Typical and Representative, Part 1

In 1973, the Bureau of Biologics (now CBER), issued a Request for Proposals (RFP) for a new endotoxin standard to replace the existing Klebsiella standard.  The intent was to establish a single common calibrant and control reference for the “new” LAL gel clot tests to provide uniformity and label claim consistency across manufacturers and lots of the reagents.  It was not intended to be a substitute for nor representative of contaminating endotoxins that could be present in a product.  The project was awarded to Dr. Jon Rudbach at the University of Montana (Rudbach, et al, 1975). 

The specific laboratory construct developed by the Rudbach lab to exhibit the desired genotypic and phenotypic attributes is E. coli O113:H10k(-):

1. It does not contain immunodominant sugars that are found in many enteric bacteria.
2. It was selected to be “k negative” meaning that it is incapable of making a capsular polysaccharide that could contaminate the extracted LPS.
3. It was grown under highly controlled laboratory conditions utilizing a well-defined medium.
4. LPS was extracted using a modified hot phenol (Westphal) method.

Portions of the resulting 77g bulk have been formulated and lyophilized as needed to replenish national and international standards’ inventories. (Rudbach, et al, 1975; Firca and Rudbach, 1982; Hochstein, et al, 1983).

In a 1982 presentation, Joseph Firca and Jon Rudbach explained the required attributes for the standard and their rationale for decisions that were made to meet them. They wrote, “… by design, the reference endotoxin subsequently produced represented a typical preparation of average biological potency with sufficient chemical purity, and excellent properties of stability and solubility.” (Firca and Rudbach, 1982, emphasis added). Each of these attributes is discussed below. However, two definitions are provided for context, consistency, and clarity in the discussions:

• Endotoxins: Endotoxins are high molecular weight complexes that originate from the outer leaflet of the outer membrane of most Gram-negative bacteria and therefore contain lipopolysaccharide (LPS), outer membrane proteins, and phospholipid (Franco, et al, 2018). However, the term, “endotoxin” is often used to describe the biologically active portion of the complex, LPS, as well as the extracted, partially purified, formulated calibrant. For clarity in this discussion, the term, “native endotoxin” as used in FDA’s Guidance on Pyrogens and Endotoxins Testing (2026) will be used to describe endotoxins from autochthonous microorganisms that could potentially contaminate a product. The terms, “calibration standard”, “calibrant”, or LPS will be used to describe the extracted, partially purified reference standard.
• Typical and Representative: “Typical” microorganisms and their associated LPS are defined as those that possess the defining characteristics of their genus or species or perhaps related genera. E. coli is a typical Gram-negative enteric. A “representative” microorganism is one that epitomizes a specific community or ecosystem of microorganisms. Pathogenic strains of E. coli can be representative of organisms associated with Gram-negative infection.

Typical Preparation

As decisions were being made on the new standard, some in the parenteral industry questioned the choice of E. coli, as Enterobacteriaceae were and still are rarely encountered in the manufacturing environment. Pharmaceutical scientists favored something more representative of their manufacturing microbiomes such as Pseudomonas or another non-fermenting Gram-negative organism commonly recovered from pharmaceutical water purification systems. However, the LAL test was also a prospective diagnostic tool and pathogenic E. coli was a typical and representative cause of Gram-negative septicemia, which was prevalent at that time. While the Rudbach lab identified E. coli as a “typical” Gram-negative organism for their purposes, they recognized that the standard was NOT intended for use as a substitute for all endotoxins encountered by any one group of users, but rather as a calibrant to standardize reagents and assays across all applications (Firca and Rudbach, 1982).

The use of LAL as a diagnostic never materialized, and the primary application of the LAL assay shifted to the manufacture of parenteral products. How might one identify a microorganism that is not only typical but also representative of a manufacturing ecosystem? Since microorganisms that produce endotoxins are principally aquatic, water purification systems would be an initial focus. Penna, et al (2002), Sandle (2015), Reid, et al (2019), and Roder and Sandle (2022), all reported on extensive testing of pharmaceutical water systems. Even accounting for differences in time, geography, sampling, culturing and identification methods across the published data, Pseudomonas appears as one of the top four isolates in each of these studies. There is no E. coli or any other enteric with a significant presence in any of these publications, strongly suggesting that E. coli is neither typical nor representative of parenteral water purification environments. These findings are consistent with the earliest publications on the sources of “injection fever” and the first endotoxin standard, which was a cell free filtrate of Pseudomonas aeruginosa (Welch, et al, 1943; McClosky, et al, 1945). Florence Seibert, in a 1963 retrospective review of pyrogens wrote, “…the Pseudomonas group was found by Banks (1934) and Welch, Calvery, McClosky, and Price (1943) to yield a typical pyrogen which could be used as a standard.” (Seibert, 1963).

Lipopolysaccharides are amphiphilic glycolipids that comprise the majority of the outer leaflet of the outer membrane of most Gram-negative bacteria. The Lipid A portion of LPS, which determines pyrogenicity, typically consists of a bis-phosphorylated glucosamine disaccharide backbone with 4-7 fatty acid (acyl) chains. It was once thought that the Lipid A moiety of LPS was static, highly conserved, and therefore the most stable portion of the LPS molecule. We now know the Lipid A chemistry can be highly variable and dynamic depending on genus, species, and growth conditions (Li, et al, 2012; Dubczak and McCullough, 2018; Simpson and Trent, 2019; Dardelle, 2024). Most importantly, LPS, as an essential structural component of the outer membrane, is subject to ongoing “remodeling” via the organism’s Two Component Systems (TCS) which detect and respond to stimuli in the organism’s environment. The TCS response is controlled and rapid and ultimately leads to changes in gene expression resulting in adaptive remodeling of the LPS. For example, in the case of low magnesium environment, the Lipid A can be modified by the addition of 4-amino-4-deoxy-L-arabinose (L-Ara4N) to the phosphate groups, which reduces the net negative charge of the Lipid A and allows the membrane to remain intact to ensure survival. (Li, et al, 2012; Bonnington and Kuehn, 2016, Bonnington and Kuehn, 2017; Simpson and Trent, 2019; Dardelle, 2024). It is important to note that remodeling is an ongoing process.

Average Biological Potency

Rudbach’s yardstick for “average biological potency” was not pyrogenicity, but rather LAL reactivity using a first generation kinetic turbidimetric reagent and a research instrument modified for LAL testing. After screening seventeen different LPS preparations, Rudbach’s lab observed that E. coli demonstrated “intermediate activity” as it was eighth among the seventeen test articles as measured by onset times of dilutions of a 1ng/mL solution. A more appropriate definition of “average biological potency” might be pyrogenicity. Testing in rabbits shows that E. coli LPS is highly potent in its ability to elicit a febrile response when administered on a ng/kg basis. In contrast, Pseudomonas LPS may be up to ten times less potent than E. coli (Greisman and Hornick, 1969; Weary, et al, 1982). By the in vivo pyrogenicity yardstick, E. coli LPS is neither typical nor representative of the LPS from Pseudomonas found in pharmaceutical water systems.

The human innate immune system is our first line of defense against infection. At a very high level, the pyrogenic response involves the binding, transport and transfer of LPS to Toll Like Receptor 4/Myeloid Differentiation Factor 2 (TLR4/MD2) complexes found on the surface of immune cells (Park, et al, 2009; Park, et al, 2013; Dardelle, 2024). This interaction of LPS and TLR4/MD2 initiates an internal signaling pathway that releases proinflammatory cytokines IL-6, IL-1β and TNF-α.

The degree of pyrogenicity of an LPS is described by Dardelle (2024) as a function of a number of Lipid A attributes: 1) the number of fatty acid (acyl) chains attached to the disaccharide backbone, 2) the length of those chains, 3) the presence of phosphate groups on the disaccharide backbone and 4) their possible substitutions with various decorations or modifications that are largely an adaptive response to environmental stress. The bis-phosphorylated, hexa-acylated C:12 and C:14 fatty acid chain length structure of the prototype E. coli Lipid A aligns optimally with the hydrophobic MD2 pocket in the TLR-4/MD-2 complex and is therefore an active initiator of the TLR4 mediated cytokine response and a potent pyrogen. Lipid A with structural and chemical attributes other than the prototype may be a less optimal fit with the MD2/TLR4 complex, making them less inflammatory and therefore less pyrogenic on a ng/kg basis. For example, Helicobacter pylori Lipid A is typically tetra-acylated and mono-phosphorylated. While this LPS will demonstrate LAL reactivity, its chemistry and structure make it a poor stimulator of the MD2/TLR4 complex which allows it to elude the MD2/TLR4 complex (Dardelle, et al, 2024).

Sufficient Chemical Purity

The Rudbach lab recognized that the ideal standard must be a biological standard, not a highly purified LPS preparation. This decision was criticized by some at the time because analytical practice was and still is to use highly purified material as the calibration standard. However, Rudbach understood that a highly purified preparation of LPS would have low activity, and would present problems with solubility and stability, so the goal was to prepare an LPS that was “free of potentially biologically active proteins, peptides, polynucleotides, or polysaccharides.” (Firca and Rudbach, 1982). This level of purity (impurity) along with formulation excipients, usually a sugar and polyethylene glycol (PEG), would serve to assure “excellent properties of stability and solubility”, which was another of Rudbach’s requirements.

Native endotoxins, in contrast, are complex structures that contain significant levels of LPS. Research since Rudbach’s work has revealed the omnipresence and clinical importance of Gram-negative Outer Membrane Vesicles (OMVs), which are nonviable structures that bud from the outer membrane under normal growth conditions. Their production, however, can be exacerbated in response to an environmental stress (Beveridge, 1999; Bonnington, 2016). Because they originate from the outer membrane, OMVs are rich in LPS. OMVs are much smaller than the cells from which they originated, and they do not possess complete cell walls, so they can slip through sterilizing grade filters, surviving as pyrogenic contaminants in sterile products.

Current scientific literature suggests that LPS from Gram-negative infection is presented, not in the form of LPS monomers as previously thought, but rather as OMVs:

• Kim, et al, (2026) observed that OMVs can be considered “PAMP delivery vehicles”, delivering LPS, peptidoglycan and other PAMPs (Pathogen Associated Molecular Patterns) to Toll Like Receptors found on immune cells.
• Mazgaeen and Gurung, in their 2020 discussion on the transport of LPS, speak of LPS that they call “micelles”. The term, “micelles” often refers to aggregates of LPS monomers, but the authors explain that in their context, micelles are secreted by Gram-negative bacteria in the form of OMVs.
• Ryu and co-workers (2017) state that “the hydrophobic part of LPS is thought to be, “…buried either in the interior of the bacterial membrane or in the hydrophobic pocket of MD2 after being transferred to the TLR4-MD2 complex. Thus, the multi-acyl chains of LPS must be extracted from the bacterial membrane and protected until being presented to TLR4-MD2.”

Ellis and co-workers summed it up in their observation that while much of the experimental literature uses purified LPS as a research tool, it is “unlikely that pure LPS is shed from bacteria in the context of an infection. Rather, LPS has been found to be shed from the bacteria in the form of outer membrane vesicles.” (Ellis, et al, 2010). It is plausible that OMVs are the predominant form of contaminating endotoxins in pharmaceutical manufacturing.

Discussion

Science is a fluid discipline, based on challenging assumptions, testing hypotheses, collecting and analyzing data, and re-formulating evidence-based theories. The published scientific literature over the last 53 years has significantly advanced our understanding of endotoxins, lipopolysaccharide structure, Lipid A chemistry, OMVs and their importance in infection, microbial adaptation, LPS remodeling and the human innate immune system. This review has documented the following:

• E. coli and its LPS, particularly the laboratory construct used for RSE, is neither typical nor representative of the native endotoxins we see as potential contaminants in the pharmaceutical industry.
• The Lipid A component of LPS is not a static structure. It is constantly changing depending on genus, species, and growth conditions.
• Endotoxins in infections likely aren’t presented as pure free-floating LPS monomers; they are presented as complexes that exist as Outer Membrane Vesicles.
• LPS is subject to constant adaptation to stressful environments by “remodeling”.
• The innate immune system responds to LPS based on its highly variable chemistry.
• The calibrant is only partially purified and formulated to retain the required properties of stability and solubility that were in the original RFP.

As technology in endotoxins and pyrogens testing changes, and as the basic research advances our understanding of pyrogenicity, fifty-year-old assumptions and decisions should be revisited, if not challenged. Given that it is neither typical nor representative of potential contaminants, is the current calibrant still an appropriate analyte for endotoxins and pyrogens testing? To be continued…

Literature Cited

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