The US Pharmacopeia and the various international pharmacopeias specify a number of biological assays for pharmaceutical raw materials and finished products. Many of these procedures are described in the sections below.

Expand All | Collapse All


• Glucagon Assay (USP)
  • 
    

    Glucagon is a critical drug for the treatment of diabetes. The potency assay for glucagon is a challenging ex vivo procedure using a primary culture of rate hepatocyte cells. PBL is perhaps the only independent laboratory in the world offering this assay.

• Insulin Assay (USP)
  • 
    

    PBL has performed quantities insulin biopotency and bioidentity tests according to USP <121> for over ten years.  It is a routine test for this company. 

    The Bioidentity Test is a qualitative test used as a batch release test.

    1. We can give you a verbal result within 3 weeks and a report within four weeks.  The timeline can be shortened if we have commitment and advanced notice so we can order and acclimate animals.
    2. The assay uses 8 rabbits assigned in two groups of four animals.
    3. The point of the bioidentity test is to determine the potency of a test article (solution or powder).
    4. To pass the USP criteria for the bioidentity test, the potency of the insulin contained in the test article must be greater than 15 mg/dL.  (e.g. if the potency of a test formulation containing 1.0% insulin (weight/volume) is 0.2 Units/mL, then the insulin potency is 0.2 Units/0.01 = 20 Units/mg insulin and the solution passes the test). 

    The Biopotency Test is a quantitative test used to attain an accurate measurement of the potency with a statistically significant 95% confidence limit.

    1. It takes about four weeks, four assays to complete a set and attain a confidence interval.  Although it usually takes four assays to attain a result within USP compliance, we cannot guarantee that that will happen.  There is the possibility of attaining a value with fewer assays.  We only charge for the assays performed.
    2. The report takes about 7-10 working days after the completion of the last replicate.
    3. The assay uses 24 animals, four groups of six each.
    4. The dosing and blood collection procedures are the same as for the 8 rabbit bioidentity test.
    5. The endpoint of the assay is a potency value with a 95% confidence limit.
    6. USP <121> requires the confidence limit to be not greater than approximately +/- 10 percent of the potency.
    7. The level of accuracy is rarely attained with one 24-rabbit assay and normally requires the combined results of several.
    8. In our experience it normally takes four 24-rabbit assays to get a potency value and confidence limits that pass the USP <121> criteria.

    In both assays animals receive subcutaneous injections of solutions of a known insulin standard or an equivalent solution of the test article on two different days, 3-7 days apart.  The solutions are set at 1Unit and 2 Units/mL.  If the animals in a group receive the high dose of the standard on one day, they receive the low dose of the test article on the 2nd day and vice versa.  On both days, blood is collected 1 hour and 2.5 hours after dose administration.  The potency is calculated by comparing the plasma glucose response to the test article versus the standard.

    1. For raw powder, 50-100 mg of sample is enough to do all testing, whether for bioidentity or biopotency.  This is because a 40 Unit/ml stock solution is prepared from the powder in USP insulin diluent and is good for six months at 2-8C and all the dosing solutions can be prepared from it.
    2. For formulations, we normally make the dosing solutions directly from the formulations each time they are needed. About 2 mL per assay is needed (1mL per dosing day x 2 dosing days), thus 2 mL for a bioidentity test or 8 mL for a four-assay quantitative test.
• Bacterial Endotoxins (LAL) Tests
  • 
    

    The Bacterial Endotoxin Test also known as Limulus Amebocyte Lysate (LAL) Test, is an in vitro assay for the detection and quantitation of bacterial endotoxin in injectable drugs or solutions for parenteral administration, including biological products.  The current USP General Chapter <85> “Bacterial Endotoxin Test” has been harmonized with the European and Japanese Pharmacopeia. Pacific BioLabs analysts are qualified to conduct the kinetic-chromogenic test. In this method the endotoxin content of the test material is determined by interpolation from a standard regression curve which is established each time the test is conducted.

    The validation of the LAL test also known as Inhibition and Enhancement Test must be conducted for each drug formulation to determine the degree of product inhibition or enhancement of the assay. The validation must be conducted before the LAL test is used to assess the endotoxin content of any drug. All validation tests should be performed on undiluted product or on appropriate dilutions that do not exceed the Maximum Valid Dilution (MVD). The endotoxin limit of the drugs to be tested must be provided to Pacific BioLabs analysts by the sponsor at the time of sample submission in units such as EU/mL or EU/mg. The analysts need this limit to calculate the MVD for the validation test.  Three production batches of each product must be tested for inhibition and enhancement.

    A pyrogen is frequently described as a fever producing substance.  The most potent pyrogens originate from gram negative bacteria, which are common water-borne organisms.  Although not entirely accurate, the terms pyrogen and endotoxin are often used interchangeably.  Detection of bacterial endotoxin contamination is essential to insure the safety of parenteral products.  The Bacterial Endotoxins Test using Limulus Amebocyte Lysate (LAL) is recommended for the detection of endotoxins in pharmaceutical products.  Human and animal injectable drugs (including biological products) must be tested for bacterial endotoxin. 

    The number of samples to be tested should be based on the manufacturing procedure and batch size. A minimum of three units, representing the beginning, middle, and end, of a production lot should be tested.  These units can be tested individually or pooled.

    The endotoxin limit for parenteral drugs, except those administered intrathecally, is defined as K/M, where K is equal to 5.0EU/Kg and M is the maximum dose per patient weight in Kg.  For parenteral drugs that have an intrathecal route of administration, K is equal to 0.2 EU/Kg.

    FDA has published guidelines outlining validation procedures for endotoxin testing of finished products using the LAL test.  This document is titled Guideline on the Validation of the Limulus Amebocyte Lysate Test for Human and Animal Parenteral Drugs, Biological Products and Medical Devices and can be downloaded at www.fda.org  

    Because water is a source of pyrogens, it is important to routinely monitor water systems using the bacterial endotoxins test.  For process water samples, the amount of sample required is 10 mL.  Please contact Pacific BioLabs if you need sample collection containers. 

    Kinetic Chromogenic tests are available at PBL:

    1. LAL Validation – Inhibition and Enhancement
    2. Pharmaceutical Powders
    3. Pharmaceutical Liquids
    4. Water
• Pyrogen and Safety Tests
  • 
    

    PBL offers pyrogen and general safety tests to comply with all the major pharmacopeias - USP, BP, EP and JP.

• Microbial Limits Tests
  • 
    

    The Microbial Limits Test consists of a microbial count for bacteria, yeast and mold, and a screen for the presence of four objectionable organisms (Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli and Salmonella species). The USP 29 General Chapter <61> titled “Microbial Limits Test” was divided into two Chapters in USP 30 to harmonize with the European Pharmacopoeia.  The implementation of the new Chapters <61> “Microbiological Examination of Nonsterile Products: Microbial Enumeration Tests” and <62> “Microbiological Examination of Nonsterile Products: Test for Specified Microorganisms” has been postponed until May 1, 2009.  The US Pharamcopeia Notice of Postponement published on December 2006 indicates that implementation of the harmonized General Chapters prior to the May 2009 date is at the discretion of the user and may be subject to regulatory consideration.

    PBL’s analysts are currently conducting the Microbial Limits and Preparatory Tests specified in USP 29. 

• Preservative Effectiveness
  • 
    

    The Antimicrobial Effectiveness Test is required to evaluate the efficacy of the preservatives added to non-sterile dosage forms to prevent the growth of microorganisms that may be introduced by the consumers when withdrawing individual doses.  In this test individual product samples are inoculated with high concentrations (10⁵ to 10⁶ CFU/gram) of specific organisms and incubated at room temperature for 28 days. The populati/uson of surviving organisms is determined at specific time intervals using the plate count method and the log reduction of each microbial strain is calculated.  Pacific BioLabs analysts conduct the USP, EP, BP and JP tests. The plate count method validation must be performed prior to the initiation of the test.

  • Product Categories, Plating Intervals and Interpretation

    • Test	USP	EP	BP
      ATCC ORGANISMS (may include client isolates)	C. albicans – 10231 S. aureus – 6538 E. coli  - 8739 P. aeruginosa – 9027 A. niger - 16404	C. albicans – 10231 S. aureus – 6538 P. aeruginosa – 9027 A. niger – 16404 E. coli – 8739 for oral preparations Z. rouxii – NCYC 381 (IP 2021-92) (for oral preparations, with high sugar concentration)	C. albicans – 10231 S. aureus – 6538 P. aeruginosa – 9027 A. niger – 16404 E. coli – 8739 (for oral preparations) Z. rouxii – NCYC 381 (IP 2021-92) for oral preparations, with high sugar concentration)
      Platings – Rechallenge as required	T = 7, 14 and 28 days for Category  1 T = 14 and 28 days for Categories 2, 3, and 4	T = 6 and 24 hours and T =  2, 7, 14 and  28 days	T = 6 and 24 hours plus T = 2, 7, 14 and 28 days
      Dilutions for: Bioburden Inhibition Inoculated Product	10-1 to 10-4 as needed 10-1 to 10-4 as needed 10-1 to 10-5 as needed	See USP	See USP
      Pass/Fail Criteria (Reference alternate criteria listed in the British and European Pharmacopeias for cases where the criteria listed here cannot be obtained.)	Category 1 Products Injections, other parenterals including emulsions, otic, sterile nasal products, and ophthalmic products made with aqueous bases or vehicles: Bacteria reduced by 1.0 log at 7 days from initial count, 3.0 logs at 14 days from initial count, and no increase at 28 day from 14 day’s count. Fungi must show no increase from initial inoculum at 7, 14 and 28 days. Category 2 Products Topically used products made with aqueous bases or vehicles, nonsterile nasal products, and emulsions, including those applied to mucous membranes: Bacteria reduced by 2.0 log at 14 days from initial count, and  no increase at 28 days from 14 day’s count. Fungi must show no increase from the initial inoculum at 14 and 28 days. Category 3 Products Oral products other than antacids made with aqueous bases or vehicles: Bacteria reduced by 1.0 log at 14 days from initial count and no increase at 28 days from 14 day’s count. Fungi must show no increase from the initial inoculum at 14 days and 28 days. Category 4 Products Antacids made with an aqueous base: Bacteria, yeast and molds must show no increase from the initial calculated count at 14 and 28 days.	PARENTERALS/OPHTHALMICS Criteria A Bacteria reduced by 2 logs at 6 hours and 3 logs at 24 hours with no recovery at 28 days. Fungi reduced by 2 logs at 7 days with no increase at 28 days. Criteria B Bacteria reduced by 1 log at 24 hours and by 3 logs at 7 days with no increase at 28 days. Fungi reduced by 1 log at day 14 no increase at 28 days. TOPICALS Criteria A Bacteria reduced by 2 logs at 2 days and by 3 logs at 7 days with no increase at 28 days. Fungi reduced by 2 logs at 14 days with no increase at 28 days. Criteria B Bacteria reduced by 3 logs at 14 days with no increase at 28 days. Fungi reduced by 1 log at day 14 with no increase at 28 days. ORAL PREPARATIONS Bacteria reduced by 3 logs at day 14 with no increase thereafter, Fungi reduced by 1 log at day 14 with no increase at 28 days.	PARENTERALS/OPHTHALMICS Criteria A Bacteria reduced by 2 logs at 6 hours and 3 logs at 24 hours with no recovery at 28 days. Fungi reduced by 2 logs at 7 days with no increase at 28 days. Criteria B Bacteria reduced by 1 log at 24 hours and by 3 logs at 7days with no increase at 28 days. Fungi reduced by 1 log at day 14 with no increase at 28 days. TOPICALS Criteria A Bacteria reduced by 2 logs at 2 days and by 3 logs at 7 days with no increase at 28 days. Fungi reduced by 2 logs at 14 days with no increase at 28 days. Criteria B Bacteria reduced by 3 logs at 14 days with no increase at 28 days. Fungi reduced by 1 log at 14 days with no increase at 28 days. ORAL PREPARATIONS Bacteria reduced by 3 logs at day 14 with no increase thereafter, Fungi reduced by 1 log at day 14 with no increase thereafter.
      Test	CTFA	ANSI/ASTM	JP
      ATCC ORGANISMS (may include client isolates)	One or more in-house isolates *at least one gram positive cocci * at least two fermentative gram negative rods *at least one non-fermentative gram negative rod in addition to   P. aeruginosa. *at least one yeast B. subtilis (optional) A. niger Penicillium luteum *Reference guidelines for list of appropriate organisms to choose form.	See USP	See USP
      Platings – Rechallenge as required	T = 1-3, 7, 14, 21 and 28 days (At least one rechallenge is recommended)	See USP	See USP
      Dilutions for: Bioburden Inhibition Inoculated Product	See USP	See USP	See USP
      Pass/Fail Criteria (Reference alternate criteria listed in the British and European Pharmacopeias for cases where the criteria listed here cannot be obtained.)	Less than or equal to 0.1% survival of bacteria at day 7. Greater than 90% reduction in yeasts/molds at day 7. Spores should be static in number throughout the test.* *For eye-area applications.	See USP	Category IB Bacteria: 1% or less of the inoculum count at 14 days, with the same or less than level after 14 days at 28 days. Yeasts & Molds:  Same or less than inoculum count at 14 and 28 days.

  
  
• Tetracosactide Testing
  • 
    

    Tetracosactide is a recombinant (synthetic) form of the naturally-occurring pituitary hormone, adrenocorticotropin (ACTH).  ACTH is produced by the pituitary gland and plays an important role in controlling levels glucocorticoids and mineralcorticoid, which are steroid hormones produced and released by the adrenal gland. 

    When stimulated by ACTH, the adrenal gland will increase production and release of  the glucocorticoid hormone, hydrocortisone and the mineralcorticoid, aldosterone.  Glucocorticoids are steroid hormones that play important roles in glucose metabolism, immune function and protein metabolism.  Mineralcorticoids plays an important role in regulating water retention by the body.

    The primary use of Tetracosactide is as a diagnostic.  If a patient does not have normal levels of corticosteroids in his/her blood, a dose of tetracosactide can be used to test whether the adrenal gland is functioning normally.  The drug may also be used to enhance steroid production if the pituitary is not producing sufficient ACTH.

    Pacific BioLabs has developed a bioassay for tetracosactide.  This test is used to determine the potency of a preparation of tetracosactide, compared to a calibrated reference standard provided by the World Health Organization (WHO).  In this assay, adrenal cells are extracted from the adrenal glands of rats.  In the assay, a comparison is made of the ability of a test sample, in comparison to the reference standard, to cause the adrenal cells to release corticosteroids.  The test is based on an EP (European Pharmacopeia) monograph.