How to read a certificate of analysis, field by field

What a certificate of analysis is

This guide takes two documents as its benchmark for a complete certificate. The first is ICH Q7, the International Council for Harmonisation’s guideline on good manufacturing practice for active pharmaceutical ingredients, which says authentic certificates should be issued for each batch on request.² The second is the World Health Organization’s model certificate of analysis, written for quality control laboratories and for trade in pharmaceutical materials.¹

In the WHO text, a certificate lists the tests performed on a particular sample, with the results obtained and the acceptance criteria applied, followed by an indication of whether the sample complies with the specification.¹ The layout is left to the issuing laboratory, so the checks below concern content, not format.¹

Both documents were written for pharmaceutical materials. A certificate for a research peptide may follow them closely, loosely or not at all, so every field is worth reading.

The header: what was tested

Product name and description

ICH Q7 asks for the name of the material and, where appropriate, its grade.² The WHO model adds a description, such as strength and package size, and the type of primary packaging.¹

For a peptide, the description is clearer when it gives the sequence and the salt form. In the dossier for a peptide medicine, the European Medicines Agency’s guideline on synthetic peptides requires the counter-ion to be indicated and, where there is none, the free base to be stated.⁵ Check the name character by character against the label: related peptides can differ by one modification, as the article on peptide nomenclature and modifications explains.

Batch or lot number

ICH Q7 defines a batch, or lot, as a specific quantity of material produced so that it is expected to be homogeneous within specified limits.² The batch number is a unique combination of numbers, letters or symbols that identifies the batch, and from which its production and distribution history can be determined.² If the number on the document is not the number on the vial, the document describes something else.

The WHO model asks for the batch number used by the original manufacturer and, where there is one, by a repacker or trader.¹

Dates

Several dates can appear, with different meanings.

• Date of manufacture, if it is available.¹
• Date of release, which ICH Q7 asks for on every certificate.²
• Expiry date or retest date. An expiry date is the date after which the material should not be used. A retest date is the date when it should be re-examined to confirm that it is still suitable for use.²
• Date the sample was received, on a certificate from a quality control laboratory.¹
• Date of signature, beside the name of the person who approved the certificate.¹

A certificate with no date cannot be placed in the life of the batch.

The results table

The centre of the document is a table. The WHO model gives it five columns: test, method reference, acceptance criteria, result and a compliance statement.¹ ICH Q7 asks for the same content: each test performed, its acceptance limits, and numerical results where the results are numerical.²

Specification and result are different columns

A specification is a list of tests, references to analytical procedures and acceptance criteria, which are numerical limits, ranges or other criteria.³ Acceptance criteria are meant to be defined before the analysis.¹ The result is what was measured on this batch. A purity entry reads properly as a pair, for example a limit of “not less than 98.0%” beside a result of “99.1%”.

A document that shows only limits, or “typical” values, is a specification sheet. It describes what a product is meant to be, not what a batch was found to be.

The method column

The WHO model’s footnote to this column reads “Reference to a pharmacopoeia or technique”.¹ The technique matters because the figure depends on it.

Chemistry study In one comparison of five reversed-phase chromatography systems on five cationic peptides, one system outperformed the others in sensitivity and resolution, and the authors warned that the traditional method may lead to erroneous results for basic peptides.⁶

Review A 2009 review of pharmacopoeial monographs for peptide drugs found their specifications barely harmonized between the European and United States pharmacopoeias, and not always consistent within one pharmacopoeia.⁷ A result is best compared with another obtained by the same method.

The identity entry

Identity is the entry that says the vial holds the molecule named in the header. For peptide medicines, the European guideline on synthetic peptides considers identification by mass, relative retention time, liquid chromatography with mass spectrometry, peptide mapping, bioactivity, amino acid analysis or nuclear magnetic resonance appropriate, and recommends at least two orthogonal methods, meaning methods that rest on different principles.⁵

Check three things. The entry exists. It names a method that can establish identity: the ICH guideline on specifications, written for chemical drug substances, does not regard a single chromatographic retention time as specific.³ And where a mass is given, the calculated value matches the molecule in the header. Reading the spectrum itself is covered in how to verify a peptide.

Chemistry study The entry matters because wrong molecules have reached laboratories. When a group at Ghent University analyzed obestatin peptides obtained from five manufacturers, one product was a totally different peptide.⁸ In a later study of quorum-sensing peptides by the same group, the main compound of one sample had a different structure from the peptide ordered.⁴

The purity entry

Where the purity entry is a percentage by HPLC, it is easier to judge when it comes with its conditions: the technique, the detection wavelength and, ideally, the chromatogram it was calculated from. What the percentage measures is explained in what 99% purity means.

Chemistry study A stated purity is a claim until it is checked. The Ghent group ordered a set of quorum-sensing peptides at a purity of at least 95% and tested them itself.⁴ It found a large discrepancy between the purity on the suppliers’ certificates and its own results, and only 44% of the peptides met the purity ordered.⁴ In the obestatin study, two thirds of the products that were the right peptide fell short of the quality the authors considered sufficient for experiments.⁸

Animal study Purity is also not content. In a mouse study, two commercial preparations of the same peptide had similar purity, yet peptide made up 45% of the mass of one and 72% of the other.⁹ The article on peptide salts and net peptide content explains the gap.

Other entries you may see

The European guideline’s list for a peptide drug substance also includes assay or content, counter-ion identity and content, water content, residual solvents, elemental impurities, bacterial endotoxins and microbiological purity.⁵ The guide to analytical methods for peptides explains what each of these measures. One rule covers them all: a test that is not on the certificate is not documented for that batch.

Who issued it

A certificate is a signed statement, so its author matters. ICH Q7 asks that certificates be dated and signed by authorized personnel of the quality unit, and that they show the name, address and telephone number of the original manufacturer.² The WHO model asks for the name and address of the laboratory issuing the certificate, and for the date and signature of the head of the laboratory or another authorized person.¹

The benchmark documents describe three kinds of certificate, and they are not equivalent.

• A manufacturer’s certificate reports the maker’s own testing of its batch.
• An outside laboratory’s certificate covers the sample that laboratory received. The WHO model records the sample’s registration number, the date it arrived and the quantity received.¹ Where the sampling plan bears on the validity of the results, it should be referenced.¹ The link between that sample and a whole batch rests on whoever drew and sent it.
• A certificate reissued by a reseller. Under ICH Q7, a new certificate issued by or for a repacker, agent or broker should show the laboratory that performed the analysis, refer to the original manufacturer and to the original batch certificate, and have a copy of that original attached.²

Results from a subcontracted laboratory should be identified as such.¹ A laboratory accredited to ISO/IEC 17025, the international standard for testing and calibration laboratories, should refer to that accreditation on the certificate if it relates to the analysis in question.¹

Batch-specific or generic?

The fields show the difference.

The WHO model asks for a certificate number and, on every page, the page number and the total number of pages.¹

Cell study Batch matters because batches can differ. When T-cell responses to one manufacturer’s peptides could not be repeated with the same peptides re-synthesized by that manufacturer, mass spectrometry found numerous differences between the batches, including missing amino acids.¹⁰ A study of laboratory-grade Cas9 proteins, a different class of reagent, found that endotoxin levels varied between lots from the same supplier.¹¹

What a certificate does not prove

Cell study It covers only the tests that were run. In an HIV vaccine research program, peptides from two custom suppliers contained contaminating peptides capable of giving false-positive T-cell results, and analysis of one stock found about 1% by weight of an unrelated viral peptide.¹² A Belgian public health institute that screened ten falsified peptide drugs bought from suspected illegal internet pharmacies ran a separate analysis for each class of impurity and found arsenic and lead, alongside low purity.¹³ A purity line alone does not report metals.

It describes a sample at one moment. The WHO model notes that a certificate used in trade may state the expected conditions for shipping, storage and distribution, deviation from which would invalidate it.¹ What happened to a vial after the sample was taken is outside the document, and the peptide storage guide covers that side.

It is not self-verifying. ICH Q7 tells manufacturers who rely on suppliers’ certificates to run a full analysis at appropriate intervals, to compare it with the certificates and to check their reliability regularly.²

Chemistry study The Ghent group reached a similar conclusion for research: relying on suppliers’ certificates might have serious consequences, and researchers should keep up their own quality control.⁴

Cell study It says nothing about biological activity. Impurities can blur a functional result: crude preparations of one peptide, at about 70% purity, triggered a contractile response in isolated guinea pig ileum, and the purified peptide, at 95% or more, had no effect.¹⁴

Human evidence

A certificate of analysis is a chemistry document. It reports no study in cells, animals or people, and even a complete and accurate certificate is not evidence that a compound has an effect, or is safe, in people. This page cites no clinical study; the T-cell assays it mentions were run in the laboratory on cells taken from people. Products on this site are supplied for laboratory research use only.