CASE STUDY

FUNCTIONAL ASSAYS

SOLUBLE BIOMARKERS

DRUG-TARGET INTERACTIONS

IMMUNOPHENOTYPING

QUALIFICATION OF AN ULTRASENSITIVE IMMUNO-ASSAY USING CLINICAL SERUM AND PLASMA SAMPLESWHAT DO ALL THERAPIES UNDER DEVELOPMENT HAVE IN COMMON?Whether in preclinical or clinical stage, they all need robust and reliable biomarker monitoring tools to track and characterize their biological effect. Active Biomarkers is a global leader in bioanalytical services, offering expert biomarker assays to advance therapies from discovery to clinical stages. Our company combines tens of years of scientific and technical experience, the highest quality environment, and the largest panel of immunoassay technologies in Europe.

Conventional ELISA (Enzyme-Linked Immuno-Sorbant Assay), although still largely used, is limited in terms of sensitivity and the number of analytes that can be analyzed. The emergence of ultra-sensitive technologies, such as the single-molecule array (SIMOATM) technology from Quanterix, offers the possibility to detect and quantify several biomarkers with up to a 100-fold gain in sensitivity. These technologies open the path to the monitoring of previously undetectable circulating biomarkers in multiple pathological processes, notably in the field of neurobiology.Neuroinflammatory and neurodegenerative disorders, whatever their origin, lead to the accumulation of specific neuronal proteins in cerebrospinal fluid and blood. Neurofilament-Light chain (Nf-L) is one of the 3 subunits, with intermediate (Nf-M) and heavy (Nf-H) chains, constituting the main components of intermediate filaments in neurons. Neurofilaments are involved in axonal growth and maintenance, as well as electric transmission in the nervous system1. While displaying low turnover in physiological non pathological conditions, neurofilaments rise up in cere-brospinal fluid and blood during neuroaxonal injury2. Nf-L is a

blood biomarker of interest in the differential diagnosis of some neurodegenerative disorders such as Amyotrophic Lateral Sclerosis3, Parkinsonian disorders4, but also of prognostic value for progression and response to therapy in Multiple Sclerosis (MS)5. Moreover, it is linked to disease activity in MS, Alzheimer’s, and Huntington’s6. In these neurological pathologies, there is a need for targeting early stages of disease and monitoring thera-peutic intervention in easily accessible biological fluids. Blood Nf-L is a valuable biomarker not only in neurological disorders7 but also in many other indications including cancer8, infectious diseases9-10), autoimmune syndromes11, or sport- related concussions12. It is then crucial that assays for monitoring this biomarker are robust, accurate, and reproducible, in other terms, reliable. Our team has qualified the NF-Light Advantage Kit from Quanterix (ref: 102258) on the SIMOATM HD1 system, evaluating its per-formance in terms of dynamic range, precision, parallelism and selectivity, lot-to-lot consistency and stability for quantifying Nf-L in both plasma and serum matrices.

INTRODUCTION

QUALIFICATION OF AN ULTRASENSITIVE IMMUNO-ASSAY USING CLINICAL SERUM AND PLASMA SAMPLES

Our team has qualified the NF-Light Advantage Kit from Quanterix (ref: 102258) on the SIMOATM HD-1 system, evaluating its performance in terms of dynamic range, precision, parallelism and selectivity, lot-to-lot consistency and stability for quantifying Nf-L in both plasma and serum matrices.

NF-Light Advantage Kit has been qualified with paired serum and EDTA-plasma samples from 8 healthy donors (HD) aged from 24 to 68 (3 > 60 years-old), and with 8 serum samples from MS patients with secondary progressive MS (SPMS) or with relapsing-remitting MS (RRMS) (Figure 8B).

After demonstrating that calibration curve, prepared according to manufacturer’s instructions, met precision (CV≤25%) and recovery (70-130%) acceptance criteria, we were able to extend the dynamic range down to 0.34 pg/mL (Figure 1).

While recombinant Nf-L protein was poorly recovered when spiked in blood matrices (Figure 2A), likely due to differences between recombinant and endogenous protein, parallelism could be demonstrated over 3 to 4 serial dilutions in serum or plasma samples from respectively healthy or MS individuals (Figure 3) and the minimal required dilution (MRD) of 1:4 was confirmed in blood matrix. In addition, selectivity was demons-trated by fractionally admixing high Nf-L with low Nf-L serum samples (Figure 2B). As a consequence, validation parameters, except accuracy, were evaluated on endogenous Nf-L.

Figure 1 - Calibration curve of Nf-L kit

Figure 2A : Recovery of recombinant Nf-L, spiked at 180 pg/mL and measured at MRD (1:4) ranges from 34 to 44% in serum and 33 to 39% in plasma, largely below the acceptance criteria (70-130 %). Recovery improves with dilutions (1:8 and 1:16), suggesting interfe-rence between reference material and blood matrix.

Figure 2B : Samples with elevated endogenous Nf-L (MS4 and MS8) were admixed with samples with low endogenous Nf-L (MS2 and MS6) at different ratios. For each ratio, measured values (blue shaded bars with ) are compared to theoretical/expected levels and are highly correlated with a coefficient of correlation r2=0.9954.

Figure 2 - Selectivity of the method tested on recombinant (Fig.2A) and endogenous Nf-L (Fig. 2B)

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CASE STUDY

Nf-L was measured in plasma and serum samples from 2 healthy do-nors in diluted matrix (1:6 to 1:16) to reach 0,5-0,7 pg/mL. Each sample was measured in 10 replicates. Individual values (blue symbols) as well as mean (black horizontal bar) are shown for each donor (1&6) in serum or plasma.

Intra- and inter-run precision were evaluated in plasma and serum from 4 healthy individuals (Donor 1 (D1), 3, 4 & 5). Coefficient of variation (CV), calculated according to “SFSTP commission reports on the harmoniza-tion of approaches in the validation of quantitative analytical procedure13” is below 25 % for each run7-10.

Serum & plasma from 5 healthy individuals <60 (HD1-5) and 3 >60 (HD6-8) and serum from 8 patients with multiple sclerosis (MS) (Neurobiotec, CRB Hospices Civils de Lyon, France) tested at MRD (1:4), MRD/2 (1:8), MRD/4 (1:16) and MRD/8 (1:32).The kit has shown a good parallelism on 3 dilutions in HD, and 4 dilutions in MS samples.

Figure 4 - Precision of Nf-L kit in blood matrix Figure 5 - Estimated LLOQ below 0,6 pg/mL

Figure 3 - Parallelism of endogenous Nf-L This method displayed good precision in both plasma and serum, when tested at the MRD, with coefficient of variation (CV) below 20% for measured levels in the range of 1-2 pg/mL (Figure 4). Lower Limit of Quantification (LLOQ) was challenged in diluted blood matrix on 10 replicates (Figure 5).

Nf-L levels were measured down to 0,55 and 0.63 pg/mL respectively in plasma (at 1:8 dilution) and serum (1:16 dilution) from HD.

In addition, Nf-L in blood matrices was shown to be stable both upon up to 3 freeze-thaw cycles as well as over a 9-month period at -80°C (Figure 6).

Finally, 2 distinct lots of the kit were evaluated on 3 samples from healthy donors and 8 samples from MS patients, showing good consistency with a recovery ranging from 77 to 91% (Figure 7).

This kit allowed us to quantify endogenous levels of Nf-L, not only in MS samples, but also in blood samples from healthy donors (Figure 8).

According to results reported by others, Nf-L levels tended to be higher in the serum of MS patients compared to healthy indivi-duals, and in individuals over 60 years-old.

Inter-run variabilityIntra-run variability

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(1) Yuan, A., Rao, M., Veeranna and Nixon, R. (2012). Neurofilaments at a glance. Journal of Cell Science, 125(14), pp.3257-3263.

(2) Petzold, A., Keir, G., Warren, J., Fox, N. and Rossor, M. (2007). A Syste-matic Review and Meta-Analysis of CSF Neurofilament Protein Levels as Biomarkers in Dementia. Neurodegenerative Diseases, 4(2-3), pp.185-194.

(3) Xu, Z., Henderson, R., David, M. and McCombe, P. (2016). Neurofila-ments as Biomarkers for Amyotrophic Lateral Sclerosis: A Systematic Re-view and Meta-Analysis. PLOS ONE, 11(10), p.e0164625.

(4) Hansson, O., Janelidze, S., Hall, S., Magdalinou, N., Lees, A., Andreasson, U., Norgren, N., Linder, J., Forsgren, L., Constantinescu, R., Zetterberg, H. and Blennow, K. (2017). Blood-based Nf-L. Neurology, 88(10), pp.930-937.

(5) Kuhle, J., Kropshofer, H., Hearing, D.A., Kundu, U., Meinert, R., Barro, C., Dahlke, F., Tomic, D., Leppert, D., Kappos, L. (2019). Blood neurofila-ment light as a biomarker of MS disease activity and treatment response. Neurology, 92:e1-e9.

(6) Byrne, L., Rodrigues, F., Blennow, K., Durr, A., Leavitt, B., Roos, R., Sca-hill, R., Tabrizi, S., Zetterberg, H., Langbehn, D. and Wild, E. (2017). Neurofi-lament light protein in blood as a potential biomarker of neurodegeneration in Huntington’s disease: a retrospective cohort analysis. The Lancet Neu-rology, 16(8), pp.601-609.

(7) Barro, C., Chitnis, T., Weiner, H. L. (2020). Blood neurofilament light: a critical review of its application to neurologic disease. Annals of Clinical and Translational Neurology, doi: 10.1002/acn3.51234.

(8) Winther-Larsen, A., Hviid, C.V. B., Meldgaard P., Sorensen B. S., Sandfeld-Paulsen B. (2020). Neurofilament Light Chain as A Biomarker for Brain Metastases. Cancers, 12:2852.

(9) Tyrberg,T., Nilsson, S., Blennow, K., Zetterberg, H., Grahn, A. (2020). Serum and cerebrospinal fluid neurofilament light chain in patients with central nervous system infections caused by varicella-zoster virus. Journal of NeuroVirology 26:719–726.

(10) Cooper, J., Stukas, S., Hoiland, R. L., Fergusson, N. A., Thiara, S., Fos-ter, D., Mitra, A., Stoessl, J.A., Panenka, W.J., Sekhon, M.S., Wellington, C.L. Quantification of Neurological Blood-Based Biomarkers in Critically Ill Patients With Coronavirus Disease 2019. (2020). Critical Care Explorations; 2:e0238.

(11) Tjensvoll, A.B., Lauvsnes, M.B., Zetterberg, H., Kvaløy, J.T., Kvivik, I., Maroni, S.S., Greve, O. J., Beyer, M.K., Hirohata, S., Putterman, C., Alves, G., Harboe, E., Blennow,K., Gøransson, L.G., Omdal, R. (2020). Neurofilament light is a biomarker of brain involvement in lupus and primary Sjögren’s syndrome. Journal of Neurology, doi: 10.1007/s00415-020-10290-y

(12) McCrea M., Broglio, S.P., McAllister, T.W., Gill, J., Giza, C.C., Huber, D.L., Harezlak, J., Cameron, K.L., Houston, M.N., McGinty, G., Jackson, J. C., Guskiewicz, K., Mihalik, J., Brooks, M.A., Duma, S., Rowson, S., Nel-son, L.D., Pasquina, P., Meier, T.B., and the CARE Consortium Investigators (2020). Association of Blood Biomarkers With Acute Sport-Related Concus-sion in Collegiate Athletes-Findings From the NCAA and Department of De-fense CARE Consortium. JAMA Network Open.,3(1):e1919771

(13) Hubert, P., et al., Harmonization of strategies for the validation of quantitative analytical procedures: a SFSTP proposal part IV. Examples of application. J Pharm Biomed Anal, 2008. 48(3): p. 760-71.Hubert, P., et al., Harmonization of strategies for the validation of quanti-tative analytical procedures. A SFSTP proposal--part III. J Pharm Biomed Anal, 2007. 45(1): p. 82-96.Hubert, P., et al., Harmonization of strategies for the validation of quantita-tive analytical procedures. A SFSTP proposal--part II. J Pharm Biomed Anal, 2007. 45(1): p. 70-81.Hubert, P., et al., Harmonization of strategies for the validation of quantita-tive analytical procedures. A SFSTP proposal--Part I. J Pharm Biomed Anal, 2004. 36(3): p. 579-86.

Despite poor recovery of spiked recombinant protein, the assay showed very good sensitivity and precision allowing to measure levels <1 pg/mL of Nf-L in blood matrices (e.g. in healthy donors <60) with good lot-to-lot consistency. Overall this method showed performance characteristics suitable for the monitoring of Nf-L in blood in the context of therapeutic intervention in neurological disorders.

CONCLUSION

REFERENCES

Serum samples from HD (n=3) and MS (n=8) individuals were tested in parallel with lots #1 and #2 of the Nf-L kit at MRD. Results show a good correlation (r2=0.9998) between both lots of the kit.

Figure 7 - Lot to lot bridging

Figure 8A : Nf-L concentration is increased in MS samples. Some of MS patients were under treatment during sampling.

Figure 8B : Description of MS patients in terms of sex, age, disease status and treatment (Yes or No), during sampling. RRMS = Relapsing Remitting MS; SPMS = secondary progressive MS; JCV = JC polyomavirus. Samples were obtained from Neurobiotec biobank, CRB Hospices Civils de Lyon, France.

Experimental plan Samples :

serum from healthy donors (HD) <60 yrs old (n=5)

serum form HD >60 (n=3)

serum from MS patients (n=8, <60)

* Age at sampling ; SPMS secondary progressive;  SPMS‐/SPMS+ : non active/active SPMSMS; RRMS : Relapsing‐Remitting MS 

Tested at MRD

Increased serum NfL in MS patients compared to healthy donors

NfL(pg/mL)

Concentration values (log scale) correspond to measured concentration multiplied by dilution factor

1

10

100

1000

Healthy donors Multiple sclerosis

Age >60 <60 <60Blood matrix SerumSamples (n=) 3 5 8Values (n=) 3 24 8Median 8,9 6,1 20,8Mean 10,1 6,5 33,9Range 7,5‐13,9 4,1‐10,4 5,0‐152,7

ID NFL (pg/mL) Sex Age* Treatment Time since 

onset (years) Stage

MS1 8,3 M 47 Y 20 SPMS+MS2 152,7 F 27 N 6 RRMSMS3 18,3 F 45 Y 7 SPMS‐MS4 5,0 F 27 Y 5 RRMSMS5 23,3 M 19 Y 3 RRMSMS6 30,8 M 53 N 23 SPMS+MS7 24,6 F 38 Y 21 SPMS+MS8 7,8 M 44 Y 13 SPMS‐, JCV‐

Experimental plan Samples :

serum from healthy donors (HD) <60 yrs old (n=5)

serum form HD >60 (n=3)

serum from MS patients (n=8, <60)

* Age at sampling ; SPMS secondary progressive;  SPMS‐/SPMS+ : non active/active SPMSMS; RRMS : Relapsing‐Remitting MS 

Tested at MRD

Increased serum NfL in MS patients compared to healthy donors

NfL(pg/mL)

Concentration values (log scale) correspond to measured concentration multiplied by dilution factor

1

10

100

1000

Healthy donors Multiple sclerosis

Age >60 <60 <60Blood matrix SerumSamples (n=) 3 5 8Values (n=) 3 24 8Median 8,9 6,1 20,8Mean 10,1 6,5 33,9Range 7,5‐13,9 4,1‐10,4 5,0‐152,7

ID NFL (pg/mL) Sex Age* Treatment Time since 

onset (years) Stage

MS1 8,3 M 47 Y 20 SPMS+MS2 152,7 F 27 N 6 RRMSMS3 18,3 F 45 Y 7 SPMS‐MS4 5,0 F 27 Y 5 RRMSMS5 23,3 M 19 Y 3 RRMSMS6 30,8 M 53 N 23 SPMS+MS7 24,6 F 38 Y 21 SPMS+MS8 7,8 M 44 Y 13 SPMS‐, JCV‐

Figure 8 - Comparison of Nf-L levels in serum of healthy patients versus MS patients

Experimental plan Samples :

serum from healthy donors (HD) <60 yrs old (n=5)

serum form HD >60 (n=3)

serum from MS patients (n=8, <60)

* Age at sampling ; SPMS secondary progressive;  SPMS‐/SPMS+ : non active/active SPMSMS; RRMS : Relapsing‐Remitting MS 

Tested at MRD

Increased serum NfL in MS patients compared to healthy donors

NfL(pg/mL)

Concentration values (log scale) correspond to measured concentration multiplied by dilution factor

1

10

100

1000

Healthy donors Multiple sclerosis

Age >60 <60 <60Blood matrix SerumSamples (n=) 3 5 8Values (n=) 3 24 8Median 8,9 6,1 20,8Mean 10,1 6,5 33,9Range 7,5‐13,9 4,1‐10,4 5,0‐152,7

ID NFL (pg/mL) Sex Age* Treatment Time since 

onset (years) Stage

MS1 8,3 M 47 Y 20 SPMS+MS2 152,7 F 27 N 6 RRMSMS3 18,3 F 45 Y 7 SPMS‐MS4 5,0 F 27 Y 5 RRMSMS5 23,3 M 19 Y 3 RRMSMS6 30,8 M 53 N 23 SPMS+MS7 24,6 F 38 Y 21 SPMS+MS8 7,8 M 44 Y 13 SPMS‐, JCV‐

Figure 6 - Nf-L stability

Nf-L stability after over up to 3 freeze/thaw cycles and up to 9 months at -80°C. Recovery between levels measured after 2 or 3F/T cycles or after 9 months storage at -80°C, and levels measured at T0 is shown.

OVER UP TO 3 FREEZE / THAW CYCLES

AND UP TO 9 MONTHS AT -80°C

Age