Biologics or recombinant protein drugs, such as therapeutic antibodies and recombinant hormones and enzymes, require an expression system for their production. As a result, manufacturing and purification requires continuous monitoring of process sterility and tight microbial control strategies. In order to obtain a sterile and patient-safe biologic drug, the manufacturing process must ensure that the final product is free from intrinsic impurities (upstream) and extrinsic contaminants (downstream). These include:
- Host-cell impurities – HCPs include residual components of the expression system. For example, the host cells (CHO cells, yeast, E. coli, etc.) and their cell components such as DNA, RNA, proteins, and notably, endotoxins in the case of E. coli expression system.
- Process contaminants – Certain downstream processes may lead to bioburden or microbial contamination, especially via non-sterile water, buffers or excipients. Often, the growth of gram-negative bacteria such as E. coli results in microbial by-product contaminants such as endotoxins.
What are Endotoxins in Biologic drugs?
Endotoxins are amphiphilic lipopolysaccharides (LPS) located in the outer cell membrane of gram-negative bacteria. Endotoxins are categorized as pyrogens (fever inducing foreign substances) since they trigger TLR response, leading to inflammation, fever, and sometimes shock and organ failure. Hence, pyrogenic contamination of biological drugs and the excipients used to formulate them are a concern for biologics manufacturers. At the time of submission of investigational new drug (IND) or biologics license application (BLA) it is important to include controls on pyrogens, including endotoxins, as a part of the chemistry, manufacturing, and control information.
LPS or bacterial endotoxin comprise three main parts: O antigen, core oligosaccharide, and the Lipid-A molecule, which is the conserved and active toxic component of bacterial endotoxin. If the bacterial cell wall is lysed after it reaches the human blood stream, Lipid-A molecule of the bacterial endotoxin activates the toll-like receptor 4 (TLR4) of the immune system, releasing different types of vasoactive peptides and cytokine mediators responsible for cell apoptosis and fever, diarrhea, vomiting, and also fatal endotoxic shock (septic shock). Therefore, bacterial endotoxin/LPS characteristics and structural aspects have been utilized for development of endotoxin detection tests and removal methods. Hence, endotoxin detection in biologics is a necessity to ensure that the therapeutic drugs are endotoxin free.
Detection of endotoxins
It is always better to design manufacturing processes that produces endotoxin-free end products, rather than include steps later on to remove endotoxins in the final product. From an FDA perspective, in order to implement Quality by Design manufacturing concepts, the endotoxins testing strategies must be tailored depending upon the specific biologic product and process development. Additionally, appropriate risk management steps are required to assure consistent final product quality. Suitable in-process testing methods should be employed to monitor the production process areas that pose a risk of endotoxins contamination.
Conventionally, the bacterial endotoxins test (BET) and the rabbit pyrogen test (RPT) have been used to detect endotoxin levels during biologic drug development.
- The RPT came into existence since rabbits were found to have a similar pyrogen tolerance to humans. RPT helps in the qualitative detection of pyrogens in-vivo, wherein pyrogens are detected by observing changes in the body temperature of rabbits. This test can also determine the presence of non-bacterial endotoxin pyrogens. Disadvantages of the test include time consumption, requirement of great quantity of rabbits to conduct the test, non-quantification of endotoxins, false-positive results due to the presence of other pyrogens, and also, false-negative results owing to the endotoxins found in an inactive form.
- The BET is also referred to as the Limulus Amebocyte Lysate (LAL) test. LAL test, an in-vitro assay, aids in the quantitative and qualitative detection of endotoxins from Gram-negative bacteria using amoebocyte lysate from the horseshoe crab (Limulus polyphemus or Tachypleus tridentatus). Here, the presence of endotoxin will activate Factor C-mediated clotting cascade in horseshoe crab blood, which is used as the detection principle. US FDA approved the LAL test as an endotoxin test method in 1983. The test can be conducted using three methods: (a) The gel-clot technique, which is based on gel formation; (b) The turbidimetric technique, based on the development of turbidity after cleavage of an endogenous substrate; and (c) The chromogenic technique, based on the development of color after cleavage of a synthetic peptide-chromogen complex. Also, the Gel-clot method and the chromogenic method are approved for all developmental phases of the therapeutic products and proved appropriate for biologic drugs such as monoclonal antibodies, vaccines, recombinant proteins, cell therapy and gene therapy. Recently, Recombinant Factor C (rFC) test has been validated as an alternative to LAL test where recombinant factor C protein is utilized for robust results. Thus, owing to the sensitivity, specificity, and simplicity of the LAL test, it retains the prime important status in parenteral drug industry. Yet, because of the interferences observed with the LAL assay and its inability to detect non-endotoxin pyrogens, LAL test has not fully replaced the RPT in the therapeutic industry.
Recently, low endotoxin recovery (LER) phenomenon has been described for LAL tests. LER is the inability to recover known amounts of purified endotoxin from biological formulations, and can be caused by the presence of chelating agents and surfactants in biological formulations. The LER phenomenon can be evaluated using suitable reference standard endotoxin, control standard endotoxin, and naturally occurring endotoxin.
Endotoxin acceptance criteria for biologic formulations
In general, endotoxin acceptance criteria are set such that endotoxin administration level does not exceed the pyrogenic threshold. Thus, the mode of administration is also an important determinative factor. In the case of intravenous and subcutaneous administration, the upper limit is set at 5 EU/Kg/hour or 100 EU/m2/hour. And for intrathecal the threshold is set at 0.2 EU/kg/hour. These values denote the maximum combined endotoxin exposure level from all agents (including concomitantly administered drugs), provided there is no clinically significant increase in body temperature at these exposures. For lyophilized drugs, the endotoxin contribution from the diluent or reconstitution fluid is to be considered while setting the acceptance threshold.
Importantly, for development of combination therapies, the combined endotoxin levels of all drugs must be evaluated. Combination therapy involves administration of two or more biologics to obtain a synergistic clinical effect. Most of these drugs are originally designed as monotherapies, therefore the individual impurity levels and mode of administration must be taken into consideration. In the case where EU exceeds the recommended levels, phase wise drug administration could be an alternative treatment option.
Reference drugs from Evidentic
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- US FDA. Setting Endotoxin Limits During Development of Investigational Oncology Drugs and Biological Products Guidance for Industry. JULY 2020.
- US FDA. Guidance for Industry Pyrogen and Endotoxins Testing: Questions and Answers. June 2012.
- Williams KL. The Biologics Revolution and Endotoxin Test Concerns. Endotoxin Detection and Control in Pharma, Limulus, and Mammalian Systems. 2019;331-402. Published 2019 Mar 18. doi:10.1007/978-3-030-17148-3_8
- Suvarna K. Endotoxin Detection Methods – Where are we now? American Pharmaceutical Review. August 25, 2015.
- Setting Endotoxin Acceptance Criteria for Biologics Intravenous (IV) and Subcutaneous (SC) Mono- and Combination Therapies. American Pharmaceutical Review. September 17, 2018.