Hello, how can we help you?

USP 85

Nano-Test® > Research > Standards > USP 85

USP <85>

USP <85>, United States Pharmacopeia (USP) – this is the general section of the ‘Bacterial Endotoxins Test’ published by the USP. The section describes test procedures using Limulus amoebocyte lysate (LAL) to determine the presence of lipopolysaccharides (endotoxins) derived from the cell walls of Gram-negative bacteria.

Harmonisation and relevant standards: Harmonised with USP <85>, EP 2.6.14 (European Pharmacopoeia) and JP 4.01 (Japanese Pharmacopoeia) (common text of the three pharmacopoeias). The principles of the method, the validation requirements and the acceptance criteria are largely identical. References outside the pharmacopoeias include ISO 10993-11 (Evaluation of material-related pyrogenicity in medical devices) and USP <161> (blood/solvent contact sets); where endotoxin determination is required for medical devices, it is carried out in conjunction with these standards.

Basic principle

LAL is a lysate derived from the amoebocytes of the horseshoe crab (Limulus polyphemus or Tachypleus tridentatus), which triggers an enzymatic coagulation cascade in the presence of endotoxins. Depending on the method used, this cascade is measured in terms of gel formation, a change in turbidity or colour development. The accuracy of the test is ensured by the specified sensitivity of the lysate (λ, EU/mL), the standard endotoxin calibration (CSE/RSE) and the interference control.

Methods

Die USP <85> definiert drei grundlegende Methodengruppen:

  • Gel-Clot method: threshold test and semi-quantitative method; visual assessment of gel formation in a lysate-endotoxin mixture. This is the most widely used method and is regarded as the reference method.
  • Turbidimetric method: Photometric measurement of the change in turbidity resulting from coagulation; kinetic or end-point mode
  • Chromogenic method: Photometric measurement of the coloured product formed by the breakdown of a synthetic substrate via the coagulation cascade; kinetic or end-point mode

Key calculations and terms

  • Endotoxin limit (EL): Limit defined in the monograph (e.g. K/M formula for parenteral products; K = pyrogenic threshold dose, M = maximum hourly dose per kg of body weight in humans)
  • Maximum Valid Dilution (MVD): MVD = (EL × sample concentration) / λ
  • λ (lambda): Specified sensitivity of the lysate (EU/ml)
  • CSE (Control Standard Endotoxin): A control standard for endotoxins calibrated against the USP Endotoxin Reference Standard (RSE)
  • PPC (Positive Product Control): Confirmation of the detection of a known quantity of endotoxin in the sample; assessment of interference (inhibition/potentiation)
  • Interference test (Test for Interfering Factors): Must be carried out for each product during the validation phase; elimination of interference through appropriate dilution or adjustment of the method

The key parameters reported in the study are as follows:

  • Product description, endotoxin limit (EL) and its basis (monograph/formula)
  • LAL method used (gel aggregate, turbidimetric, chromogenic) and mode
  • Lysate batch, λ value and manufacturer’s specifications
  • CSE/RSE calibration and validation data
  • MVD and applied dilutions
  • Results of the interference test (PPC recovery, acceptance range)
  • Results of the negative and positive controls
  • Test result and comparison with EL
  • Edition of the USP-NF and chapter revision used
  • Measurement uncertainty and repeatability data

What is this for?

USP <85> wird für die folgenden wissenschaftlichen und regulatorischen Zwecke angewendet:

  • Safety of parenteral products: Evidence that endotoxin contamination in injectable preparations, infusion solutions, eye drops and other pharmaceutical products is within the specified limits
  • Compliance with the Pharmacopoeia: carrying out the prescribed test for products for which an endotoxin test is specified in the USP-NF monographs, and issuing a declaration of compliance for marketing on the US market
  • Assessment of medical devices: Assessment of material-related pyrogenicity and endotoxins in products that come into contact with blood or solutions (e.g. infusion sets, catheters, implants) (in accordance with ISO 10993-11 and USP <161>)
  • Raw Material and Process Control: Monitoring the endotoxin load in water (purified water, water for injection), raw materials and packaging components in pharmaceutical manufacturing
  • Sterilisation and process validation: Demonstration of the efficacy of depyrogenation processes
  • Quality control and release: Assessment of production batches for endotoxins prior to market launch
  • FDA inspections and submissions: Provision of endotoxin data in 510(k), NDA/ANDA, IND and BLA submissions; demonstration of compliance with the pharmacopoeias during inspections
  • Animal welfare (3Rs): Use of the LAL test as an alternative to the pyrogen test on rabbits (USP <151>)
  • Evidence-based approach: A contribution to the holistic safety assessment of the product in conjunction with data on sterility, biocompatibility and pyrogenicity

USP <85> detects bacterial endotoxins only; pyrogenicity arising from the material itself (non-endotoxic pyrogens) and microbial contamination fall outside the scope of this test and must be assessed using the appropriate methods (USP <151> rabbit pyrogen test, USP <71> sterility test, monocyte activation test).

Als Nano-Test führen wir die bakteriellen Endotoxintests gemäß USP <85> im Rahmen unserer akkreditierten Laborinfrastruktur und unseres Qualitätsmanagementsystems mit wissenschaftlicher Sorgfalt durch.

Für Informationen zur Methodenauswahl, Validierung und Erstprüfung, zur MVD-Berechnung, zu den Probenannahmebedingungen, zum Prüfplan und zu den Berichtsprozessen für Ihre bakteriellen Endotoxintests gemäß USP <85> können Sie sich gerne an Nano-Test wenden.

Standards