CC8 - Biotech

Question 1

What are the three main areas of application for biotechnology?

Answer 1

The main areas of application for biotechnology are agricultural, medical, and industrial.

Question 2

Describe the fundamental difference between biopharmaceuticals (biologics) and pharmaceuticals.

Answer 2

Biopharmaceuticals are produced by cells and generally have a high molecular weight (100s of kDa). They are sensitive to manufacturing processes, mostly protein-based and comparatively complex, less stable, and potentially immunogenic. Pharmaceuticals are produced by chemical reactions, have a low molecular weight, undergo robust manufacturing, are more stable, and mostly non-immunogenic.

Question 3

What are some examples of protein-based biotherapeutics?

Answer 3

Examples include monoclonal antibodies (and derivatives), hormones, growth factors, fusion proteins, enzymes, and cell-based drugs.

Question 4

What are the key factors to consider when designing protein-based drugs?

Answer 4

Factors include mechanism of action, characteristics of good/bad biotherapeutics (efficacy, safety—toxicity, off-target effects—and immunogenicity), and design strategies.

Question 5

Name three common recombinant protein expression systems used in biotechnology and list a key characteristic of each.

Answer 5

E. coli: simple, cheap, fast, no glycosylation. S. cerevisiae: eukaryotic, intracellular/secreted proteins, yeast-type glycosylation. CHO cells: mammalian, human-like glycosylation, biotech workhorse.

Question 6

What is the role of glycosylation in biotherapeutics, particularly monoclonal antibodies?

Answer 6

Glycosylation (especially N-glycosylation) impacts stability, solubility, bioavailability, pharmacokinetics, and immunogenicity. In mAbs, N-glycans at CH2 domain influence ADCC and CDC.

Question 7

Describe the basic structure of an IgG monoclonal antibody and the function of its main domains.

Answer 7

Fab domain (light and heavy chains with CDRs) binds antigen; Fc domain (CH2, CH3) binds Fc receptors, activates complement, and induces ADCC.

Question 8

How can monoclonal antibody functions be modulated through engineering?

Answer 8

Fc-mediated functions can be enhanced (↑ADCC for oncology) or disabled (for immune checkpoint blockade) via amino acid mutations or glycosylation modulation.

Question 9

What is an antibody-drug conjugate (ADC)?

Answer 9

A monoclonal antibody chemically linked to a cytotoxic drug, delivering the drug specifically to cancer cells, reducing off-target effects.

Question 10

What are bispecific antibodies (bsAbs)?

Answer 10

Engineered antibodies recognising two different epitopes/antigens, enabling new mechanisms like tumour-immune cell engagement.

Question 11

How was insulin, the first biotech drug, produced?

Answer 11

Human insulin was produced by recombinant DNA technology in E. coli.

Question 12

Why is glycosylation important for the therapeutic protein Erythropoietin (EPO)?

Answer 12

Glycosylation, especially sialylation, determines EPO’s serum half-life. Non-sialylated EPO is rapidly cleared by hepatocytes.

Question 13

What are nucleic acid-based drugs and what is their potential in treating disease?

Answer 13

Nucleic acid drugs treat disease via gene inhibition, replacement, or editing, using gene transfer or molecules like ASOs and siRNA.

Question 14

What are some key considerations when evaluating if a biotherapeutic is “good” or “bad”?

Answer 14

Efficacy, safety (toxicity, off-target effects), and cost.

Question 15

What is a display library in the context of biotechnology?

Answer 15

A large collection of molecules displayed on a system linking the binder to its gene, used to screen for desirable properties.

Question 16

What types of molecules might be found in a display library?

Answer 16

Antibodies, nanobodies, artificial binders, nucleic acids, or small chemical compounds.

Question 17

What are some common display technologies used in library screening?

Answer 17

mRNA display, phage display, and yeast display.

Question 18

Describe the general process of screening a library.

Answer 18

Selection of binders to a target protein, elution and replication, multiple enrichment cycles, followed by binder gene isolation and production.

Question 19

What are some methods used for selecting binders from a library?

Answer 19

Surface panning, pull-down techniques (e.g., mRNA-protein fusion), magnetic bead sorting, and flow sorting.

Question 20

What is the core principle of directed evolution?

Answer 20

Introducing diversity and selecting for improved traits iteratively, leading to molecules with enhanced properties.

Question 21

Name three methods for introducing diversity into a library for directed evolution.

Answer 21

Mutagenesis, error-prone PCR, and synthetic DNA library construction.

Question 22

How does error-prone PCR contribute to creating diversity in directed evolution?

Answer 22

By using low-fidelity polymerases to introduce random mutations during amplification.

Question 23

What is DNA shuffling and how is it used in directed evolution?

Answer 23

Recombination of gene fragments to create diverse, chimeric genes for improved functionality.

Question 24

How can the stringency of selection be increased during directed evolution?

Answer 24

Tougher conditions (e.g., weaker binding buffers, fewer beads, altered gating) favour higher-affinity binders.

Question 25

What is de novo protein design?

Answer 25

Designing entirely new proteins with sequences and structures not found in nature.

Question 26

What are the two main categories of de novo protein design methods?

Answer 26

Energy minimisation approaches and artificial intelligence-based approaches.

Question 27

How are energy optimization-based approaches (like Rosetta) used in protein design?

Answer 27

Using physics-based energy functions to identify sequences with minimum energy for a desired structure.

Question 28

How are artificial intelligence-based methods used in de novo protein design?

Answer 28

Neural networks predict sequences or generate novel structures, e.g., AlphaFold, ProteinMPNN, RFdiffusion.

Question 29

Provide an example of how de novo protein design has been used to create a novel protein topology.

Answer 29

Rosetta was used to design Top7, a novel protein fold confirmed by X-ray crystallography.

Question 30

How are display libraries, directed evolution, and de novo protein design integrated to create new biomolecules?

Answer 30

De novo libraries are screened by display tech, and best molecules are improved by directed evolution (e.g., Adalimumab).

Question 31

Describe how protein design can be used for thermal stabilization of proteins.

Answer 31

Using tools like PROSS to analyse sequence alignments, mutate variable sites, and select stabilising mutations via energy scoring.

Question 32

How can protein design be used in vaccine development, specifically for Virus-Like Particles (VLPs)?

Answer 32

Designing VLPs by assembling trimeric proteins into precise geometries, mimicking viral surfaces to elicit strong immunity.

Question 33

What is template grafting in protein design for vaccine development?

Answer 33

Grafting an epitope onto a matching template protein backbone, followed by design optimisation for proper folding.

Question 34

What is topology building in protein design, and why is it used?

Answer 34

Building new protein folds around challenging epitopes when grafting fails, often followed by yeast display and selection.

Question 35

How is rational design combined with directed evolution in enzyme engineering?

Answer 35

Rational active site design is followed by random mutagenesis and shuffling to evolve enhanced catalytic function.

Question 36

What are the four main stages of pharmacokinetics (ADME)?

Answer 36

Absorption, Distribution, Metabolism, and Excretion.

Question 37

What are some challenges that biotherapeutics face in the human body?

Answer 37

Proteolysis, pH denaturation, crowded molecular environments, and localisation difficulties.

Question 38

Why is subcutaneous (SC) administration often preferred over intravenous (IV) administration for biologics?

Answer 38

SC is more convenient, faster, cheaper, enables self-administration, and allows fixed dosing.

Question 39

How can nanoparticles be used to improve the delivery of biologics?

Answer 39

Nanoparticles encapsulate biologics, enhancing stability, cell uptake, circulation time, and enabling targeted delivery.

Question 40

Name four main types of nanoparticles used for drug delivery.

Answer 40

Lipid-based nanoparticles (LNPs), polymeric nanoparticles (PNPs), inorganic nanoparticles (e.g., gold), and carbon-based nanoparticles.

Question 42

What are adeno-associated viruses (AAVs) and why are they used for gene delivery?

Answer 42

AAVs are small, non-pathogenic viruses with a simple structure that can package genetic material (recombinant AAVs or rAAVs). They are used for gene delivery due to their low toxicity, non-pathogenic nature, and ability to target specific tissues, although they require a helper virus for replication.

Question 43

What are some challenges associated with using AAVs for gene therapy?

Answer 43

Challenges include the presence of pre-existing neutralizing antibodies (NAbs) that can block receptor binding, premature degradation in endosomes triggering immune responses, degradation via the proteasome, and the potential for innate immune responses triggered by dsRNA formation in the nucleus.

Question 44

What are some strategies to improve the stability and longevity of protein-based drugs?

Answer 44

Strategies include mutagenesis to create more stable variants, PEGylation (attachment of polyethylene glycol) to protect against proteolysis and immune recognition and increase half-life, using excipients to prevent surface adsorption and aggregation, supercharging proteins to enhance stability, glycoengineering to modify glycosylation patterns for better activity and half-life, and Fc fusion to increase half-life through increased size and FcRn recycling.

Question 45

What are CAR T cells and how do they work in cancer immunotherapy?

Answer 45

CAR T cells are T cells engineered to express a chimeric antigen receptor (CAR). This CAR has an antigen recognition site (e.g., for a cancer-specific antigen) and intracellular signaling domains that activate the T cell upon binding. CAR T cells re-enable the patient’s own immune system to target and kill cancer cells.

Question 46

What are some limitations and challenges associated with CAR T cell therapy?

Answer 46

Limitations include the risk of cytokine release syndrome ("cytokine storm"), challenges in targeting solid tumor microenvironments, and the high cost of therapy.

Question 47

What are some potential future directions for CAR T cell therapies?

Answer 47

Future directions include tuning the ratio of CD4+ and CD8+ T-cell populations to improve efficacy, developing CAR-NK (natural killer) cells and allogeneic ("off-the-shelf") CAR T cells to reduce cost and time, and advancing 3rd and 4th generation CAR protein designs to enhance functionality.

Question 48

What are some key considerations for the regulation of biotherapeutics by agencies like the FDA and EMA?

Answer 48

Regulatory agencies like the FDA and EMA aim to ensure studies are conducted under applicable laws and regulations and to protect the rights, safety, and welfare of human research subjects. They evaluate clinical data to assess safety and efficacy before approving drugs for commercialization.

Question 49

Briefly outline the 5-step drug development and approval process in the US.

Answer 49

The process includes: 1) Discovery and Development (identifying new drug candidates), 2) Preclinical Research (testing for safety and toxicity in the lab), 3) Clinical Research (testing in human clinical trials across multiple phases), 4) FDA Review (evaluation of clinical trial data to determine safety and efficacy), and 5) FDA post-market safety monitoring (ongoing surveillance after approval).

Question 50

What are Good Laboratory Practices (GLP) and Good Manufacturing Practices (GMP) in the context of biopharmaceutical development?

Answer 50

GLP includes guidelines and principles for laboratory operations to ensure the quality and integrity of preclinical research data, covering aspects like personnel, premises, equipment, and procedures. GMP encompasses all aspects of making a drug to ensure its safety, quality, and consistency, including production, quality control, and packaging.

Question 51

What is bioprocessing in the context of industrial-scale protein production?

Answer 51

Bioprocessing is any manufacturing process involving living cells to generate a product, such as biopharmaceuticals. It occurs in highly controlled environments and typically includes upstream (cell growth and cultivation), downstream (purification), and fill-finish stages.

Question 52

Briefly describe upstream bioprocessing.

Answer 52

Upstream bioprocessing refers to the steps related to cell growth and cultivation before purification. It involves optimizing media, cell inoculation, and growth kinetics in bioreactors, often scaling up from small to large volumes.

Question 53

Briefly describe downstream bioprocessing.

Answer 53

Downstream bioprocessing encompasses the steps required to purify and formulate the product from the cell broth after upstream processing. This typically involves clarification (removing cells and debris), isolation/capture (recovering the product, often using chromatography), purification (removing remaining impurities), and polishing/formulation (preparing the drug for clinical administration with excipients and sterilization).