MOII Lecture: Proteins, Peptides and Biologics

• Lecture MOII covers biologics, specifically proteins and peptides, with an emphasis on size considerations within Fundamental Pharmaceutical Science Module 1: Molecules.
• The lecture aims to help understand the process of pharmaceutical development.
• Another aim is to evaluate and recommend the suitability of a dosage form, administration route, storage, and appropriate use in relation to how well a medicine works.

Assessable Tasks

• Understand the production of recombinant therapeutic proteins along with their advantages.
• Recognize the different categories of protein therapeutics.
• Understanding recombinant DNA technology.
• Describe the steps to produce recombinant insulin.
• Describe the advantages of recombinant proteins compared to animal-sourced proteins.
• Be able to compare small molecule pharmaceuticals with biologics.

Biologics Defined

• A biologic, biological product, or biopharmaceutical is a drug product manufactured in, extracted from, or semisynthesized from biological sources like animals, plants, or microorganisms.
• Biologics include vaccines, blood and blood components, allergenics, cells, gene therapy, tissues, and recombinant therapeutic proteins.

Small Molecules vs. Biologics

• Synthesis: Small molecules are chemically synthesized, while biologics are genetically engineered in living organisms or cells.
• Size: Small molecules are small; biologics are large.
• Structure: The structure of small molecules is well-known, but the structure of biologics is complex and often partially unknown.
• Contamination: Small molecules have low susceptibility to contamination, but biologics have high susceptibility.
• Uniformity: Small molecules are single substances, whereas biologics are mixtures of variants.
• Molecular Structure: Small molecules have relatively simple spatial structures determined through analytical technology, while biologics exhibit complex spatial structures that are difficult to determine.
• Complexity: Small Molecules are relatively pure, while biologics contain complex ingredients.
• Physical factors: Small molecules have low sensitivity compared to the sensitivity of biologics to physical factors.
• Clinical behaviour: Small molecules have well understood processes whiles the processes for biologics are complicated.
• Manufacturing: Small molecules have straightforward and simple processes but biologics are more comples to produce.
• Species: Small molecules are interdependant but biologics are specific.
• Immunogenicity: Small molecules are non-antigenic whereas biologics are antigenic if there molecular weight (MW)>10kDa.
• Absorption: Absorption for small molecules is faster where as absorption for biologics is slower.
• Distribution: There is high distribution for smaller molecules as opposed to low or limited distribution for biologics
• Metabolism: Small molecules are metabolised where as biologics are catabolised
• Testing for biologics takes 250 times more during production compared to 40-50 times during small molecule testing
• Examples: Aspirin is a small molecule and Bevacizumab is a biologic.
• The average cost in the US per day is $2 for small molecules and $45 for biologics.

Proteins in Humans

• Proteins are macromolecules.
• Approximately 20,000 genes in the human body code for proteins.
• Proteins include enzymes that catalyze chemical reactions, receptors and ion channels, and transport proteins.
• Proteins are non-branching polymers that form macromolecules.
• They are composed of a linear sequence of chiral amino acids covalently linked via amide (peptide) bonds.
• Short chains of amino acids are peptides.

Protein Secondary Structure

• Protein secondary structures include α-helices and β-sheets.

Sources of Therapeutic Proteins

• Humans: Therapeutic proteins can be sourced from humans.

  • Antithrombin III, Factor VIII, Factor IX, Plasmin, and β-glucocerebrosidase are examples of substances sourced from human blood or placenta for therapeutic use.
  • Risks include pathogen transmission (Hepatitis, HIV) and limited tissue availability.

• Non-Human: Therapeutic proteins can be sourced from non-human sources.

  • Insulin derived from cattle/pig pancreas is used to treat diabetes.
    • Such sources raise issues like supply vs demand, immunogenicity, ethics, and cultural beliefs.
  • Sources include bacteria.
  • Recombinant protein is a non-human source that diminishes these challenges.

Recombinant Protein

• Recombinant DNA is recombined DNA that does not occur naturally in that organism.
• Production involves inserting DNA from a different species (or altered DNA from the same species) into the genome.
• Insulin production is done from the combining of human gene into bacteria.
• "Recombinant DNA technology" – Biotechnology produces protein therapeutics.
• Insulin Production in Bacteria:
  • Involves producing insulin in bacteria from bacterial "plasmids," which are circular self-replicating DNA molecules.
  • Human DNA is inserted into the bacterial plasmid using a restriction enzyme.
  • The plasmid is inserted into bacteria, and an inducer signals protein synthesis.
  • The protein is then produced and purified
• Expression Hosts:
  • Bacteria (E. coli)
  • Yeast (Saccharomyces cerevisiae)
  • Insect cells
  • Mammalian cells
  • Transgenic animals and plants
  • Bacteria do not carry out glycosylation, whichcan affect activity and half-life.
• Expression host advantages:
  • Bacteria: Inexpensive, easy to cultivate, rapid growth, ease of modification, high yield, easy scale-up, cost-effective, and virus-free but they cannot form disulfide bonds.
  • Yeast: Inexpensive, easy to cultivate, rapid growth, ease of modification, and easy scale-up but can cause hyperglycosylation.
  • Mammalian Cells: Capable of humanized glycosylation and proper folding, but are difficult to cultivate, have low yield, and are expensive
• Advantage of recombinant biologics over animals is there is less of a chance for immune rejection.
• Additional benefits include translation of a “exact” human gene/protein, efficiency, lower cost compared to animal, and high quality.

Transgenic Animals

• These animals secrete protein in their milk.
• Antithrombin (an anticoagulant) is produced from the milk of genetically modified goats.
• Advantages include: large amounts produced, cheaper than mammalian cell culture, easy to scale up or down, proteins are folded and fully functional, and proteins can be readily purified from the milk.
• Disadvantages: long generation/validation time, harvesting time, affects animal.

Advantages of Proteins over Small-Molecule Drugs

• Proteins carry out highly specific functions that small molecules cannot mimic.
• Highly specific (human) proteins have less potential for adverse side effects
• Therapeutic proteins well-tolerated and less likely to produce an immune response and are naturally produced.
• In diseases with mutated or deleted genes, proteins are an effective replacement treatment.
• Approval time (FDA) is potentially faster than small-molecule drugs.

Classification of Protein Therapeutics

• Group I: With enzymatic or regulatory activity.
  • Replacing a deficient or abnormal protein, exemplified by insulin for diabetes.
  • Augmenting an existing pathway
  • Providing a novel function or activity, like Botox for medical and cosmetic applications.
• Group II: With special targeting activity (monoclonal antibodies)
  • Interfering with drug targets like proteins, DNA, and cells
  • Delivering other compounds or protein
  • Anti-body drug conjugate -Includes mAb - Trastuzumab, and anti-body drug conjugate mAB + emtansine.
• Group III: Protein vaccines like Gardasil (HPV vaccine)
  • Clinical trials are ongoing.
• Group IV: Protein diagnostics
  • Includes hormones and growth hormones
  • Utilizes imaging agents like mAB for prostate cancer detection.
  • Labels peptides for imaging acute venous thrombosis and diagnoses Hep C exposure via antigens.

Protein Therapeutic Strengths

• Strengths include the good efficacy, selectivity, and synthetic protoocls
• Predictable metabolism
• Shorter time to market

Key Approvals

• Recombinant human insulin (E. coli expression) was approved by the US FDA in 1982.
• The first therapeutic monoclonal antibody was approved in 1985 for renal transplant rejection.
• In 2019, the FDA approved 10 biologics and 17 in 2018.