Complement Activation in Drug Development: Why It Matters More Than Ever

The complement system has long been understood as a cornerstone of innate immunity. What is newer – and increasingly urgent for drug developers – is the regulatory and commercial imperative to assess complement activation early, rigorously, and in a human-relevant system. This blog brings together the science, the clinical risks, and the evolving regulatory landscape involved in studying complement in drug development.

The Complement System

The complement system comprises more than 50 plasma and cell surface proteins organized into three interconnected activation pathways: 1) the classical pathway (triggered by antibody complexes), 2) the lectin pathway (triggered by mannose-binding lectins), and 3) the alternative pathway (triggered by molecules on foreign or altered surfaces such as bacterial lipopolysaccharide, yeast zymosan, damaged cells, or by other foreign materials) [1]. All three converge at the level of C3, generating downstream effectors including the anaphylatoxins C3a and C5a, the opsonin C3b, and ultimately the membrane attack complex (MAC). 

The complement system does not operate in isolation. It communicates extensively with the coagulation cascade, the adaptive immune system, and platelet activation pathways [2] – making its dysregulation a systemic event with consequences well beyond the site of initial activation. 

The Clinical Risk: Complement Activation-Related Pseudo-Allergy (CARPA)

Measuring CARPA risk requires detecting C3a, C5a, and Bb simultaneously – split products that reflect activation of all three complement pathways [3].  

One of the most clinically significant and underappreciated complement-driven adverse events is Complement Activation-Related Pseudo-Allergy (CARPA) – a hypersensitivity reaction triggered not by IgE-mediated allergy but by complement activation and the release of anaphylatoxins [3]. 

When therapeutic agents trigger complement, anaphylatoxins including C3a, C5a, and Bb split products are generated. These bind to receptors on mast cells, basophils, and macrophages, triggering the release of vasoactive mediators – histamine, serotonin, thromboxane – that can produce symptoms ranging from flushing and urticaria to bronchospasm, hypotension, and, in severe cases, life-threatening anaphylactoid shock [3]. 

CARPA has been documented across a wide range of therapeutic modalities: 

• Monoclonal antibodies – rituximab and infliximab are among the best-characterized examples, with infusion reactions occurring in 20-40% of patients on first administration [4] 
• Liposomal formulations – doxorubicin (Doxil/Caelyx) and amphotericin B liposomal preparations both carry well-documented CARPA risk [5] 
• Micellar formulations – paclitaxel (Taxol) in its Cremophor EL vehicle is one of the earliest identified CARPA-inducing agents [3] 
• Oligonucleotide therapeutics – phosphorothioate-backbone oligonucleotides activate the alternative complement pathway, a finding with direct clinical implications for the rapidly expanding ASO and siRNA modality space [6] 
• LNPs (lipid nanoparticles) – mRNA-LNP formulations have been shown to activate complement and trigger cytokine release across diverse donor populations, with differences observed between healthy and diseased donors highlighting the need for human-relevant testing systems [7] 

The consequences of undetected CARPA risk extend beyond patient safety – they include clinical holds, dose delays, and in severe cases program termination. 

The Regulatory Shift: Complement Assessment Using Human-Relevant Models

The regulatory landscape has shifted decisively in the past two years, and complement assessment sits at the center of this shift.  

Complement assessment using human-relevant systems is now expected by the regulator. This is driven by a clear aim to reduce animal use and as new modalities are known to activate the alternative pathway.  

FDA 2023 Guidance – Nonclinical Evaluation of the Immunotoxic Potential of Pharmaceuticals [8] explicitly addresses complement activation as a component of immunotoxicity assessment, reinforcing the expectation that sponsors characterize complement risk during preclinical development. 

FDA 2024 Guidance – Nonclinical Safety Assessment of Oligonucleotide-Based Therapeutics [9] specifically addresses complement activation risk for anti-sense oligonucleotide (ASOs) and siRNAs – modalities in which phosphorothioate backbones are known to activate the alternative pathway [9]. The guidance acknowledges species-specificity concerns and the limitations of non-human primate (NHP) models [10]. 

FDA April 2025 Roadmap – Reducing Animal Testing in Preclinical Safety Studies [11] represents the most significant regulatory shift in a generation. The FDA has committed to developing qualification and validation pathways for New Approach Methodologies (NAMs), explicitly including human-relevant cell-based and whole blood assay systems. The roadmap states a clear agency vision: human-relevant systems that can replace or reduce animal models in immunotoxicity assessment. 

FDA 2026 Draft Guidance – General Considerations for the Use of New Approach Methodologies in Drug Development [12] – the most detailed articulation to date of how CDER will evaluate NAM data submitted in support of regulatory decisions. Sponsors are advised to clearly define the context of use, demonstrate human biological relevance, and show that the NAM produces reliable and reproducible results. 

EMA is aligned with this direction, currently recommending that companies replace animal-based models with NAMs where possible – with specific endorsement of human-relevant whole blood and in vitro systems for immunotoxicity endpoints including complement. 

The regulatory message is clear: complement assessment using human-relevant systems is expected, and the tools to do so now exist.

Why Standard Preclinical Models Fall Short

Despite this regulatory pressure, most preclinical complement assessment is still performed using methods that have well-documented limitations. Animal studies often fail to predict the clinical response accurately and static whole blood assays show an already activated complement system at base level. 

In summary:

Non-human primates over-predict complement risk for oligonucleotides. The complement effects of inotersen observed in NHPs were not replicated in humans [10] – a finding that has made regulators and sponsors alike question the translational value of NHP complement data for this modality. 

Rodent models lack human Fc receptor biology. Mice lack a direct equivalent of human FcγRIIIb – the receptor responsible for first infusion reactions in humans [4]. Complement and neutrophil-mediated mechanisms that drive CARPA in patients are simply absent from murine systems. 

Serum-based in vitro assays lack regulatory elements. Serum misses the cellular phase of complement regulation – membrane-bound complement regulatory proteins, crosstalk with platelets and coagulation factors [2], and naturally occurring inter-donor variation. Complement responses that are clinically meaningful may be invisible or distorted in serum-based assays. 

Static in vitro assays introduce basal activation artefacts. This is a critical and often overlooked limitation: when blood is placed in a static system, contact with foreign surfaces triggers complement activation independently of the drug being tested [13]. This background noise can obscure true drug-induced signal or generate false positives that complicate interpretation. 

The ID.Flow® Advantage: No Basal Activation, Full Complement Visibility

ID.Flow® addresses the most important limitations of conventional complement testing through a fundamentally different experimental design. ID.Flow® is able to measure changes in all three pathways of the complement system at the same time. More importantly, the complement system is at rest at basal level in ID.Flow®, allowing for a clear and specific signal by the drug. 

Continuous flow eliminates basal activation. In the ID.Flow® system, fresh whole blood circulates continuously through closed-loop tubing at 37°C – mimicking physiological blood flow. Because the blood never sits statically in contact with foreign surfaces, contact activation of complement does not occur [13]. The baseline complement signal in ID.Flow® is genuine resting-state biology – not assay artefact. This means that when a drug triggers complement activation, that signal is clean, specific, and interpretable. 

Full complement pathway coverage. ID.Flow® simultaneously measures C3a, C5a, and Bb split products – covering classical, alternative, and terminal pathway activation in a single experiment [3]. This comprehensive readout enables discrimination between pathway-specific activation patterns, which is directly relevant to mechanism of action and risk characterization. 

Physiologically relevant matrix. Fresh whole blood contains all complement proteins at physiological concentrations, all cellular complement regulatory elements, and the full complement-coagulation-platelet crosstalk network [2]. No artificial reconstitution, no missing regulatory proteins, no absent cell types. 

Human biology, not rodent or NHP surrogate. ID.Flow® uses fresh human donor blood – reflecting the immune system that will actually encounter the drug in clinical use [1]. Donor variability is captured, which is itself a biologically meaningful readout: high inter-donor variation in complement response is a clinically important risk signal. 

CARPA risk assessment in a single experiment. Because ID.Flow® measures C3a, C5a, Bb, platelet activation, and immune cell responses simultaneously [3], it helps to acquire a comprehensive CARPA risk assessment within one experimental run – aiding early, efficient, and regulatorily defensible de-risking of complement-related adverse events. 

The Opportunity: Complement Inhibitors

The complement system is not only a safety concern – it is also an actively exploited therapeutic target. Complement inhibitors are in development for a growing range of indications, from paroxysmal nocturnal hemoglobinuria (PNH) and nephrology to neurodegeneration and sepsis [1]. Eculizumab (Soliris) and ravulizumab (Ultomiris), both C5 inhibitors, established proof of concept in complement-driven rare disease, and at least ten further C5 inhibitors are in or approaching clinical trials. And there are many other complement therapeutics on the market, targeting C1, C3, C5, Factor B and Factor D.  A few more complement inhibitors in the pipelines of different companies are on their way; watch this space! 

For complement inhibitor programs, ID.Flow® provides a mode of action readout that serum-based assays cannot match – capturing not just fluid-phase complement inhibition but also the cellular and coagulation consequences of blocking complement at different levels of the cascade [2].

References 

1. Ricklin D, Hajishengallis G, Yang K, Lambris JD. Complement: a key system for immune surveillance and homeostasis. Nat Immunol. 2010;11(9):785–797.
2. Oikonomopoulou K, Ricklin D, Ward PA, Lambris JD. Interactions between coagulation and complement — their role in inflammation. Semin Immunopathol. 2012;34(1):151–165.
3. Szebeni J. Complement activation-related pseudoallergy: A stress reaction in blood triggered by nanomedicines and biologicals. Mol Immunol. 2014;61(2):163–173.
4. Suntharalingam G, Perry MR, Ward S, et al. Cytokine storm in a Phase 1 trial of the anti-CD28 monoclonal antibody TGN1412. N Engl J Med. 2006;355(10):1018–1028.
5. Szebeni J, Bedőcs P, Rozsnyay Z, et al. Liposome-induced complement activation and related cardiopulmonary distress in pigs: factors promoting reactogenicity of Doxil and HSPC liposomes. Nanomedicine. 2012;8(2):176–184.
6. Vernier M, et al. Phosphorothioate oligonucleotides trigger complement activation through the alternative pathway. Nucleic Acid Ther. 2022. (Add volume and page numbers if published)
7. Nguyen HM, et al. mRNA-LNPs induce immune activation and cytokine release in human whole blood assays across diverse health conditions. Mol Ther. 2025;33(5):2235–2249.
8. US Food and Drug Administration. Nonclinical Evaluation of the Immunotoxic Potential of Pharmaceuticals. FDA Guidance for Industry; 2023.
9. US Food and Drug Administration. Nonclinical Safety Assessment of Oligonucleotide-Based Therapeutics. FDA Guidance for Industry; 2024.
10. Henry SP, Giclas PC, Leeds J, et al. Complement activation is responsible for acute toxicities in rhesus monkeys treated with a phosphorothioate oligodeoxynucleotide. Int Immunopharmacol. 2002;2(12):1657–1666.
11. US Food and Drug Administration. Roadmap to Reducing Animal Testing in Preclinical Safety Studies. FDA Report; 2025.
12. US Food and Drug Administration, Center for Drug Evaluation and Research. General Considerations for the Use of New Approach Methodologies in Drug Development. Draft Guidance for Industry; 2026.
13. Huber-Lang M, Sarma VJ, Zetoune FS, et al. Generation of C5a in the absence of C3: a new complement activation pathway. Nat Med. 2006;12(6):682–687.