Chapter 32 — Quiz

How Peptides Are Made — Solid-Phase Synthesis, Recombinant Production, and Why Peptide Drugs Cost What They Cost

22 questions. Answer all of them before opening the key. A calculator is permitted; questions 7–10 require one.

1. Merrifield's central insight in 1963 was to:

  • A. Automate the addition of amino acids using a programmable machine
  • B. Anchor the growing peptide chain to an insoluble solid support so that excess reagents and byproducts could be washed away at every step
  • C. Replace acid-labile protecting groups with base-labile ones
  • D. Use enzymes rather than chemical activation to form peptide bonds

2. Merrifield received the Nobel Prize in Chemistry in:

  • A. 1963
  • B. 1972
  • C. 1984
  • D. 1993

3. In the Fmoc strategy, the N-terminal protecting group and the side-chain protecting groups must respond to different chemical triggers because:

  • A. Side-chain groups are larger and require harsher conditions
  • B. The N-terminal cap is removed once per cycle while side-chain caps must survive every cycle and come off only at the end
  • C. Fmoc groups are unstable in the presence of activating reagents
  • D. It reduces the amount of solvent required for washing

4. Why does the incoming amino acid in a coupling step arrive already carrying its own N-terminal protecting group?

  • A. To improve its solubility in the reaction solvent
  • B. To prevent racemization during activation
  • C. To prevent the chain from growing by more than one residue in a single cycle
  • D. To make it easier to wash away the excess

5. "Difficult sequences" fail to couple efficiently primarily because:

  • A. The activating reagent degrades over long syntheses
  • B. The growing chains associate into aggregates on the resin, physically burying the reactive amino group
  • C. The resin loses its linker over many cycles
  • D. Long chains diffuse out of the bead into solution

6. Racemization during activation produces:

  • A. A chain missing one residue
  • B. A chain that has stopped growing
  • C. A molecule of nearly identical mass containing a mirror-image residue
  • D. A branched peptide

7. At 99% coupling efficiency per step, the approximate theoretical yield of correct full-length product after 30 couplings is:

  • A. 30%
  • B. 54%
  • C. 74%
  • D. 90%

8. At 99% coupling efficiency per step, the approximate yield after 50 couplings is:

  • A. 36%
  • B. 50%
  • C. 61%
  • D. 78%

9. At 98% per step, the approximate yield after 50 couplings is:

  • A. 13%
  • B. 36%
  • C. 55%
  • D. 66%

10. Comparing your answers to questions 8 and 9, the most accurate statement is:

  • A. Per-step efficiency matters little once a process is above 95%
  • B. One percentage point of per-step efficiency roughly halves or doubles the full-length yield over a 50-step synthesis
  • C. Yield falls linearly with chain length
  • D. The relationship depends mainly on which amino acids are used

11. A deletion sequence forms when:

  • A. A chain is cleaved prematurely from the resin
  • B. A coupling fails and the chain then accepts the following residue on the next cycle
  • C. A side-chain protecting group is removed too early
  • D. Two chains couple to each other

12. Compared with a deletion sequence, a truncated sequence is:

  • A. Harder to remove, because it is longer
  • B. Easier to remove, because it differs more from the product
  • C. Identical in difficulty, since both are peptides
  • D. Not produced by Fmoc chemistry

13. Capping after a failed coupling:

  • A. Increases the yield of correct full-length product
  • B. Converts deletion sequences into truncated sequences, which are easier to separate
  • C. Removes the failed chains from the resin entirely
  • D. Prevents racemization on subsequent cycles

14. Semaglutide's substitution at position 34 exists principally to:

  • A. Block cleavage by DPP-4
  • B. Increase receptor affinity
  • C. Ensure the fatty acid attaches at exactly one site rather than producing a mixture of attachment isomers
  • D. Improve solubility in the injection buffer

15. "Purity by peak area" on an HPLC certificate is:

  • A. A statement of what fraction of the vial's mass is the labeled peptide
  • B. A ratio among UV-detected species, which does not establish identity and does not include non-absorbing components
  • C. A measurement of sterility and endotoxin
  • D. Equivalent to peptide content when the method is validated

16. Which of the following would contribute mass to a lyophilized peptide vial but little or no peak area to a UV-detected chromatogram?

  • A. A deletion sequence
  • B. A racemized full-length chain
  • C. Trifluoroacetate counterion and residual water
  • D. A truncated fragment containing tyrosine

17. Trifluoroacetate is the usual counterion on synthetic peptides because:

  • A. It stabilizes the peptide against proteolysis
  • B. Trifluoroacetic acid is used both in cleavage from the resin and as an HPLC mobile-phase additive
  • C. It is required by pharmacopeial monographs
  • D. It improves solubility in cold water

18. Human insulin, approved in 1982, is significant because it was:

  • A. The first peptide drug of any kind
  • B. The first drug produced by solid-phase synthesis
  • C. The first approved recombinant DNA drug
  • D. The first drug delivered by injector pen

19. The genuinely hard problem in producing recombinant insulin was:

  • A. Inserting the gene into E. coli
  • B. Achieving correct folding and disulfide bond formation
  • C. Growing bacteria at sufficient scale
  • D. Obtaining the human insulin sequence

20. No purely ribosomal process can produce semaglutide because:

  • A. It is too long for a ribosome to assemble
  • B. It contains Aib at position 8, which is not one of the twenty encoded amino acids
  • C. Its disulfide bonds cannot form inside a cell
  • D. Bacteria degrade GLP-1 analogs

21. The binding constraint on GLP-1 supply during the shortage was:

  • A. Peptide synthesis capacity
  • B. Availability of the non-natural amino acid Aib
  • C. Aseptic fill-finish capacity and injector-pen assembly
  • D. Cold-chain shipping containers

22. The most accurate account of why a gray-market research vial costs far less than a prescription product is:

  • A. Gray-market manufacturers have found genuine production efficiencies
  • B. The prescription price is pure profit margin
  • C. The vial reflects synthesis and basic purification and essentially nothing else — it is a different product, missing formulation, aseptic fill-finish, device, cold chain, quality systems, regulatory compliance, clinical evidence, and liability
  • D. Research vials contain less peptide by mass
Answer key **1. B.** The insight was the insoluble anchor, which converted purification between steps into filtration and rinsing. Automation (A) followed from it and was not the insight itself; the Fmoc/Boc distinction (C) came later; enzymatic coupling (D) is a different technology entirely. §32.1 **2. C — 1984.** The method was published in 1963; the Nobel Prize in Chemistry followed in 1984, unshared. §32.1 **3. B.** The N-terminal cap is removed once per cycle; the side-chain caps must survive every cycle and come off only at final cleavage. If both responded to the same trigger, the first deprotection would strip the side chains and the chain would start branching. §32.2 **4. C.** The incoming residue arrives capped so that it cannot immediately accept another residue in the same pass. Racemization (B) is a real concern but is addressed by the choice of activation chemistry, not by the N-terminal cap. §32.2 **5. B.** Aggregation on the resin buries the reactive amino group. The reagents are present and the chemistry is sound; the site is simply inaccessible. §32.2 **6. C.** Racemization flips the configuration of the residue's central carbon, giving a mirror-image residue — a different molecule at essentially the same mass. §32.2 **7. C — 74%.** 0.99³⁰ ≈ 0.740. §32.3 **8. C — 61%.** 0.99⁵⁰ ≈ 0.605. §32.3 **9. B — 36%.** 0.98⁵⁰ ≈ 0.364. §32.3 **10. B.** Sixty-one percent versus thirty-six percent over fifty steps, from a single percentage point of per-step efficiency. This is why process chemistry effort concentrates there. §32.3 **11. B.** The chain that missed a coupling is deprotected again on the next cycle and accepts the following residue, producing an internal gap rather than a stopped chain. §32.3 **12. B.** A truncated chain differs more from the product — different length, different terminus — so chromatography separates it more readily. §32.3 **13. B.** Capping permanently blocks failed chains, converting a hard separation problem into an easier one. It does nothing for yield, which is exactly why the trade has to be argued. §32.3 **14. C.** Position 8 (Aib) is the DPP-4 answer; position 34 is the attachment-isomer answer. Both substitutions exist, and they solve different problems — one pharmacological, one manufacturing. §32.3, Chapter 4 §4.5 **15. B.** Area percent is a ratio among detected peptide species. It says nothing about identity, about non-absorbing components, or about anything that co-elutes with the product. §32.4 **16. C.** Counterion and water carry mass and do not produce meaningful UV peaks. A, B, and D are all peptides and would appear in the chromatogram (though a co-eluting one might hide under the main peak — which is a different problem). §32.4 **17. B.** Trifluoroacetic acid appears in both the cleavage step and the HPLC mobile phase, so the peptide comes out as its trifluoroacetate salt unless a deliberate exchange step is performed. §32.4 **18. C.** First approved recombinant DNA drug, 1982. Insulin as a therapy dates from 1922 (Chapter 11), and the recombinant milestone is a manufacturing one. §32.5 **19. B.** Expression was tractable; folding and correct disulfide pairing were not. Six cysteines, fifteen possible pairings, one correct answer. §32.5 **20. B.** Aib is not encoded by any codon, so no ribosome can install it. This makes chemical steps mandatory regardless of any other consideration. §32.6 **21. C.** Synthesis capacity was expanded successfully. The constraint was the sterile fill line and the pen assembly — both regulated physical plant with multi-year build-and-qualify timelines. §32.7 **22. C.** The price gap is largely a measure of what has been removed. Note that this is not a claim that prescription pricing is justified — Chapter 12 is genuinely critical of it — nor that gray-market vials contain no peptide. It is a claim about what a price difference is evidence *of*. §32.8, §32.9