Chapter 34 — Exercises

Thirty-two exercises. Items marked † are the harder ones — they require holding two ideas together rather than recalling one. No answers are provided here; several of these have more than one defensible response, and the reasoning is the assessed thing.

A standing constraint on every exercise in this set. Nothing here asks you to evaluate whether a product is acceptable to obtain or use, and no answer to any of these questions produces that conclusion. These exercises train you to read an analytical claim precisely. Per §34.9, reading analytical claims precisely does not substitute for a supply chain, and an exercise answered well still leaves that entire problem untouched.

Part 1 — The three questions (§34.1, §34.4)

A. In your own words, state the difference between identity, purity, and content, in one sentence each, without using the word "quality."

B. For each of the three questions in exercise A, name the analytical method or methods that answer it.

C. A supplier document reports a single number: "Purity: 99.2%." List everything that number leaves undetermined about the material.

D. † A vial is labeled as containing 5 mg of a peptide. Analysis of its contents finds HPLC purity of 98.4%. Explain how it is possible for both of the following to be true simultaneously: the purity figure is accurate, and the vial contains meaningfully less than 5 mg of peptide.

E. Draw, from memory, the mass-balance diagram from §34.4 showing what occupies the mass of a lyophilized peptide vial. Label which region HPLC purity examines.

F. Explain why a peptide with many basic residues will, all else equal, carry more counterion mass than one with few.

G. † A person reasons: "The certificate says 99% purity, so at most 1% of what I'm getting is something other than the peptide." Identify the two distinct errors in that inference — one about what "percent" is a percent of, and one about what the detector saw.

H. Explain why a determination of peptide content requires either a reference standard or amino acid analysis, whereas a purity figure requires neither.

Part 2 — Identity and mass spectrometry (§34.2, §34.6)

I. State, in one sentence each, what a mass spectrometer measures and what a tandem mass spectrometer additionally measures.

J. Why does a single peptide often produce several peaks in a raw electrospray spectrum? What does software do about it?

K. Explain the difference between monoisotopic mass and average mass, and describe a situation in which confusing the two would lead someone to a wrong conclusion about a sample.

L. † A peptide has been substituted at one position with a D-amino acid instead of the intended L-form. Explain why mass spectrometry — at any resolution, in tandem mode or not — cannot detect this. What property of the molecule has changed, and what property has not?

M. Leucine and isoleucine have identical residue masses. What does this imply about the strength of an identity claim based on MS/MS sequencing alone?

N. Explain why signal intensity in a mass spectrum is not a reliable measure of how much of a species is present.

O. † Rank the following identity claims from weakest to strongest and justify each step in the ranking: (i) the container was labeled with the compound name; (ii) amino acid composition matched the claimed sequence; (iii) the correct mass was observed; (iv) the sequence was confirmed by MS/MS and independently by an orthogonal method.

P. Amino acid analysis of a peptide claimed to contain one tryptophan reports no tryptophan. Explain why this is not necessarily evidence that the peptide is wrong.

Q. Why can amino acid analysis distinguish neither asparagine from aspartate nor glutamine from glutamate? What does an analysis report instead?

R. † Explain, using the combinatorial argument from §34.6, why establishing composition is a much weaker claim than establishing sequence. Then explain why composition analysis is nonetheless a primary method for something else entirely.

Part 3 — Purity and chromatography (§34.3)

S. Define co-elution and explain why it is a limitation of every separation method rather than a defect of any particular one.

T. A purity method monitors ultraviolet absorbance at 280 nm. Name a class of species this method would count as an impurity, and a class it would not count at all.

U. † Chapter 32 identified deletion sequences as the characteristic impurity of solid-phase peptide synthesis. Explain why they are also the impurity class that area-percent purity is least likely to detect. What does the coincidence of those two facts imply about how much reassurance a high purity figure should provide for a synthetic peptide specifically?

V. Explain what a response factor is and why area percent is not a mass percent.

W. Define orthogonal methods and explain why running two of them addresses a limitation that running one very good method cannot.

X. † A supplier reports purity by reversed-phase HPLC only. Name a specific type of impurity that this configuration might separate poorly, and name a different separation principle that would be more likely to reveal it.

Part 4 — Sterility, endotoxin, and process (§34.5, §34.9)

Y. State the Chapter 19 formulation distinguishing sterility from endotoxin, and explain what physical property of lipopolysaccharide makes the distinction necessary.

Z. Explain why passing a sterility test provides no information about endotoxin content.

AA. † A preparation is sterile-filtered. Explain, in terms of relative sizes, why this removes bacteria but not endotoxin. Then explain why autoclaving does not solve the problem either — and why autoclaving might in one respect make the endotoxin situation worse rather than better.

AB. Endotoxin has no visual signature. Explain why this makes it a different kind of hazard from, say, visible particulates, and describe the clinical presentation associated with it.

AC. † Reconstruct the argument in §34.9 for why sterility cannot be tested into a product. Your answer should include (i) why the test is destructive, (ii) why that forces sampling, and (iii) why sampling has low power against low-level contamination.

AD. Name three properties other than sterility that §34.9 identifies as properties of a process rather than of a material, and explain what "property of a process" means in this context.

Part 5 — Certificates, programs, and standards (§34.7, §34.8, §34.10)

AE. List the six things a properly conducted certificate of analysis can establish, and the four things it cannot.

AF. † State the general principle about certificates in the form given at the end of §34.7, then explain in your own words why each of the four limitations follows from it.

AG. Explain what a specification is and why a document reporting results without specifications cannot be a release decision.

AH. A friend says a product is "third-party tested." Write the three questions you would ask to convert that phrase back into a checkable claim.

AI. † Explain, using all four reasons from §34.8, why this book declines to quote a failure rate for unregulated peptide products. Then address the follow-up objection: "But surely an approximate number is better than no number at all." Is it? Under what conditions would it be, and are those conditions met here?

AJ. † Distinguish a pharmacopeial monograph from GMP, then explain why a material can meet a monograph on the batch tested and still tell you nothing about the manufacturer's next batch. Connect your answer to the sterility argument in exercise AC — the two arguments have the same shape, and naming the shape is the point of the exercise.