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Volume 28, Issue 2
The recognition that microbial artifacts are capable of modulating the mammalian immune system is an emerging view of biologic drug contamination control testing.
Article submitted: Aug. 20, 2015.
Article accepted: Nov. 24, 2015.
AbstractThe recognition that microbial artifacts are capable of modulating the mammalian immune system is an emerging view of biologic drug contamination control testing. The term IIRMI, or “innate immune response modulating impurity,” has been coined. It is important to recognize that pyrogenicity is only one potential risk of endotoxin contamination and that immune activation is an inherent property of endotoxin, even in the absence of pyrogenicity. Immune stimulation of biologics is undesirable, as it can stimulate anti-drug antibodies against administered recombinant proteins. Historically, many methods have been used to “detoxify” endotoxin to remove the pyrogenicity of endotoxin while retaining its immune stimulation properties for adjuvant use in vaccines. This article gives a broad perspective for understanding potential risks from low endotoxin recovery (LER) and other potential detoxification methods and presents a new paradigm to help drive future testing.
Given the safety concerns associated with the presence of microbial impurities in therapeutic proteins, the preclusion of impurities at more sensitive levels has been suggested (1). The detection of endotoxin as an innate immune response modulating impurity (IIRMI) would occur at levels that may be well below the currently prescribed limits for endotoxin as a pyrogen-as per United States Pharmacopeia (USP) <85> and <151> (2). The term “IIRMI” is contained in the FDA Center for Drug Evaluation and Research/Center for Biologics Evaluation and Research (CDER/CBER) guidance document on Assessment of Immunogenicity in Therapeutic Proteins (3). Manufacturers of therapeutic proteins seek to preclude microbial contaminants because they can increase immunogenicity risks (4); however, this preclusion is made from a pyrogen perspective rather than an immunogenicity perspective. While fever is a type of immune response that typically occurs as a result of infection, there are many possible additional immunogenic responses that occur in the absence of fever or prior to the development of fever. This paper elaborates several ways in which the two perspectives differ and how they may merge over time. Adjuvant-type activity (immune stimulation from added impurities) is desirable for vaccines (5, 6) but is undesirable for therapeutic proteins (see Figure 1) because it can make the associated therapeutic protein a target of antidrug antibodies (ADA) and can result in either immunogenicity or the neutralization of antibody efficacy (7, 8, 9).
Figure 1: The basic mechanism of the innate immune response modulating impurity (IIRMI) adjuvant effect. MPLA is monophosphoryl lipid A. The immunogenicity phenomenon has been seen historically in mild to severe adverse reactions (10,11,12).
Section 5 of the 2014 FDA guidance document, Immunogenicity Assessment for Therapeutic Protein Products (3), states the following:
“Impurities with adjuvant activity
Adjuvant activity can arise through multiple mechanisms, including the presence of microbial or host-cell-related impurities in therapeutic protein products (Verthelyi and Wang 2010; Rhee et al. 2011; Eon-Duval et al. 2012; Kwissa et al. 2012). These innate immune response modulating impurities (IIRMIs), including lipopolysaccharide (LPS), β-glucan and flagellin, high-mobility group protein B1 (HMGB1), and nucleic acids, exert immune-enhancing activity by binding to and signaling through toll-like receptors (TLR) or other pattern-recognition receptors present on B-cells, dendritic cells, and other antigen-presenting cell populations (Iwasaki and Medzhitov 2010; Verthelyi and Wang 2010). This signaling prompts maturation of antigen-presenting cells and/or serves to directly stimulate B-cell antibody production.
It is very important for manufacturers to minimize the types and amounts of such microbial or host-cell-related impurities in therapeutic protein products.”
In terms of immunogenicity, therapeutic proteins have become increasingly safe over time with the realization that natural animal-derived proteins may be recognized as non-self, and recombinant human proteins can become aggregated to bring about immunogenic reactions. The “humanization” of previously animal-based and chimeric monoclonal antibodies has also lowered immunogenicity rates. But a low level (and some not so low levels) of persistent proclivity toward immunogenicity remains and can be seen in clinical studies and marketed package inserts (13).
Establishing endotoxin as an IIRMI
The FDA guidance document (3) references a study by Verthelyi and Wang (1), which shows that even low levels of microbial artifacts such as LPS, peptidoglycan, and deoxyribonucleic acid (DNA) fragments can induce the immunogenicity of therapeutic proteins. Researchers have shown both in vitro and in vivo that synergistically, IIRMIs are active at lower levels than when present alone:
“This synergistic effect was then confirmed in vivo, as studies showed that the combination of 10 ng of LPS and 500 ng of cytosine-phosphate guanine oligodeoxynucleotides (CpG ODN), which do not induce an immune response when present individually, were sufficient to promote the immunogenicity of proteins and contribute to a clinically relevant break in tolerance to self” (1).
Verthelyi and Wang noted that while low levels of multiple impurities present in a product can synergize to act as adjuvants in mice, the levels are not expected to predict the levels that might be relevant in humans, whom they state “are likely to be much more sensitive to TLR agonists than rodents” (1, 14). They discuss the relevant levels of endotoxin viewed as an IIRMI to those standardized for testing of pyrogens, by either Limulus-based methods (limulus amebocyte lysate [LAL] and recombinant factor C [rFC]) or rabbit pyrogen tests (RPT). The authors write, “Of note, the current guidelines for setting limits on these impurities are not based upon their potential impact on product immunogenicity” (1). The response to IIRMIs (here using LPS and bacterial DNA) is thus amplified by the engagement of multiple receptors, reminiscent of an engine firing on multiple cylinders rather than a single cylinder.
The effects of low pyrogenic potency, “detoxified” endotoxins, administered with therapeutic proteins, can be seen in the realm of vaccines-specifically, the use of monophosphoryl lipid A (MPLA) as an FDA-approved adjuvant that stimulates the immune sensing of co-administered or subsequently administered proteins: “MPLA is a heterogeneous mixture of lipid A derivatives created by successive acid and base hydrolysis of lipid A from Salmonella minnesota 595. The predominant species created from that process is 3-O-deacyl-4-monophosphoryl lipid A. MPLA possesses attractive biological characteristics as an immunoadjuvant such as augmentation of T helper 1 (Th1) activity and antigen-induced T cell clonal expansion. Yet, MPLA possesses approximately 1/1000th of the systemic proinflammatory activity of native E. coli [Escherichia coli] lipid A in humans” (15).
Detoxified endotoxin is used (or being developed for use) in several vaccines including malaria (16, 17), hepatitis B (18), human papilloma virus (19), and various cancer vaccines (20). MPLA has low activity with LAL and rabbit pyrogen while retaining its adjuvant activity. While the LAL reduction associated with O-deacylation and dephosphorylation has long been known, what is less recognized is that after “detoxification,” both LAL and RPT activity are greatly muted (“when MPLA and lipid X were similarly tested, they showed very low pyrogenicity” ), and the adjuvant activity remains (21). The mechanism of adjuvant type response versus the historically recognized proinflammatory response to LPS is believed to be due to activation of the TRIF versus MyD88-dependent pathway (22, 23).
MPLA is not the only vaccine adjuvant using LPS being developed (24, 25, 26). There is a widespread interest in developing nontoxic LPS types for use as adjuvants of peptide and protein components of disease-causing organisms to complement proteins that typically elicit low levels of immune stimulation (unlike live attenuated vaccines), yet “adding MPLA to vaccine preparations boosts serum antibody titers by 10-20 fold compared to vaccine alone” (15). The growing importance of nonpyrogenic LPS structures can be seen in the development of nontoxic lipid A derivative drugs (27). An example is the anti-sepsis drug candidate Eritoran, which has been shown to block the TLR4 receptor by displacing active lipid A with the inactive lipid A form. Eritoran is a synthetic molecule derived from natural Rhodobacter sphaeroides lipid A (28). Despite providing valuable information on the interaction of the antagonist with the endotoxin receptor TLR4 and co-receptor MD-2 (29), the drug candidate failed its Phase III trial, as it did not provide a clear survival benefit (30).
Control of contaminants from an IIRMI vantage
The IIRMI view is one of endotoxin and other artifacts of microorganisms being able to elicit an immune response in mammalian systems at very low levels. The relevance to the administration of therapeutic proteins is seen as an adverse event producing capability that mirrors the effect of an adjuvant as paired with a clean recombinant protein. Thus, the occurrence of immunogenicity can be viewed as a problem of the past that is not entirely in the past. Biologics are lifesaving drugs, but some (depending upon the dose and indication), still have infusion reaction incidences approaching 25%, with half of those being said to be Grade 3 (severe) or 4 (life-threatening) (12). A caveat is that endotoxin control is only one aspect of the overwhelmingly complex issue of immunogenicity. The importance of general microbiological control in the manufacture of biologics can be seen from many references (31). Such control largely revolves around traditional efforts to control bioburden during processing, process validation that includes more extensive testing, cleaning validation, and the assurance of the quality of high-purity water systems.
The emerging view of endotoxin as an IIRMI--while straightforward in concept--has ramifications extending across a broad spectrum of current activities associated with the manufacture and administration of a therapeutic protein drug compound. Some items that seem common place today may require review in light of this emerging paradigm, including:
The levels of endotoxin Verthelyi and Wang identified as significant for adjuvant activity of LPS was stated to be as low as 1–10 ng/mL in the presence of sub-stimulatory levels of bacterial DNA (CpG), which does not easily translate from mouse models. An endotoxin unit (EU) is 1/5th the activity needed by E. coli reference standard to bring about the threshold pyrogenic response (K=5 EU/kg=1 ng/kg) (32). The 1–10 ng/mL range is likely much lower due to the known differences between mice and human response. Relative to the human response, mice are highly resilient to inflammatory challenge. For example, the lethal dose of endotoxin is 5–25 mg/kg for most strains of mice, whereas a dose that is 1,000,000-fold less (30 ng/kg) has been reported to cause shock in humans (33). The purpose here is not to suggest specific levels, but rather to point to a characteristic of the IIRMI view, which is that relevant IIRMI levels are expected to be lower than the levels precluded by traditional pyrogen and bacterial endotoxin test (BET) testing. Further experimentation will be needed to authoritatively inform manufacturers and regulators of relevant levels for humans.
Historical pyrogen and BET testing always considers the dose to be a critical parameter of drug administration as it pertains to endotoxin test preclusion. A large dose should contain less endotoxin than a small dose. This relationship is described in the tolerance limit (TL) calculation expressed as TL=K/M where K is the threshold pyrogenic response constant (K=5 EU/kg/hr for parenterals) and M is the relevant dosage of a specific drug (34). Today’s biologic drugs are expected to be cleaner than that required by historical pyrogen standards. This requirement can be seen in FDA biologics license application (BLA) requests to lower BET limits as well as the FDA Q&A Guideline expectation that drugs be tested at a “…dilution just above the level that neutralized the interference” (35).
The practice of pre-dosing before therapeutic protein administration with anti-fever and a steroid drug prior to some monoclonal therapy shows the expectation of adverse responses that includes fever (36). The large amounts of various solutions being administered to patients can be seen in the use of one and sometimes more than one LVP infusion. The expectation of lower-than-calculated TL specifications in BET is built into the administration of such large volumes of solutions and may point to an already occurring encroachment of the IIRMI-based view onto traditional BET preclusion activities. This mixing or blurring of lines of expected endotoxin exclusion levels for contaminants (the pyrogen versus immunogenic potential of contaminants) can be expected to continue as the IIRMI view advances.
The use of MPLA here as an example of LPS adjuvant activity is analogous because it is not naturally occurring; however, there are many natural, low pyrogenic LPS types that are associated with waterborne-type Gram-negative bacteria of the non-hexaacyl type (that differ from the prototypically pyrogenic E. coli LPS) (37). According to Darveau and Chilton, “Naturally occurring low biologically reactive lipopolysaccharide (LBR LPS) forms are known to function through TLR4, which directly activates B cells and indirectly activates naive T cells through APCs [antigen-presenting cells]. Therefore, LBR LPS forms are attractive candidate molecules for future adjuvant study. Although various structure/function studies have established key components of the lipid A structure required for potent immunostimulatory activity without toxicity, it is still not possible to reliably predict how a specific alteration in the LPS structure might affect the ability to function as an effective immune adjuvant” (38).
The basic assumption that the effect of a detoxified endotoxin adjuvant may equate to “low potency natural endotoxin” (LPNE) activity should be explored experimentally. If LPNE possesses adjuvant activity, then testing for such varieties of LPS (e.g., from genuses that include Pseudomonas and Burkholdaria) could be done by testing at levels well below current standards if these types are shown to be prevalent in a particular process. Such efforts would represent a significant change that would not be enacted lightly. IIRMI testing, however, could be advantageous for select processes and products based on risk assessment, for example, processes containing LPNE from such bioburden types.
Given that the types of bacteria likely to proliferate in water systems include Gram-negative bacteria with LPNE, such as Pseudomonas (which is 50–70 times less pyrogenic than E. coli ) and Burkholderia (some species were previously classified as Pseudomonas), it is worth exploring the preclusion of these less potent types. For example, overgrowth of a specific LPNE in bioburden or water purification systems may not be detected by conventional testing but could present a significant amount of LPS by mass. This level of “cleanliness” is not an issue in nonbiologics if they are not co-administered with a therapeutic protein. Munford lists LPNE types (39) occurring in soil, water, or plant habitats as including those from Burkholderia, Acinetobacter, Enterobacter, Chromobacterium, Erwinia, Rhodobacter, Rhizobium, Xanthomonas, and Pseudomonas. Potent types listed are largely members of Enterobacteriaceae (e.g., E. coli, Salmonella, etc.) and are natural inhabitants of the human gut. General methods used in processes to remove LPS regardless of the bacterial type would be unaffected. However, for processes relying solely upon LAL to gauge the efficacy of endotoxin removal, for example, this philosophy could change depending upon tools developed to gauge a wider spectrum of LPS types.
It is an unsettling prospect that LPNE could add to adjuvant activity of therapeutic proteins (38). Historically, there has been a singular focus on precluding the bacteria that produce proinflammatory, “endotoxic” endotoxins (Enterobacteriaceae, i.e., E. coli) as per USP <151> and <85> (2), rabbit pyrogen, and bacterial endotoxin testing, respectively. This fits an underlying, longstanding theme that microbial artifacts injected into the blood stream may have significant effects that do not necessarily correlate with our ability to “see” them, analytically speaking, or correlate with their ability to produce fever. The mammalian physiological view of endotoxin is ultra-sophisticated when it comes to the detection of microbe invaders and their artifacts. The basic Lipid A PAMP should be viewed as a set of dials (phosphate, sugar, number and types of acyl chains-either symmetrical or asymmetrical, substitutions, etc.) rather than an “on-off” button (pyrogenic or non-pyrogenic) (39, 40, 41). The activity of LPS at low levels is being borne out in studies of the low-dose effect of endotoxin in various disease states-such as sepsis (42), inflammation (43), cancer (44), and cardiovascular disease (45).
Detoxification does not remove the adjuvant effect of MPLA, but rather, significantly diminishes the proinflammatory effect. This is seen in other kinds of “detoxification” efforts, as Gamma irradiation of Salmonella typhimurium is known to remove its pyrogenicity, while allowing it to retain its immunogenicity inducing capability (46). Similarly, irradiated LPS retains the adjuvant activity of LPS, and it serves as a good adjuvant for inactivated virus vaccines. A wide variety of historically accumulated means of detoxification are shown in Figure 2. References include chemical (47, 48, 49) ionizing radiation (46, 50) use of surfactants (51) (reversible), enzymatic (52, 53) mutation (54, 55, 56) (natural and induced), antimicrobial peptides (57), natural low pyrogenic forms, and LER. A review of practices that do not incinerate or completely remove the functional LPS PAMP would be in order from the “endotoxin as IIRMI” view for therapeutic protein processing.
Figure 2: Low endotoxin recovery (LER) can be viewed as one of a dozen general methods of “detoxifying” lipopolysaccharide, historically performed for the purpose of adjuvant research.
Pairing biologics with LVP/SVPs governed bydiffering historical requirements
The most common symptoms associated with mAb infusions are endotoxin-like, dose-dependent, and include a fever component (with chills, aches, and neutropenia). Package inserts often recommend including a pre-infusion regimen of acetaminophen, antihistamine, and steroid in preparation for the initial mAb dose. Historically, too many drugs being administered at once would be considered potentially pyrogenic; however, in a recent FDA Q&A document (35), testing is recommended just over the level of interference (below tolerance limit).
Many mAbs are administered in a LVP infusion. LVPs have a rather permissive limit of 0.5 EU/mL, although often tested at much lower levels. SVPs also may have permissive historical limits that may not be updated to be in line with biologics drug expectations that are often assigned lower limits as part of the BLA review. A new quality designation of “for use with therapeutic proteins” for LVPs and/or SVPs to be used with biologics might improve current safeguards. Such a designation would also allow specific preclusion of some synergistic IIRMIs. Additionally, BET limit calculations are based on a one-hour criteria for K and M, where K is the threshold pyrogenic response =5 EU/kg/hr and M is the maximum human dose as dosed in either mg or mL. Here, the 350 EU/dose value is given using the routinely applied patient weight of 70 kg (5 EU/kg X 70 kg/dose=350 EU/dose limit. BET limit calaculations may have little relevance to immunogenic concerns.
From a quality perspective, the practices of some compound pharmacies seem out of line with regulatory agency-approved biologics. Given the new FDA draft guideline on Mixing, Diluting, or Repackaging of Biologics Products Outside the Scope of an Approved Biologics License Application (58), there are many recent warnings associated with compound pharmacy testing in which no endotoxin testing had been performed (59). Also often cited are the poor aseptic conditions present.
Using solutions of low quality or from low quality compounding environments in a co-administered or concurrent manner with painstakingly manufactured and tested therapeutic proteins seems incongruent from an IIRMI perspective.
Low endotoxin recovery (LER)
Endotoxin subjected to LER solutions can be considered a type of “detoxified” endotoxin by the IIRMI view. The LER discussion is an active one with industry participants split on the characteristics of potential endotoxin contaminants that could come from processes subjected to LER-causing conditions. The concept of “detoxification” with residual immune activation potential could help inform the LER debate. The search for compounds that utilize the immune stimulation property of LPS without the induction of proinflammatory effects is ongoing, as many subunit vaccines do not have the ability to stimulate the immune system (38). In the realm of endotoxin testing, if one is singularly worried about the pyrogenicity of a sample, then it may come to play out that LER subjected drug formulations are not particularly pyrogenic, although there is conflicting rabbit pyrogen data (60, 61). However, if one is worried that a given LER-prone protein formulation could increase the therapeutic protein immunogenicity if such LPS monomers are present, then one would want to detect and preclude the presence of LPS monomers or otherwise “detoxified” endotoxin solutions that retain the potential to be recognized by mammalian immune systems.
What may the IIRMI view mean for the use of LAL?
One might assume that, given the IIRMI view, cytokine-based tests such as a monocyte activation test (MAT) or human toll-like receptor test (h-TLR) would enhance current LAL testing. A couple of facets of LAL, however, may be viewed as critical to its continued use. The first is the sensitivity of LAL. It is more sensitive than any cytokine-based test available commercially. The recognition of endotoxin as an IIRMI is to acknowledge that pyrogenic activity does not equate to immunogenic potential. And the preclusion of LPS, by far the most potent of IIRMIs, could serve to preempt the possibility of synergism with another low-level IIRMI that may not be able to be readily precluded.
Secondly, LAL has been found to be active to under-acylated LPS in a way that mammalian-based cytokine assays are not (62). This has been touted as an advantage, as it is thought that these tests respond only to what a human would respond to. But this view only considers the proinflammatory pathway of LPS and not the potential for adjuvant-induced immune stimulation. Under-acylated LPS is one type of LPS being studied for its low pyrogenicity but high immunogenicity potential. Because LAL is better, although not perfect, at detecting these types, the use of mammalian-based assays that cannot detect them present a flawed strategy.
The emerging view of endotoxin as an IIRMI highlights several new concerns to consider, including: (a) the level and types of endotoxin contaminant required to produce fever versus the level and types capable of stimulating the immune system, (b) the pairing of therapeutic proteins with large-volume or small-volume drugs possessing lower-quality standards as compared with biologics requirements, and (c) the delivery of biologics with or without additional external handling, such as compound pharmacy manipulation.
The last thing biologics manufacturers intend is to introduce impurities with an adjuvant effect to therapeutic proteins. As illustrated is this article, endotoxin adjuvants (including detoxified endotoxin) administered with vaccine proteins are capable of eliciting nonpyrogenic endotoxin responses. The need for an updated view on immunogenicity is well stated by Haile, et al.: “It is only the more recent understanding of the innate immune system’s biology that dictates the need of assessing a broader spectrum of known and unknown IIRMIs in order to control or reduce the risk of unwanted immunogenicity by therapeutic proteins” (63). These biologics manufacturing concerns contrast with historical, purely pyrogen-centric activities that represent an important--but more minimal--standard that is typically associated with nonbiologic medications.
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ALL FIGURES ARE COURTESY OF THE AUTHORS
About the Author
Kevin Williams is senior R&D scientist at Lonza.
Article DetailsBioPharm International
Vol. 29, No. 2
Citation: When referring to this article, please cite it as K. Williams, "The Emerging View of Endotoxin as an IIRMI," BioPharm International 29 (2) 2016.