explain the advantages of using recombinant human proteins to treat disease, using the examples insulin, factor VIII and adenosine deaminase

Genetic Technology – Recombinant Human Proteins

Objective

To explain why recombinant human proteins are advantageous for treating disease and to describe the complete pipeline – from gene cloning to final drug formulation – using insulin, factor VIII and adenosine deaminase as case studies. The notes also cover the core techniques, vector design, host‑cell systems, screening methods, gene‑editing tools, broader biotechnological applications and the relevant biosafety, ethical and regulatory considerations required by the Cambridge International AS & A Level Biology (9700) syllabus.

1. Core Molecular‑Biology Techniques (Syllabus Topic 19.1)

• Polymerase Chain Reaction (PCR) – exponential amplification of a specific DNA fragment using sequence‑specific primers, a thermostable DNA polymerase and thermal cycling.
• DNA Sequencing – Sanger (chain‑termination) method or next‑generation platforms to confirm the exact nucleotide order of a cloned gene.
• Gel Electrophoresis – separation of DNA fragments by size in agarose (for large fragments) or polyacrylamide (for small fragments) gels; visualised with ethidium bromide or safe‑green dyes.
• Southern, Northern and Western Blotting – transfer of nucleic acids (Southern, Northern) or proteins (Western) from gels to membranes for hybridisation with labelled probes or antibodies.
• DNA Fingerprinting – use of highly variable microsatellite loci or restriction‑fragment length polymorphisms (RFLP) to generate a unique pattern for an individual.

2. Vector Design (Syllabus Topic 19.2)

3. Gene Cloning Pipeline (Syllabus Topic 19.3)

3.1 Gene Isolation & Amplification

1. Extract genomic DNA or cDNA from human tissue.
2. Amplify the therapeutic gene with PCR using primers that introduce restriction‑enzyme sites compatible with the vector MCS.
3. Verify the PCR product by agarose‑gel electrophoresis.

3.2 Restriction Digestion & Ligation

• Digest both PCR product and plasmid vector with the same pair of restriction enzymes to generate compatible “sticky” ends (or blunt ends for certain enzymes).
• Purify fragments (gel extraction or column).
• Ligate insert into vector with T4 DNA ligase; optimise insert:vector ratio (typically 3:1).

3.3 Transformation / Transfection (Syllabus Topic 19.4)

3.4 Screening & Selection (Syllabus Topic 19.5)

• Antibiotic selection – only cells that have taken up the plasmid survive on selective medium.
• Blue‑white screening – insertion disrupts lacZ; white colonies indicate successful cloning.
• Colony PCR – rapid check of insert presence directly from bacterial colonies.
• Restriction analysis – mini‑prep DNA digested with the same enzymes used for cloning; pattern confirms orientation.
• Sequencing – final verification of the exact nucleotide sequence and absence of mutations.

3.5 Amplification of the Clone

1. Inoculate a single verified colony into a small liquid culture (seed).
2. Scale‑up to large‑volume fermentation or cell‑culture bioreactors under controlled temperature, pH, dissolved oxygen and nutrient feed.

4. Expression Systems (Syllabus Topic 19.6)

5. Protein‑Engineering Tools (Syllabus Topic 19.7)

• Site‑Directed Mutagenesis – introduces precise amino‑acid changes; used to create insulin analogues (e.g., rapid‑acting lispro) or extend half‑life of factor VIII.
• Fusion Tags – His‑tag, GST, MBP aid purification by affinity chromatography and can increase solubility.
• Glyco‑Engineering – modifies glycosylation pathways (e.g., knock‑out of α‑1,3‑galactosyltransferase in CHO) to reduce immunogenicity and improve pharmacokinetics.
• Domain‑Swapping / PEGylation – attachment of polyethylene glycol or fusion with albumin‑binding domains to prolong circulating half‑life.

6. Purification, Formulation & Quality Control

1. Capture step – affinity chromatography (Ni‑NTA for His‑tag, protein A for antibodies).
2. Intermediate purification – ion‑exchange (DEAE‑Sepharose) and hydrophobic‑interaction chromatography to remove host‑cell proteins.
3. Polishing – size‑exclusion chromatography to eliminate aggregates and ensure monodispersity.
4. Removal of contaminants – endotoxin depletion (e.g., polymyxin B columns), nuclease treatment for residual DNA, viral clearance (nanofiltration, low‑pH treatment).
5. Formulation – buffer optimisation (pH, excipients, stabilisers), lyophilisation or sterile liquid for injection; delivery devices may include pens, pumps or inhalers.
6. Quality‑control assays – SDS‑PAGE, HPLC, mass spectrometry, bio‑assays for activity, sterility testing, endotoxin (LAL) assay.

7. Why Use Recombinant Human Proteins? (Syllabus Requirement 19.8)

• Exact human amino‑acid sequence – minimises immune reactions and allergic responses.
• Unlimited, reproducible supply – independent of animal or human donors; scalable to meet global demand.
• High purity & safety – removal of pathogens (prions, viruses), endotoxin and host‑cell contaminants.
• Controlled post‑translational modifications – essential for activity, stability and half‑life.
• Ability to engineer improvements – longer half‑life, altered receptor affinity, reduced aggregation.
• Ethical advantage – reduces reliance on animal‑derived products and on plasma from donors.

8. Therapeutic Case Studies

8.1 Recombinant Human Insulin (r‑hINS)

• Disease: Type 1 diabetes (absolute insulin deficiency).
• Expression system: E. coli (inclusion‑body expression) or S. cerevisiae (secreted).
• Key advantages:

  1. Identical to native human insulin – negligible immunogenicity.
  2. Massive, cost‑effective production; eliminates reliance on animal pancreas extracts.
  3. Purity > 99 % reduces injection‑site reactions.
  4. Platform for analogue design (lispro, aspart, glargine) via site‑directed mutagenesis.

8.2 Recombinant Human Factor VIII (r‑hFVIII)

• Disease: Haemophilia A (deficiency of functional factor VIII).
• Expression system: Mammalian CHO or BHK cells – required for complex N‑glycosylation and correct folding.
• Key advantages:

  1. Virus‑free product – removes risk of HIV, hepatitis B/C transmission associated with plasma‑derived factor.
  2. Consistent activity units (IU) enable accurate dosing.
  3. Reduced batch‑to‑batch variability improves clinical reliability.
  4. Engineering of extended‑half‑life FVIII (e.g., Fc‑fusion, PEGylated) reduces injection frequency.

8.3 Recombinant Human Adenosine Deaminase (r‑ADA)

• Disease: Severe Combined Immunodeficiency (ADA‑deficient SCID).
• Expression system: CHO cells – provide necessary N‑glycosylation for enzyme stability.
• Key advantages:

  1. Supplies the exact human enzyme, restoring purine metabolism and lymphocyte development.
  2. Avoids risky bone‑marrow transplantation or gene‑therapy procedures.
  3. Improves survival and quality of life for infants.
  4. Glyco‑engineered forms (e.g., PEG‑ADA) have prolonged plasma half‑life and lower immunogenicity.

9. Comparative Summary of Therapeutic Recombinant Proteins

10. Emerging Gene‑Editing & Genome‑Engineering Tools (Syllabus Topic 19.9)

• CRISPR‑Cas9 – RNA‑guided nuclease creates double‑strand breaks at a defined locus; repaired by non‑homologous end joining (knock‑out) or homology‑directed repair (knock‑in). Used to generate high‑producing CHO cell lines.
• TALENs & Zinc‑Finger Nucleases (ZFNs) – protein‑based DNA‑binding domains fused to a nuclease; enable precise genome edits with lower off‑target rates in some contexts.
• Base Editing & Prime Editing – introduce single‑base changes without double‑strand breaks, useful for correcting point mutations in therapeutic cell lines.
• Delivery methods: plasmid transfection, ribonucleoprotein (RNP) electroporation, viral vectors (AAV, lentivirus).
• Ethical considerations: germ‑line editing, off‑target effects, equitable access.

11. Applications of Recombinant DNA Beyond Therapeutics (Syllabus Topic 19.10)

• GM Crops – herbicide tolerance (e.g., glyphosate‑resistant soy), insect resistance (Bt toxin).
• Industrial Enzymes – cellulases for bio‑ethanol, lipases for detergents, amylases for food processing.
• Bioremediation – bacteria engineered to degrade pollutants (e.g., oil‑spill hydrocarbonases).
• Vaccine Production – recombinant subunit vaccines (HBsAg, HPV L1 capsid protein) produced in yeast or insect cells.
• Model‑Organism Research – transgenic mice, Drosophila, zebrafish for functional genomics.

12. Biosafety Levels & Containment (Syllabus Topic 19.11)

13. Ethical, Legal and Social Implications (ELSI) (Syllabus Topic 19.12)

• Patents & Access – Proprietary recombinant technologies can raise drug‑price issues, especially in low‑income countries.
• Biosafety & GMO Regulation – Compliance with national and international guidelines (e.g., Cartagena Protocol).
• Animal Welfare – Recombinant production reduces the need for animal‑derived raw materials.
• Equity of Investment – Decisions on which diseases receive biotech investment may reflect socioeconomic priorities.
• Gene‑Therapy Ethics – Balancing potential cures against long‑term safety and societal concerns.

14. Regulatory Pathway for Recombinant Therapeutics (Syllabus Topic 19.13)

1. Good Manufacturing Practice (GMP) – validated, documented processes; batch‑release testing for identity, purity, potency, sterility.
2. Pre‑clinical Studies – acute/chronic toxicity, pharmacokinetics, immunogenicity in at least two animal species.
3. Clinical Trials:

   • Phase I – safety and dose‑finding in healthy volunteers or patients.
   • Phase II – efficacy, optimal dosing, side‑effect profile.
   • Phase III – large‑scale confirmation of benefit and monitoring of rare adverse events.

4. Regulatory Submission – Dossier (CTD format) to MHRA, FDA, EMA; includes CMC (Chemistry, Manufacturing, Controls), non‑clinical and clinical data.
5. Post‑marketing Surveillance – Pharmacovigilance, periodic safety update reports (PSUR), risk‑management plans.

15. Key Points to Remember

1. Recombinant DNA technology enables the production of proteins that are chemically identical to their natural human counterparts.
2. Safety is markedly improved by eliminating animal or human donor sources and by achieving > 99 % purity.
3. Scalable, controllable expression systems (bacterial, yeast, mammalian) provide a reliable supply for global patient populations.
4. Protein‑engineering tools allow the creation of improved therapeutics – longer half‑life, reduced immunogenicity, tailored pharmacokinetics.
5. A thorough understanding of the full pipeline – from gene isolation, vector design, host‑cell expression, purification, to regulatory approval – is essential for both scientific competence and responsible practice.