Source: Singota Solutions

By Ken Chomistek, Singota Solutions

Compendial monograph (pharmacopeial) testing applied to pharmaceutical raw materials and finished drug products is a critical element of drug development. A testing record is created in the form of a Certificate of Analysis (CoA) for manufacturing release and distribution. However, the testing required for a given product can be a source of consternation for biopharmaceutical sponsors and their partners.

Some tests/assays are mandatory, while others are supplemental, optional additions that can make a CoA more robust. Still other tests are product specific. For early-stage injectables or IV-infusion therapies — the focal point of this article — between 5 and 30 tests could appear on a CoA, though that number differs depending on applicable regulations where the therapy is to be manufactured and/or distributed.

United States Pharmacopeia (USP) chapter one (USP <1>) defines broad guidelines for injectables and implanted drug products, but leaves it to the sponsor to determine which specific tests meet the evidentiary burden outlined in the chapter. Further, testing requirements evolve as development advances: moving from Phase 1 toward commercialization, the CoA usually grows to include more testing.

A drug product formulation includes the active pharmaceutical ingredient (API) and, typically, salts, preservatives, and/or other buffers in the solution, an acid or base to adjust the formulation’s pH, and/or a surfactant (or co-surfactants working together). Each component of the formulation (raw material) requires its own CoA, as does the final drug product. Accordingly, some tests need not be repeated at the drug product level because their requirements have been satisfied at the raw material level.

Mandatory Assays

Three microbiology tests are among those the U.S. Food and Drug Administration (FDA) and most other regulators expect to see completed during the development of early-stage injectables and IV-infusion therapies:

  • Bioburden — Bioburden testing occurs on a sample aliquot taken from the end of formulation activities, acting as a microbiology checkpoint before the drug product is filter sterilized and filled.
  • Sterility — It must be established that the material/drug product is sterile before it can be injected into a patient or IV bag. These samples are taken from the finished drug product lot.
  • Bacterial Endotoxin — Endotoxin testing also occurs post-manufacture and ensures no undesirable organisms have been incorporated into the final drug product.

Endotoxin and sterility results are recorded on the CoA, while bioburden documentation is part of the batch record (i.e., it does not appear on the CoA, but is required testing).

Mandatory chemistry tests introduce more options and variability, as they comprise either identity or stability-indicating methods. These methods must be in place to demonstrate identification or how/when a product degrades over time. Typically, potency testing, purity, and characterization of impurities are part of the CoA via a high-performance liquid chromatography (HPLC) assay. These HPLC tests are stability-indicating and will continue to be executed while expiry dates are developed for the drug (stability studies). The tests listed below are also used to characterize identity or stability:

  • pH — A product’s pH may shift in the vial or in the syringe, and that shift could be caused by product degradation over time.
  • Osmolarity — Osmolarity characterizes type of concentration in a solution and an ability for parts of the solution to dissociate. A shift in osmolarity can indicate stability problems with a formulation and its numerical value can also be related to isotonicity, as hypertonic or hypotonic solutions (e.g., a formulation with high salt content or very low pH) can be painful when injected or infused.
  • Physical appearance — A solution is compared to a standard and examined for color and/or clarity, as well as visual evidence of particles forming in the solution.
  • Container content — This testing confirms all delivery apparatuses (e.g., syringes) accurately deliver the value reported on the label.
  • Identity — Identity tests generally are executed in every phase of development to quantify the drug substance present in the drug product. HPLC-mass spectrometry or Fourier-transform infrared (FTIR) spectroscopy can be used, comparing the drug product against a standard to ensure the API peak matches the reference standard peak.
  • Particulate matter — During sterile injectables manufacturing, everything passes through a 0.22 μm filter (i.e., a sterilizing filter with pores smaller than the size of bacteria). Post-filling, the filled units are enumerated to characterize potential particulates. The testing also can occur during stability studies to characterize aggregation or flocculation over time, if applicable.

It warrants mention that the FDA affords sponsors in early phases some leniency if documented justifications exist around bracketing different concentrations of the same drug product or leveraging data from the drug substance CoA before that material is made into drug product. For example, if microbiology testing has been conducted on a therapy delivering a 100 mg/mL dose and the sponsor introduces a second dosage at 50 mg/mL. The latter dose does not necessarily need to be requalified, because the drug substance may have been essentially replaced with sterilized water. The highest concentration of the drug product will require qualification, assuming no other components or component concentrations of the solution change (e.g., the diluent, other raw materials). Organizations regularly build a development and submission strategy around these bracketing strategies.

Optional Testing to Bolster a CoA

Supplemental tests often are considered optional, as they are not required for Phase 1, but they enable the sponsor to illustrate to regulators a more complete view of their drug product and delivery system. These tests may be necessary later in development, closer to commercialization, or they may be omitted as unnecessary or prohibitively expensive:

  • Break-loose force — This test gauges the amount of force required to dislodge a syringe plunger from its resting position in the syringe’s barrel.
  • Glide force — Also called “sustaining force,” this test measures the force required to maintain plunger movement once the plunger has broken loose from its static position.
  • Antimicrobial effectiveness — This test measures the efficacy of antimicrobial preservatives in the drug product to prevent growth over time.
  • Preservative Content — This test gauges the effectiveness of multi-dose products. For example, if a client produces 10 mL of drug product in each vial and it is used for ten individual 1 mL doses, preservatives must be added to the drug product to ensure that, when the stopper is punctured by a fresh syringe each time, no bacteria or other environmental contaminants enter and contaminate the solution.
  • Burkholderia cepacia complex (Bcc) — This test detects whether Bcc microorganisms in a nonsterile, aqueous-based pharmaceutical product or component exceed pharmacopeia-established limits.
  • Viscosity — This test gauges and ensures consistency in delivery system (e.g., syringe) functionality.
  • Container closure integrity (CCI) – This test confirms that the container closure used (e.g., a stopper in a vial with a crimp or press-fit closure) provides an adequate seal to prevent foreign substances from entering the unit.
  • Dosage uniformity – This test gauges homogeneity of the manufacturing to confirm that doses were filled uniformly.
  • Extractables and leachables (E&L) — This test, examines that no element of the packaging or delivery system has seeped into the drug product; it will become mandatory closer to commercialization. It generally does not apply to early development, as it is a costly test, unless the sponsor needs information to determine the appropriate class of vial, stopper, syringe, etc., for their product.

Product- and Market-Specific Testing

These tests can be considered good recommendations to prove a drug product is safe and compliant for early-stage clinical trials or toxicology studies. Often, their use is a business decision. While the U.S. market is key because of its size and the prices it can command for drug products, sometimes it can be more difficult from an evidentiary standpoint to secure approval in other markets. For example, the EU or Japan may require testing the FDA does not.

One example is that the FDA generally accepts an appearance test, which comprises viewing a clear solution in a vial against a white background and a black background to identify any visible particulates, if applicable. The EU’s version of this assay is more specific, characterizing the appearance of the solution against color or clear/opalescence standards (rather than using black/white backgrounds).

Final Thoughts

Ultimately, sponsors are responsible for ensuring all necessary testing has been performed on their drug substance/drug product to satisfy a given regulator’s CoA requirements. However, a knowledgeable, well-established partner can help sponsors through the testing process, saving them time and expense. Such a partner will have experience with different molecules, container configurations, and excipients, as well as be able to provide general guidance.

To learn more, visit us at

About The Author

Ken Chomistek is the Director of Quality Control and Development at Singota Solutions. Ken received his BS and MS degrees in Chemical Engineering from the University of Michigan. He has over 15 years of experience in the pharmaceutical and biotech industries, including roles in product development, cGMP analytical testing, laboratory management, new product launches, R&D, equipment validation, and ICH stability programs.

About Singota Solutions

Singota Solutions is a contract development and manufacturing organization (CDMO) focused on helping clients in the pharmaceutical, animal health, and biotechnology industries move their products through the drug development pipeline faster by being agile, accountable, and transparent.