Chapter 32 — Exercises

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

These exercises are for working through, not for looking up. No answers are provided here; the instructor guide holds them. Items marked † go beyond what the chapter states directly — they ask you to extend an idea, argue a position, or reason about a case the chapter did not cover.

A calculator is useful for Set B. Nothing else is required.

A standing rule for this chapter, and for the book: none of these exercises asks you to obtain, prepare, or use any peptide. Several ask you to evaluate documents and claims. That is the skill being trained.

Set A — Merrifield's idea and the synthesis cycle

a. In two sentences, state the problem with classical solution-phase peptide synthesis that Merrifield's method solved. Your answer should name what happened between reactions, not what happened during them.

b. Explain why a synthesis resin needs to swell. What would go wrong if the beads were hard, non-porous spheres?

c. A protecting group has to satisfy two requirements that pull against each other. Name both, and explain why a group that satisfied only the first would be useless.

d. In Fmoc chemistry, the N-terminal cap is removed by a base and the side-chain caps by an acid. Describe, step by step, what would go wrong on cycle three of a synthesis if both responded to the same base.

e. The incoming amino acid in each coupling step arrives already carrying its own N-terminal protecting group. Name the specific error this prevents, and describe what the product would look like if it were not prevented.

f. † Solid-phase synthesis builds the chain from the C-terminus toward the N-terminus; ribosomes build from N to C. Argue both sides of the following question: does this difference matter to the final molecule? Be precise about what "the molecule" means in your answer.

Set B — The arithmetic of stepwise yield

Show your work. Round to the nearest whole percent unless told otherwise.

a. A process couples at 99% efficiency per step. Calculate the theoretical yield of correct, full-length product for peptides of 8, 16, and 24 couplings.

b. Repeat (a) at 98% per step. Then state, in one sentence, what the comparison shows about the value of a single percentage point of coupling efficiency.

c. A 40-residue peptide is being made. What per-step efficiency is required for the theoretical full-length yield to reach 80%? Solve it rather than guessing, then sanity-check your answer against the table in §32.3.

d. A manufacturer reports that a 30-residue peptide came out of the reactor at 74% full-length product before purification. What per-step coupling efficiency does that imply, assuming every step performed identically?

e. Explain why the assumption in (d) — that every step performed identically — is known to be false, and name the phenomenon from §32.2 that breaks it. What does this imply about which position in a sequence is likely to contribute most of the deletion content?

f. A 15-residue peptide is made at 99.5% per step. A 45-residue peptide is made at the same efficiency. Calculate both yields. Then calculate the yield if the 45-residue peptide were instead made as three 15-residue fragments, each purified separately and then joined in two ligation steps that are each 70% efficient. Compare, and comment on what the comparison explains about why fragment condensation exists.

g. † A vendor claims a 34-residue peptide was synthesized at "greater than 99.9% purity, crude." Using the arithmetic of §32.3, state what per-step efficiency this would require. Is your answer physically impossible, merely implausible, or unremarkable? Defend your classification.

Set C — Deletion sequences and what purification leaves

a. Define a deletion sequence in one sentence, and explain the specific mechanism by which one forms. Your answer must explain why the chain does not simply stop.

b. A 30-residue peptide has a mass of roughly 3,400 daltons. A deletion sequence missing a single glycine differs from it by 57 daltons. Express that difference as a percentage of the total mass, and explain in one sentence why that number matters for detection.

c. Explain why a deletion sequence is harder to remove by preparative HPLC than a residual synthesis solvent. Frame your answer in terms of what chromatography actually separates on.

d. Capping does not increase the yield of correct product. Explain precisely what it does instead, and state the trade a manufacturer is making when they decide to cap.

e. Rank these impurities from easiest to hardest to remove from a 30-residue synthetic peptide by preparative HPLC, and justify your ranking: (i) a residual organic solvent; (ii) a 12-residue truncated fragment; (iii) a 29-residue deletion sequence missing an internal leucine; (iv) a full-length chain with one residue racemized.

f. † Chapter 4 explained that semaglutide carries an arginine at position 34 where GLP-1 has a lysine. Argue that this is a manufacturing decision rather than a pharmacological one — and then argue the opposite. Which argument do you find stronger, and what fact would settle it?

Set D — Purity, content, and what a document establishes

a. State, in your own words, the difference between "purity by peak area" and "peptide content." Give an example in which both numbers are truthfully reported and yet a reader who conflates them draws a badly wrong conclusion.

b. Name four things present in a lyophilized peptide vial that contribute mass but contribute little or no peak area to a UV-detected HPLC chromatogram.

c. A certificate of analysis reports: "Purity: 99.1% (HPLC)." List every question you would need answered before you could interpret that number. Aim for at least six.

d. Explain why "98% pure by HPLC" does not establish that the vial contains the molecule named on the label. What measurement would establish identity, and why is it a different kind of measurement?

e. Why is "pharmaceutical grade" not a checkable claim, while "manufactured under GMP to a named pharmacopeial monograph" is? Your answer should say what makes a claim checkable in general.

f. † Write the three questions you would put to a seller who uses the phrase "pharmaceutical grade." Each question must be answerable with a specific document or fact, not with an adjective. Then predict what a seller who means something real would answer, and what a seller who does not would answer.

Set E — Recombinant production and route selection

a. Explain, in three sentences, why insulin's disulfide bonds made recombinant production harder than getting E. coli to express the gene.

b. Six cysteines can pair in fifteen ways. Explain how proinsulin's connecting segment changes the odds, and state in one sentence what the segment's "job" is.

c. For each of the following, state whether you would expect synthetic or recombinant production, and give the single most decisive reason: (i) a 9-residue peptide with no unusual residues; (ii) a 191-residue protein hormone; (iii) a 31-residue peptide containing one non-natural residue; (iv) a 5-residue cosmetic ingredient; (v) a 51-residue two-chain molecule with three disulfide bonds.

d. Explain, using a specific structural feature, why no purely ribosomal process can produce semaglutide.

e. A colleague says "recombinant is always cheaper, so everything should be made that way." Identify the two distinct errors in that sentence.

f. † A hypothetical new drug is a 45-residue peptide with two disulfide bonds, no non-natural residues, and a fatty acid attached to a single lysine. Design a route for it in outline — which parts would you express, which would you synthesize, and in what order would you attach the fatty acid relative to forming the disulfides? Defend each choice, and name the step you would expect to be hardest.

Set F — Manufacturing scale, cost, and price

a. Name the two final manufacturing steps that constituted the binding constraint on GLP-1 supply, and explain in one sentence each why neither could be expanded quickly.

b. Why can peptide injectables generally not be sterilized in their final sealed container? What does that force the manufacturer to do instead?

c. Explain the phrase "you cannot buy your way out of a constraint whose limiting factor is time" in the context of §32.7. Then name one thing money could do on that timeline, and one thing it could not.

d. List the eleven cost components in the §32.8 stack from memory, grouped into the four blocks the chapter uses. Then mark which components a gray-market vial's price includes.

e. The same pen, from the same facility, sells at very different prices in different countries. Explain why this single observation is fatal to the claim that manufacturing cost explains price.

f. Restate in your own words the chapter's central sentence about research vials — "it is cheaper because it is a different product, not because someone found efficiencies" — and then state what the sentence does not claim. Be careful; most readers overstate it in one direction or the other.

g. † Construct the strongest good-faith argument you can that a peptide medicine's price is unjustified, using only claims this chapter supports. Then construct the strongest good-faith argument that a large price gap between a prescription and a research vial is not evidence of overcharging. Note where the two arguments actually disagree — it is narrower than it first appears.

Set G — Synthesis across the book

a. † Chapter 1 §1.5 gave the peptide/protein convention as roughly 50 residues. §32.3 shows that synthetic yield becomes punishing in roughly the same region. Argue that the coincidence is accidental, then argue that it is not entirely accidental. Which position requires fewer assumptions?

b. Chapter 19 described documented harm in the unregulated peptide market arising from contamination, wrong identity, and wrong concentration rather than from exotic peptide toxicity. For each of those three, name the specific manufacturing stage in §32.8 whose absence permits it.

c. † Chapter 30 will discuss cosmetic peptide ingredients, which are characteristically three to eight residues long. Give the §32.3 explanation for that length, then give a second, independent explanation based on Chapter 1 §1.6. Which explanation would you lead with when talking to someone who is not a chemist, and why?

d. † Chapter 34 covers analytical testing. Before you read it, write down the three tests you would most want run on an unknown peptide preparation, in priority order, with one sentence on what each would establish and one sentence on what it would leave open. Keep this and compare it with Chapter 34.

e. † Someone argues: "Peptide synthesis is automated, so making peptides is easy, so peptide drugs should be cheap." Identify the three separate inferential steps in that argument and evaluate each one against this chapter. State which step is weakest.

f. Add the Field 1+ block from this chapter's dossier section to two of your dossier compounds — one approved product and one that is not. Then write one sentence naming the single most important thing you cannot say about the second one.