

Gyros / Gyrolab

Answer 23

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.

Question 24

Alexa Fluor Dyes

Answer 24

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.

Question 25

Sandwich ELISAs

Answer 25

Sandwich ELISA:

Purpose: Sandwich ELISAs are widely employed for detecting specific antigens in samples.

Antibody Pairs: These assays require the use of matched antibody pairs. Each antibody recognizes a different and non-overlapping region (epitope) of the antigen molecule.

Capture Antibody: The capture antibody binds to the antigen, which can then be detected.

Advantages:
High Sensitivity: Sandwich ELISAs are 2-5 times more sensitive than direct or indirect ELISAs.

High Specificity: Two antibodies are used to detect the antigen.
Flexibility: Both direct and indirect methods can be employed.

Disadvantages:
Antibody Optimization: If a standardized ELISA kit or tested antibody pair is not available, optimization is needed to reduce cross-reactivity between the capture and detection antibodies.

Question 26

Streptavidin-Biotin Detection: Sandwich ELISA

Answer 26

Streptavidin-Biotin Detection:

In a sandwich ELISA with streptavidin-biotin detection:

The detection antibody is biotin-labeled.

An extra streptavidin-HRP (horseradish peroxidase) is used for signal amplification.

Biotin-Streptavidin Interaction: Streptavidin has a strong affinity for biotin, making it an excellent detection system.

Streptavidin binds to the biotin-labeled detection antibody, enhancing the signal output.

Remember, this approach provides both sensitivity and specificity, making it useful for various applications in research and diagnostics.

Question 27

Streptavidin-biotin detection is a technique commonly used in sandwich enzyme-linked immunosorbent assays (ELISAs):

Answer 27

Sandwich ELISA:

Sandwich ELISAs are the most common type of ELISA.

They require the use of matched antibody pairs, where each antibody is specific for a different and non-overlapping region or epitope of the antigen molecule.

The capture antibody binds the antigen, which can then be detected.

The key advantage of a sandwich ELISA is its high sensitivity, being 2-5 times more sensitive than direct or indirect ELISAs.

It also offers high specificity since two antibodies are used to detect the antigen.

Sandwich ELISAs are particularly suited for analysing complex samples, as the antigen does not need to be purified prior to the assay.

There are different forms of sandwich ELISA based on the detection method.

Streptavidin-Biotin Detection:
In this form of sandwich ELISA, the detection antibody is biotin-labeled.

Biotin is a small molecule that binds strongly to streptavidin.

Streptavidin is a protein with an exceptionally high affinity for biotin (with a dissociation constant in the femtomolar range).

Streptavidin acts as a bridge between the biotin-labeled detection antibody and an enzyme (usually horseradish peroxidase, HRP).
The extra streptavidin-HRP complex is added to amplify the signal.

This method enhances sensitivity and allows for accurate detection of low-abundance targets in the sample.

So, in summary, sandwich ELISA with streptavidin-biotin detection combines the specificity of sandwich ELISAs with the signal amplification provided by streptavidin-biotin interactions, making it a powerful tool for detecting antigens!

Fleet Examples: Gyros FRD11 job with biotinylated capture antibodies and conjugated detection antibodies with AF.
Wellfish project.

Question 28

What is an Immunoassay?

Answer 28

Immunoassays use antigen antibody recognition to detect and quantify an antigen in a sample
ELISAs are the most common examples.

Analyte: The molecule detected by the immunoassay is often referred to as an “analyte”. It can be a protein, although it may also be other types of molecules, as long as the proper antibodies with the required properties are developed for the assay.

Antibody-Antigen Interaction: Immunoassays rely on the ability of an antibody to recognize and bind to a specific macromolecule (referred to as an antigen).

Signal Detection: The other key feature of immunoassays is the production of a measurable signal in response to the antibody-antigen binding.

Most immunoassays involve conjugating antibodies or antigens with detectable labels. These labels emit radiation, produce colour changes, fluoresce under light, or emit light, allowing for detection through various means.

In summary, immunoassays play a crucial role in medical diagnostics and research, allowing us to detect and quantify specific molecules in biological samples through antigen-antibody interactions.

Question 29

What is an ELISA?

Answer 29

An ELISA, or enzyme-linked immunosorbent assay, is a laboratory technique that can detect and measure the presence of specific molecules, such as proteins, antibodies, or hormones, in a liquid sample.

ELISA is based on the principle of antigen-antibody binding and enzyme-mediated colour change. ELISA can be used for various purposes, such as diagnosing diseases, testing for pregnancy, or monitoring immune responses.

Question 30

Immunoassay - simple

Answer 30

Immunoassays use antigen antibody recognition to detect and quantify an antigen in a sample

ELISAs are the most common examples.

Question 31

TNF - alpha sandwich ELISA example

Answer 31

First, we bind the anti TNF alpha capture antibody to the microplate wells, next the wells are washed and blocked with albumin to cover any exposed plastic and prevent unspecific binding!

Now we add the standard or serum sample, neat and diluted! This leaves the antigens containing the TNF alpha cytokine to bind to the capture antibodies in the well.

We then add a secondary anti TNF alpha antibody conjugated with horse radish peroxidase.

TMB: tetramethylbenzidine (or ‘Sureblue’) is used a chromogenic substrate to the peroxidase. It acquired a blue colour in the presence of the enzyme, proportional to the amount of antigen present in the well.
The intensity of the colour is proportional to the concentration of TNF alpha in the sample.

Sulphuric acid is used to stop the HRP reaction over developing and to stop it reaching saturation, turning the blue liquid yellow.

Lastly, measurements determine the presence and quantity of antigen in the sample. Require a plate reader and statistical analysis.

Question 32

What is an assay?

Answer 32

Test of an analyte to get a dose response.

Question 33

How do immunoassays work?

Answer 33

There are many different types of immunoassays, but they typically include three main components:

Target analyte: The antigen you want to detect within a sample.
Antibody: Y-shaped proteins that bind specifically to the target analyte.
Detection label: A label, usually coupled to the antibody or antigen, that generates a measurable signal (e.g., colour, fluorescence or luminescence).

When the target analyte and antibodies are mixed together, they bind specifically to each other like a lock and key. The unbound antibodies are “washed” off or separated from the antigen-bound antibody.

Either the free antibody or antigen-antibody complexes are then measured by detecting the signal generated from the detection label.