Monocyte Activation Test: From Validation to GMP Lab testing

Pyrogens comprise a heterogeneous group of fever inducing compounds derived from microorganisms and non-microbial substances. This kind of contamination is considered a serious public health issue and can result in symptoms ranging from vascular alterations to shock and death.¹

The most widely known pyrogen is the Lipopolysaccharide (LPS) from Gram-negative bacteria contamination.² Other pyrogen sources could potentially derive from Gram-positive bacteria, like Lipoteichoic Acid (LTA), viruses and fungi particles.³ Non-microbial pyrogenic substances are microscopic plastic particles, rubber or metal compounds in elastomers.⁴ Health Authorities required absence of pyrogens in injectable drugs for health and safety reasons. The pyrogen test aims to prove that the concentration of pyrogens contained in the product does not exceed a certain threshold, known as the contaminant limit concentration (CLC), that will preserve patient safety.

Nowadays, the most used tests are the Rabbit Pyrogen Test (RPT) and/or the Bacterial Endotoxin Test (BET). However, both tests have their disadvantages. The BET detects endotoxins through amoebocyte lysate coagulation from the horseshoe crab hemolymph, a principle developed more than 50 years ago.⁵ In the United States, Limulus crabs are generally released back into the wild after drawing about 20% of their blood. Nevertheless, this procedure highly increases horseshoe crabs mortality, even more severe then fisheries, habitat loss and pollution.⁶ Although BET is highly sensitive, non-endotoxin pyrogens are not detected. Furthermore, the BET is not able to provide any information about interactions and synergetic effects or the pyrogenic activity of the endotoxin, as well as its susceptibility to interference, known as Low Endotoxin Recovery (LER) effect, prevent the full rabbit pyrogen test replacement.^7,8 Besides, the issue to convert the rabbit test results in potential human fever reactions is still present. Hence, an estimated 200,000 rabbits per year are still involved for pyrogen testing in the European Union.⁹ Moreover, some reports on biological products have reported pyrogenic episodes in patients although the products were valid after RPT and BET testing. Consequently, these tests do not always provide a sufficient safety warranty.^10,11 Additionally, both methods are based on animals or animal-derived products countering the 3Rs principles (Replacement, Reduction and Refinement) regarding animal welfare.

Thus, the interest in in-vitro alternative methods has risen for ethical, safety and business reasons since 1984. In particular, scientists have focused on the development and validation of in-vitro tests,^9,11,12 such as recombinant Factor C (rFC) test and Monocyte Activation Test (MAT).

The MAT allows the detection of the full range of pyrogens including endotoxins and non-endotoxin pyrogens (NEPs). It could be performed as a quantitative test (Method A), semi-quantitative test (Method B) or as a Reference lot comparison test (Method C).13 Based on what the US Interagency Coordinating Committee stated for the Validation of Alternative test Methods (ICCVAM),14 the method can be used in replacement of the Rabbit Pyrogen test after suitable product-specific validation.

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MAT was introduced in the European Pharmacopoeia (Ph. Eur.; Chapter 2.6.30)¹³ among the compendial methods for pyrogen detection in 2010. Moreover, this method was also mentioned by the FDA “Guidance for Industry – Pyrogen and Endotoxins testing: Questions and Answers” as an alternative method to the Rabbit Pyrogen Test, which should be validated according to USP <1225>.^15,16 Additionally, the USP <151> Pyrogen Test suggests that “A validated, equivalent in vitro pyrogen or bacterial endotoxin test may be used in place of the in vivo rabbit pyrogen test, where appropriate”.¹⁷

Following the European Pharmacopoeia and USP trend, other regulatory agencies have introduced the MAT chapter into their guidelines, such as Russian and Indian Pharmacopoeias.

To reduce the animal-based test and anticipate the Health Authority requirements for pyrogens detection, a MAT based method has been developed in EMD Serono Ivrea Biological Quality Control laboratories, based in the North-West of Italy. The MAT results will be compared with the currently used methods (RPT and LAL tests) performed in other EMD Serono laboratories.

The method has been developed using a cell-based MAT kit and following the Method A protocol described in the European Pharmacopeia.¹³ To quantify the pyrogenic load in the samples, MAT system uses a cryo-preserved Mono-Mac-6 (MM6) cell line as monocyte source and detects the Interleukin-6 (IL-6) concentration in response to pyrogenic substances through an ELISA assay.

Despite MAT already introduced as a compendial method in Eur. Ph., a full GMP method validation will be carried out to satisfy USP requirements too.

Following the ICH Q2(R1) guideline,¹⁸ the Monocyte Activation Test is classified as a Quantitative test for impurity content. Based on this classification, the parameters to be checked are: Robustness, Accuracy, Precision, Specificity, Detection Limit (not mandatory), Quantitation Limit, Linearity and Range of applicability.

The Robustness has to be checked by introducing some changes into critical steps of the method. Based on the FDA’s Guidance for Industry “Analytical Procedures and Methods Validation for Drugs and Biologics” issued in July 2015, it is recommended to evaluate the Robustness in advance of the validation. The experimental design has been established through statistical software. This tool allows combining the critical parameter variations optimizing the number of tests to be carried out and evaluating the interactions between them. A risk assessment approach was used to evaluate the method workflow in each phase. Severity, detectability and probability were the parameters scored in such evaluation.

Consequently, the steps that can have a significant impact on the collected results should be evaluated in the robustness exercise. In the MAT method workflow, three critical parameters were identified:

1. Plate incubation time from 20 to 24 hours (22 hours ± 10%)
2. ELISA Antibody incubation time for 2 hours (2 hours ± 10%)
3. ELISA Substrate incubation time for 30 min (30 min ± 10%)

By combining these parameters, a total of 16 tests will be executed to verify the method robustness.

The Specificity has to be verified using both clean and contaminated samples with known quantities of endotoxin (Reference Standard Endotoxin - LPS) and non-endotoxin pyrogens (NEPs) such as Flagellin or HKSA. RSE and NEP must be identified and quantified.

The Accuracy will be determined exploiting data produced during Specificity experiments. The Accuracy parameter will be assessed comparing four RSE concentrations (e.g. 0.05, 0.1, 0.2, 0.3 EU/mL) tested in the same analytical session. All data will be further analyzed estimating the Coefficient of Variation (CV%). CV% should not exceed 25%.

Precision is evaluated at three different levels: repeatability, intermediate precision and reproducibility.

The Repeatability will be verified testing four contaminated samples at known concentrations for three replicates within the range of application of the method (e.g. 0.05, 0.1, 0.2, 0,3 EU/mL).

The Intermediate precision will be performed comparing endotoxin standard curves performed by two different operators on different days.

The Reproducibility will be assessed comparing the results achieved in parallel from tests performed in two different EMD Serono laboratories.

The Limit of Detection (LOD) expresses the slightest pyrogen concentration detected through a qualitative and/or quantitative analysis. The LOD will be assessed by the reference standard endotoxin curve. To validate the LOD of the test system, the LOD claimed by the supplier (0.05 EU/mL) will be verified by checking that the cut-off value obtained with the endotoxin standard curve is below the signal obtained with a number of solutions of endotoxin at 0.05 EU/mL.

The limit of concentration up to which it is possible to obtain a quantitative measure with relative confidence in a sample is defined as Quantitation Limit. In some cases, the quantification limit can be defined as the lowest value in the application range and will be calculated in a test correlated way during the validation exercises.

The Linearity refers to the linear “dose-response” correlation between the analyte concentration and its measurement and is expressed by a mathematical function. A minimum of five pyrogens concentrations is recommended to investigate the linearity.

The Range of applicability is usually obtained from the linearity tests. This parameter defines the range by which the results are characterized by an appropriate level of linearity, accuracy and precision.

After the method validation, a comparability exercise will be designed to confirm that animal testing can be replaced by this invitro pyrogen test.

References

1. da Silva CC, Presgrave OAF, Hartung T, de Moraes AML, Delgado IF. Applicability of the Monocyte Activation Test (MAT) for hyperimmune sera in the routine of the quality control laboratory: Comparison with the Rabbit Pyrogen Test (RPT). Toxicol Vitr. 2016;32:70-75. doi:10.1016/j.tiv.2015.12.004
2. Cavaillon JM. Exotoxins and endotoxins: Inducers of inflammatory cytokines. Toxicon. 2017;1888:1-9. doi:10.1016/j.toxicon.2017.10.016
3. Gimenes I, Caldeira C, Presgrave OAF, Moura WC de, Villas Boas MHS. Assessment of pyrogenic response of lipoteichoic acid by the monocyte activation test and the rabbit pyrogen test. Regul Toxicol Pharmacol. 2015;73(1):356-360. doi:10.1016/j.yrtph.2015.07.025
4. Di Cristo L, Movia D, Bianchi MG, et al. Proinflammatory effects of pyrogenic and precipitated amorphous silica nanoparticles in innate immunity cells. Toxicol Sci. 2016;150(1):40-53. doi:10.1093/toxsci/kfv258
5. Levin, J., Bang FB. A description of cellular coagulation in the limulus. Bull Johns Hopkins Hosp. 1964:115, 337.
6. The Horseshoe Crab. http://www.horseshoecrab.org.
7. Roslansky PF, Novitsky TJ. Sensitivity of Limulus amebocyte lysate (LAL) to LAL-reactive glucans. J Clin Microbiol. 1991;29(11):2477-2483.
8. Fennrich S1, Fischer M, Hartung T, Lexa P, Montag-Lessing T, Sonntag HG, Weigandt M WA. Detection of endotoxins and other pyrogens using human whole blood. Dev Biol Stand. 1999:101:131-9.
9. Hoffmann S, Peterbauer A, Schindler S, et al. International validation of novel pyrogen tests based on human monocytoid cells. J Immunol Methods. 2005;298(1-2):161-173. doi:10.1016/j.jim.2005.01.010
10. Perdomo-Morales R, Pardo-Ruiz Z, Spreitzer I, Lagarto A, Montag T. Monocyte activation test (MAT) reliably detects pyrogens in parenteral formulations of human serum albumin. ALTEX. 2011;28(3):227-235.
11. Hasiwa N, Daneshian M, Bruegger P, et al. Evidence for the Detection of Non-Endotoxin Pyrogens by the Whole Blood Monocyte Activation Test. Vol 30.; 2013. doi:10.14573/altex.2013.2.169
12. Gaines Das RE, Brügger P, Patel M, Mistry Y, Poole S. Monocyte activation test for proinflammatory and pyrogenic contaminants of parenteral drugs: Test design and data analysis. J Immunol Methods. 2004;288(1-2):165-177. doi:10.1016/j.jim.2004.03.002
13. EUROPEAN PHARMACOPOEIA. Monocyte-Activation Test. 92 Chapter 2630. 2010;(1):4299-4304.
14. ICCVAM ICC on the V of AM, NTP NTP, NICEATM IC for the E of ATM. ICCVAM TEST METHOD EVALUATION REPORT Validation Status of Five In Vitro Test Methods Proposed for Assessing Potential Pyrogenicity of Pharmaceuticals and Other Products Interagency Coordinating Committee on the Validation of Alternative Methods. 2008;(08).
15. FDA. Guidance for Industry Pyrogen and Endotoxins Testing: Questions and Answers. 2012;1(June):1-10. doi:papers3://publication/uuid/EB3D7BA7-FBEC-4877-9FD7-883AFD0481B2
16. USP. Validation of Compendial Procedures <1225>. Rockville, MD USP. 2007;USP 41–NF:1-5. https://hmc.usp.org/sites/default/files/documents/HMC/GCs-Pdfs/c1225_1SUSP40.pdf%0Aw.
17. USP 41. <151> Pyrogen Test. 2018;(August):6083-6085.
18. ICH - Q2(R1). Validation of Analytical Procedures: Text and Methodology. 2005;1994(November 1996).