A Certificate of Analysis (COA) sums up what was tested and what was found. Using the last three posts, here's how to read one section by section, and what to cross-check. 1. Header and identification • Product name, sequence and/or CAS number: Make sure they match what you expected to receive. • Molecular formula and molecular weight: You'll use these to check the MS result. • Lot/batch number: This should match the label on your vial. A COA only applies to its own lot. • Test date and the lab or analyst responsible for the analysis. 2. Appearance Usually something like "white lyophilized powder." Compare it to your vial. A collapsed, discolored or melted-looking cake is worth noting, since it says something about the drying process (see the lyophilization post). 3. HPLC purity • Look for the reported purity and the chromatogram itself. • Good signs: one dominant, sharp main peak; a flat baseline; clearly labeled retention time and detection wavelength (around 214–220 nm). • Questions to ask: Are impurity peaks shown and integrated? Is the method described (column type, gradient)? • Remember that area % reflects UV-detectable, peptide-related material only (see the HPLC/MS post). 4. Mass spectrometry (identity) • Compare the expected and observed mass. They should agree within the stated tolerance. • Check whether the values are monoisotopic or average, and whether charge states (e.g., [M+H]⁺, [M+2H]²⁺) were accounted for. • A spectrum image is more informative than a single number on its own. 5. Content and composition This is the section that most often gets skipped: • Net peptide content: How much of the powder mass is actually peptide. • Counter-ion: TFA, acetate or another salt, and sometimes the measured amount. • Water content: Often measured by Karl Fischer titration. • Excipients: Any bulking agents or lyoprotectants present (see the excipients post). • Stated quantity: Is it peptide amount or total fill weight? 6. Other tests (if present)

Some vials hold a tiny wisp of powder, and others hold a full, solid cake. The difference often comes down to excipients, the non-peptide ingredients added before freeze-drying. Here's what they do and how they affect a COA. Why add anything at all? A small amount of peptide on its own can dry into a thin film or a barely visible fleck that's fragile and hard to see. Excipients can: • Give the cake structure and bulk • Protect the peptide during freezing and drying • Raise the temperature the formulation can tolerate during drying, which makes the cycle more robust Two main roles 1. Crystalline bulking agents • Mannitol and glycine are the classic examples. • They crystallize during freezing and form a rigid scaffold. That supports an elegant cake and lets primary drying run at relatively warm product temperatures. • Once crystallized, they do little to protect the peptide on their own. • If they don't crystallize fully, or crystallize later during storage, that can affect cake quality and stability. This is why freezing steps such as annealing are sometimes used. 2. Amorphous lyoprotectants • Trehalose and sucrose are the standard choices. • They stay amorphous (glassy) and protect the peptide by replacing water's hydrogen bonds and immobilizing it in a rigid glass. • Their downside is a relatively low Tg′, so drying has to stay colder to avoid collapse. Many formulations combine both: a crystalline agent for structure and a sugar for protection. How excipients show up on a COA HPLC purity: Usually largely unaffected. These excipients absorb little UV at 214–220 nm and generally aren't integrated as peptide-related peaks. A peptide with excipient can show the same area % purity as the same peptide without it. Peptide content: This is where the difference shows. Excipients add mass, so the peptide makes up a smaller fraction of the total solid. A COA should make clear whether a stated quantity means peptide content or total fill weight. Why some vials look "fuller"