MICROBIAL SOLUTIONS Endotoxin Calculations and …

1 EVERY STEP OF THE SOLUTIONSE ndotoxin Calculations and Validations: Frequently Asked QuestionsEndotoxin testing is a major component in product manufacturing. Organizations are continuously testing for endotoxins to ensure their concentrations do not meet or exceed limits that would prevent release. Below are frequently asked questions and answers that arise when performing routine Endotoxin testing. For more information, watch our webinar, Calculating Endotoxin Limits, to learn more about: BET guidance and regulations for Endotoxin Calculations Endotoxin limits and how to calculate them for different products and materials Maximum valid dilution/maximum valid concentration Calculations and inhibition/enhancement screening tests How maximum valid dilutions and Endotoxin limits fit into product validationWhen we have specific products, where only 1 batch is made, how can we validate the product?

2 Some lots of products are manufactured once every few months or years. This is not typical, but in this case it would not be acceptable to wait to validate on subsequent lots, or the validation could take many years. In a case like this, and with appropriate risk assessments in place, you could validate the one lot on three separate days, with three different analysts performing the testing. After that, every time this product is tested, you are basically gathering more data that give you more confidence in the validation over time. While you are performing validation testing on one lot of product, keep in mind that other testing is involved, such as in-process or raw material the formula K/M, what would M be for a medical device?

3 For medical devices, you will not be using the formula K/M. This applies to parenterals, radiopharmaceuticals, and intraocular injectables that have a dosage. The given Endotoxin limit for medical devices is 20 EU/device for a non-intrathecal device or EU/device if intrathecal. If the Endotoxin limit desired units are EU/mL and not EU/device, then you would have to calculate the limit for the extract solution using the formula K x N / V. The MVD can be calculated two ways: based on the extract solution or based on the maximum extraction volume. The MVD for medical devices indicates the total extraction volume that can be used. Note that intraocular devices may have a lower limit, depending on the device itself. K= Amount of Endotoxin allowed per deviceN= Number of devicesV= Total extraction volumeEndotoxin Calculations : FAQ DocumentWhen is testing the pH of a sample necessary?

4 If the pH of a sample is not within the LAL manufacturer s recommended range, regulatory guidelines such as USP Chapter <85>, EP , and ANSI/AAMI ST72:2019 all suggest the pH of equal parts sample and lysate mixture should be tested. LAL offers some buffering capacity, therefore a pH adjustment may not be necessary. If the pH of a sample does not need to be adjusted during validation, the pH does not need to be measured for each lot of product during subsequent release testing. Testing the pH on its own will give you basic information such as how acidic or basic the sample is. Keep in mind, further dilution of the sample may be necessary to overcome sample interference. If you are using LAL cartridges, you can check the pH using a micro pH probe or pH paper.

5 This can be accomplished at the end of the successful cartridge test by tipping the cartridge towards the sample well reservoirs and recovering 50 to 100 L volume using the same pipettor and pipette tips. A micro pH probe or pH paper strips can be used to measure the pH. See the cartridge package insert for more details. How do you calculate the Maximum Valid Dilution (MVD) or Minimum Valid Concentration (MVC) for a sample?Before calculating your MVD or MVC, depending on whether you are testing a dilution or concentration, the Endotoxin Limit (EL) must be calculated. MVD = Endotoxin Limit*Potency of Product/ (EU/mL)MVC= /EL When validating a product with Endotoxin specific buffer, are there any differences in the validation processes?

6 No, the validation process will not change. The Endotoxin Specific buffer is used to rehydrate the LAL. For the PTS cartridge technology you would dilute your sample in a 50% ES Buffer/LAL Reagent Water (LRW) solution instead of just LRW, not to exceed a 1:4 dilution of the ES buffer. The use of ES buffer to overcome Beta-Glucan must be documented in your written validation procedure. Which calculation would you use for products that are administered by subcutaneous injection?For subcutaneous administration, you would use the standard EL=K/M calculation for the Endotoxin threshold pyrogenic dose per kg of body weight. Intravenous: 5 EU/kg. Intrathecal: EU/kg M= maximum total dose administered in a single hour 2020, Charles River Laboratories International, using a buffer to overcome inhibition/enhancement during testing, would you also test the buffer as your negative control on the curve?

7 No, you would not use the buffer as your negative control. The purpose of the negative control is to ensure that the LAL Reagent Water (LRW) used to make your standard curve and rehydrate your LAL reagent is not contaminated. Your negative control must be prepared from the same vial of LRW used in the standard curve preparation and LAL rehydration. Buffers used in the preparation of product dilutions should be tested as part of an incoming materials control process, and therefore would not need to be tested additionally with each use during bacterial Endotoxin testing (BET). If it is suspected that the buffer is contaminated, it should be tested during the investigational process. Is it true that each required routine test assesses the validity of the sample due to the positive product control requirement (PPC)?

8 Yes, when you routinely test your product at the validated dilution, it must be within that acceptance criteria of 50-200% for your PPC. In order to determine which dilution you will be testing at the time of routine testing, you must perform validation testing including the inhibition/enhancement test phase prior to routine testing. This determines the appropriate dilution (not exceeding the maximum valid dilution (MVD)) that recovers the best PPC percentage. Also, although the LAL method is considered compendial, it still must be verified for each product requiring BET. The Product Validation, or suitability testing, is a verification of accurate and reliable process test methods for your products. All routine testing must include a PPC for each sample tested, as this is used to verify that the validated conditions are still valid, and the sample did not interfere with the routine analysis.

9 This is a pharmacopoeia requirement. Spike recovery can be used for investigational purposes and provides a health check for the manufacturing process. Typically, if something occurs in the manufacturing process it will be observed in the spike recovery and past data will validate your is the product validation procedure when performing the Inhibition/Enhancement (I/E) screen?When performing the Inhibition/Enhancement screen, it is important to first calculate the Endotoxin Limit and the MVD or the MVC for the product. The I/E screen allows you to determine the most robust dilution within your maximum valid dilution or minimum valid concentration. This can be performed using the method of your choice. To determine the dilution to validate, perform serial dilutions up to and including your MVD.

10 For example, if the MVD is 1:2,000, an option would be to make a 1:10, 1:100, 1:1,000, and 1:2,000. Ten-fold or two-fold dilutions are an easy starting point, depending on your MVD; however, it may be easier to perform a different dilution screen that is more reliable for your product. The dilution that produces the most reproducible spike recovery, and does not exceed your MVD, should be chosen to validate. When determining the concentration to validate, ten-fold or two-fold dilutions can also be made that do not exceed the MVC, depending on how low the MVC is. Ideally, the spike recovery would be closest to 100%, however this might not be the case for certain products. Once the I/E screen has completed and a dilution is chosen, (remembering this can be a dilution not included in the screen, but one the falls between two from the screen that returned valid recovery), select three lots of the same product to run on the selected dilution.