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. 2026 Feb 2;11(6):9138–9144. doi: 10.1021/acsomega.5c08058

Immunocapture-LC-MS/MS Method for Quantification of the Anti-Alzheimer’s Monoclonal Antibody Donanemab in Human and Mice Serum

Yuhang Deng †, Fenghao Xie †, Wenqing Wu †, Di Wang †, Yongjie Guan †, Qitong Zhang †, Fengmei Hu ‡, Haoqin Jiang †,*, Ming Guan †,*
PMCID: PMC12917701  PMID: 41726622

Abstract

Monoclonal antibodies hold significant promise for the treatment of Alzheimer’s disease (AD), and donanemab is the latest therapeutic human IgG1 antibody approved for clinical use. The development and pharmacokinetic evaluation of antibody drugs necessitate accurate quantification. Currently, immunoassays are mainly used by clinical trials to measure those antibody drugs for AD. However, immunoassays often face limitations such as cross-reactivity, insufficient specificity, and poor interlaboratory comparability. Therefore, we developed a robust liquid chromatography–tandem mass spectrometry (LC–MS/MS) method incorporating immunoaffinity enrichment for serum donanemab. This LC–MS/MS method can effectively distinguish donanemab from endogenous immunoglobulins in serum and have sufficient sensitivity. This developed LC–MS/MS method involved: (1) immunoaffinity capture and enrichment of serum donanemab and its internal standard using protein G-conjugated magnetic beads; (2) tryptic digestion of the purified antibodies; and (3) targeted quantification of unique signature peptides via LC–MS/MS for quantification. In conclusion, this method demonstrated acceptable performance, including a lower limit of quantification of 0.1 μg/mL, satisfactory precision (total CV < 8%), high accuracy (95–100% recovery), and a wide linear range (0.2–200 μg/mL), and is the first reported LC–MS/MS method for donanemab offering an alternative for therapeutic antibody monitoring of monoclonal antibody drugs.

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Introduction

According to Alzheimer’s Disease International (2018), the global prevalence of dementia was estimated at approximately 50 million cases and is projected to triple by 2050. Notably, Alzheimer’s disease (AD) accounts for the majority of these dementia cases and once AD-related dementia is diagnosed, the reported median survival period is only 6.2 years (95% CI: 6.0–6.5 years). , Therefore, given the high prevalence, mortality rate, and substantial socioeconomic burden, AD is emerging as one of the most costly and debilitating diseases of the 21st century. However, currently, treatment for AD remains limited due to the incomplete understanding of its pathological mechanisms. And most available treatments provide only short-term symptomatic relief without halting disease progression.

Notably, monoclonal antibodies (mAbs) targeting amyloid β (Aβ) oligomers (soluble aggregates) and plaques (insoluble extracellular fibrillar aggregates) show increasing therapeutic potential in AD, especially in modifying amyloid-β pathology. − Donanemab is a humanized IgG1 mAb that targets N 3pG-modified Aβ, which has demonstrated significant efficacy in reducing the neuropathological hallmarks of ADcerebral amyloid plaques. Clinical trials have shown that donanemab slows cognitive and functional decline by 35% in patients with early stage AD, with even greater benefits observed in those with low to intermediate tau pathology. , These findings position donanemab as one of the most promising disease-modifying therapies for AD. And in 2023, it received accelerated approval from the U.S. FDA, becoming the second commercially available mAbs treatmentafter lecanemabto directly target AD’s underlying amyloid pathology.

Notably, despite FDA approval for Alzheimer’s treatment, donanemab’s risk–benefit profile remains controversial. − It did show modest clinical efficacy, but significant safety concerns persist and no studies to date have demonstrated improvements in other meaningful outcomes, such as reduced functional dependence, delayed nursing home placement, decreased caregiver burden, or mitigation of behavioral symptoms. Meanwhile, the inherent complexity of ADsuch as variability in tau pathology burden, disease stage, and individual risk of cognitive/functional decline or adverse eventsposes significant challenges in predicting treatment responses across different patient subgroups. − Therefore, more comprehensive studies incorporating real-world evidence beyond controlled clinical trials are urgently needed. Such studies should include broader and more representative patient cohorts to better evaluate the real-world effectiveness and safety of the donanemab treatment. Therefore, reliable quantification of the donanemab concentrations is required to support these studies. Meanwhile, as a commercial treatment for AD, measuring donanemab levels is also necessary in specific clinical scenarios, such as suspected hypersensitivity reactions, a suboptimal therapeutic response, or the development of potential neutralizing antibodies.

Notably, conventional measurement of mAbs, including donanemab, has predominantly relied on immunoassays such as the enzyme-linked immunosorbent assay (ELISA). , However, the variability in sensitivity and specificity among different ELISA kits often leads to inconsistent and noncomparable results. For example, the antibodies used in ELISA may lack sufficient specificity and bind nonspecifically to endogenous serum IgG instead of the target monoclonal antibodies. Additionally, developing customized antibody reagents for specific mAbs is time-consuming and expensive. Currently, different studies often employed custom ELISA platforms with varying antibodies, calibration standards, and detection reagents for donanemab measurement. As a result, the performance characteristics are not directly comparable across publications, and there is no widely accepted standardized ELISA method for donanemab. Therefore, it is crucial to develop new methods to overcome these limitations. Liquid chromatography–tandem mass spectrometry (LC–MS/MS) provides high analytical specificity and selectivity, which are particularly advantageous for therapeutic antibody quantification. When coupled with immunocapture, this approach enables the effective enrichment of target antibodies prior to analysis. Protein G magnetic beads provide a cost-effective and broadly applicable approach for IgG capture, offering sufficient specificity for humanized IgG1 antibodies, particularly given the already high selectivity of LC–MS/MS detection. −

In this study, we successfully developed an immunocapture-LC–MS/MS quantification method to quantify donanemab in serum. Briefly, donanemab was selectively captured and purified from serum using protein G magnetic beads, followed by rapid tryptic digestion to generate unique peptide fragments for LC–MS/MS analysis. And cetuximab mAbs was used as an internal standard throughout the whole quantification process to ensure analytical accuracy. To our knowledge, this is the first reported LC–MS/MS method for quantifying donanemab in biological matrices. The developed method demonstrates an excellent accuracy (88.4–114.3% recovery) and precision (CV < 7%), with sensitivity (LLOQ = 0.1 μg/mL) meeting clinical requirements, which can be used for both clinical applications and mechanistic studies of donanemab.

Methods and Materials

Method Development

Chemicals and Equipment

Donanemab injection (Kisunla, 17.5 mg/mL, 20 mL) was purchased from Eli Lilly & Co. (Indianapolis, IN, USA). The internal standard (IS), cetuximab solution for infusion (5 mg/mL, 20 mL), was obtained from Merck KGaA (Darmstadt, Germany). LC–MS grade acetonitrile, methanol, and Tween-20 were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Formic acid and bovine serum albumin (BSA, >98%) were purchased from Sigma-Aldrich (Darmstadt, Germany). Low protein-binding Eppendorf tubes were obtained from Eppendorf (Hamburg, Germany). Ammonium bicarbonate (NH4HCO3) was purchased from MedChemExpress (Shanghai, China). Phosphate-buffered saline (PBS) was obtained from Cytiva (Massachusetts, USA), and recombinant trypsin was purchased from Solarbio (Beijing, China). Protein G Mag Sepharose Xtra beads were purchased from BEAVER (Suzhou, China). Ultrapure deionized water (≥18.2 MΩ·cm) was prepared using a Milli-Q water purification system (Millipore, Billerica, MA, USA). LC–MS/MS analysis was performed on a 6500 Plus Triple Quadrupole Mass Spectrometer (AB Sciex, Framingham, MA, USA) coupled to a Jasper HPLC system.

Preparation of Calibration Curve and Quality Control

The commercial donanemab injection (17.5 mg/mL) was used as the stock standard solution to prepare 6-point calibration curve with concentrations spanning the expected therapeutic range of donanemab (0.2–200 μg/mL). The highest calibration standard (S6 200 μg/mL) was prepared by spiking 20 μL of stock solution into 865 μL of drug-free serum. The remaining five calibration standards, S1 (0.2 μg/mL), S2 (0.4 μg/mL), S3 (2 μg/mL), S4 (10 μg/mL), and S5 (50 μg/mL), were prepared by serial dilution of S6 using drug-free serum. To ensure homogeneity, each dilution step involved vortexing for 30 s followed by centrifugation at 10,000g for 5 min. Similarly, the cetuximab IS stocking solution (1 mg/mL) was prepared by diluting the commercial cetuximab injection (20 mg/mL) with 0.1% BSA PBS. The IS working solution of 100 μg/mL was subsequently prepared by diluting 100 μL of the stock solution with 900 μL of drug-free serum. Standard stock solution and IS stock solutions were stored at 4 °C and −20 °C, respectively, while their working solutions were freshly prepared before each use.

Sample Preparation

Immunoaffinity extraction and enzymatic digestion was used in this study: (1) for the immunoaffinity extraction, briefly, 40 μL of protein G bead suspension (20% w/v) was first washed twice with 500 μL of PBS and then resuspended in 250 μL of PBS containing 0.05% (v/v) Tween-20. Subsequently, 20.0 μL of the calibrator or serum sample and 10.0 μL of the IS working solution were added to the bead suspension. The mixture was incubated at room temperature for 1 h with constant mixing on a roller mixer (80 rpm) to promote antibody–antigen binding. After that, the bead-antibody complexes were washed sequentially with (i) 500 μL of PBS containing 0.1% (w/v) BSA (twice) and (ii) 500 μL of deionized water (twice). Bound antibodies were then eluted by adding 100 μL of 0.25% (v/v) aqueous formic acid followed by vigorous vortexing (2000 rpm, 10 min).

For tryptic digestion, 90 μL of eluate was mixed with 17 μL of 1 M NH4HCO3 aqueous solution prior to thermal denaturation (95 °C, 30 min, 600 rpm). Following cooling to room temperature, proteolytic digestion was initiated by adding 15 μL of sequencing-grade trypsin (0.5 μg/μL), and the mixture was incubated at 37 °C for 2 h with continuous shaking (600 rpm). The digestion was quenched by adding 23.3 μL of 10% (v/v) formic acid in 50% (v/v) acetonitrile/water. All the above operations were carried out using low protein-binding Eppendorf tubes. After vortex mixing (2000 rpm, 1 min) and centrifugation (15,000 rpm, 5 min, 4 °C), the supernatant was transferred for LC–MS/MS analysis.

LC–MS/MS Analysis

LC–MS/MS analysis was performed on a 6500 Plus Triple Quadrupole Mass Spectrometer (AB Sciex, Framingham, MA, USA) coupled to a Jasper HPLC system. Chromatography was performed after injection of 5 μL of eluate into an XSeleted Peptide CSH C18 column (3.5 μm, 100 mm × 2.1 mm, Waters), maintained at 40 °C. Automatic sampler temperature was set at 7 °C. Mobile phase A consisted of 0.1% formic acid in water, and mobile phase B contained 0.1% formic acid in acetonitrile. The initial mobile phase composition was 90:10 (v/v) A:B at a flow rate of 0.3 mL/min with the following linear gradient steps: 1 min, 10% B; 7.5 min, 35% B; 8 min, 90% B; 10 min, 90% B; 10.1 min, 10% B; and 12 min, 10% B.

The mass spectrometer was operated in positive electrospray (ESI) mode, with the source temperature of 600 °C and an ion spray voltage of 5500 V. Multiple reaction monitoring (MRM) mode was used to monitor the mass transitions of unique peptides of donanemab and cetuximab. Nitrogen was used as the curtain gas (CUR), nebulizer gas (GS1), auxiliary gas (GS2), and collision gas (CAD), and the pressures of the gases were set at 30, 65, 60, and 8 psi, respectively.

Method Validation

Matrix Effect, Limit of Quantification, and Limit of Detection

Four types of serum samplesnormal, hemolytic, lipemic, and ictericwere used to evaluate potential interference from common abnormal serum conditions, since these four types of serum represent the most common abnormal specimens encountered in clinical practice. First, spiked samples at identical concentrations (10 and 20 μg/mL) were prepared by adding a donanemab stock solution to the four different types of serum. Then, the donanemab concentrations in the spiked serum samples were measured by using the developed LC–MS/MS method. The matrix effect was calculated as the ratio of the donanemab concentration in each abnormal serum (hemolytic, lipemic, or icteric) to that in the healthy, drug-free serum (normal). The limit of quantification (LOQ) and limit of detection (LOD) were determined using spiked serum samples at progressively decreasing concentrations. LOQ was defined as the lowest concentration with a signal-to-noise (S/N) ratio ≥10, accuracy within 85–115%, and precision (CV) ≤ 10%. LOD was defined as the lowest concentration with an S/N ratio ≥3. Because it is technically difficult to prepare samples that generate exact S/N values of 10 or 3, the LOQ and LOD were estimated by extrapolation from experimentally measured low-concentration samples exhibiting stable S/N ratios and acceptable precision.

Trueness, Analytical Recovery

A spiking recovery experiment was conducted using both human and mouse serum to assess the trueness of the LC–MS/MS method. Drug-free serum was spiked with standard solutions to prepare four concentration levels (2, 20, 50, and 100 μg/mL). Each level was measured in triplicate across three analytical runs over five consecutive days. The mean results from the three runs were used to calculate the average recovery at each concentration.

Imprecision Evaluation

The imprecision of the LC–MS/MS method was evaluated following the CLSI EP15-A3 guidelines using both human and mice serum. Four serum pools containing donanemab at different concentrations (2, 20, 50, and 100 μg/mL) were analyzed in triplicate across three runs over five consecutive days. Within-run, between-run, and total imprecision were calculated by using analysis of variance (ANOVA).

Real-World Sample Measurements

The applicability of the newly developed LC–MS/MS method was further assessed by using actual biological samples collected from AD model mice and AD patients. A serum sample was collected from an AD patient treated with donanemab. And a total of ten APP/PS1-dE9 mice were injected with donanemab via tail-vein injection at a human-equivalent dose (10 mg/kg). Serum samples were collected at multiple postdose time points (4, 12, 24, 48, 72, 96, 120, 144, 168, and 192 h), and each sample was analyzed in triplicate using the established LC–MS/MS assay. And a noncompartmental pharmacokinetic (PK) analysis was performed to characterize the systemic exposure and disposition of donanemab in the AD model mice. PK parameters were estimated using a noncompartmental analysis (NCA) approach. The area under the serum concentration–time curve from time zero to the last measurable concentration (AUC0–t) was calculated using the linear trapezoidal method. The terminal elimination rate constant (λz) was determined by linear regression of the natural logarithm of serum concentrations versus time over the terminal phase. The terminal half-life (t 1/2) was calculated as ln (2)/λz. The AUC extrapolated to infinity (AUC0–∞) was calculated as AUC0–t plus C_last/λz. Total systemic clearance (CL_tot) was estimated as Dose/AUC0–∞, and the apparent volume of distribution associated with the terminal phase (V_z) was calculated as CL_tot/λz. All PK parameters were expressed on a per kilogram of body weight basis.

The human serum used in this study was collected from the left-over serum samples in clinical laboratory. All samples were anonymized and coded, and the study was approved by the institutional Ethics Committee (KY2025–1365). Sample collection was performed in the morning (8:00–10:00 am). Peripheral venous blood was drawn by venipuncture from the median cubital vein into serum separator tubes, and 1 mL of leftover serum was collected for this study after clinical routine tests.

Statistical Analysis

Calibration curves (weighted 1/x 2) plotting the concentration against analyte/IS area ratios were established by linear regression. Sample concentrations were calculated by interpolation from these curves. Data were analyzed using Microsoft Excel 2016 (Microsoft, Redmond, WA, USA) and Analyst 1.7 software (AB Sciex, USA).

Results and Discussion

Unique Peptides of Donanemab and Cetuximab

For standard protein analysis using triple quadrupole MS, identification of a unique peptide from the target proteins was necessary. These unique peptides serve as quantifiable surrogates that specifically distinguish the protein of interest from other homologous or structurally similar proteins within the sample matrix. In this study, unique peptides of donanemab and IS (cetuximab) were identified via in silico tryptic digestion using Skyline (MacCoss Lab, University of Washington). The light and heavy chain amino acid sequences of both antibodies were imported, with parameters including digestion enzyme (trypsin), background proteome library (human IgG), peptide length (6–25 residues), and charge states (2+ to 3+) defined. All potential surrogate peptides (5–25 amino acids) were subjected to a protein BLAST search using the National Center for Biotechnology Information database (Bethesda, MD) to identify unique peptides. Ultimately, six unique candidate peptides were selected. To evaluate optimal peptides for quantification, the commercial donanemab injection was first digested and the resulting candidate peptides was then analyzed by LC–MS/MS, monitoring ion responses of 18 transition pairs derived from two charge states ([M + 2H]2+ and [M + 3H]3+), as suggested by Skyline software. Collision energy (CE) for each transition was optimized accordingly. At last, based on sensitivity and selectivity, we selected three key transition sets from three unique peptides for donanemab and one key transition set from a unique peptide for the cetuximab IS. The detailed information for the quantitative and qualitative peptide segments is presented in Table and the typical chromatograms for donanemab quantitative peptide are shown in Figure . Data are analyzed using Analyst 1.7 software (AB Sciex, USA).

1. Selected Specific Peptide Sequences and Parent-Fragment Ion Pairs .

name Q1 (2+) Q3 DP (V) CE (V) peptide amino acid sequence
donanemab 507.3 687.4 80 23.9 YYINWVR (quantitative)
1114.5 660.3 80 53.6 QAPGQGLEWMGWINPGSGNTK (qualitative)
429.7 630.3 80 20.1 LDSGVPDR (qualitative)
cetuximab 597.2 652 80 15.0 ASQSIGTNIHWYQQR
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a

Note: DP, declustering potential and CE, collision energy.

1.

1

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Chromatograms illustrating the selected quantitative peptides of donanemab. (A) Drug-free human serum (double blank). (B) Internal standard in drug-free human serum. (C) 0.2 μg/mL in drug-free human serum. (D) The one AD patient serum (24.6 μg/mL) chromatogram.

Reproducibility and Linearity of the Standard Curve

Bioanalytical methods that employ immunoaffinity enrichment coupled with LC–MS/MS have been routinely used to analyze protein biomarkers and therapeutic antibodies, offering enhanced efficiency, specificity, and sensitivity. For donanemab, Protein A/G-, antihuman IgG antibody-, or antihuman IgG1 antibody-coated magnetic beads are available. Although antihuman IgG and IgG1 antibodies theoretically offer higher specificity, considering its higher production cost compared to Protein G-coated magnetic beads, and given that the already high inherent specificity of LC–MS/MS detection, we ultimately selected Protein G-coated magnetic beads for donanemab immunocapture in this study, which was also used in many similar studies. −

To test the linearity and reproducibility of this LC–MS/MS method, the calibration curve ranging from 0.2 to 200 μg/mL was measured in three runs on five consecutive days, and the mean accuracy at each day was calculated. The results demonstrated excellent performance, with all calibrators exhibiting accuracy between 95% and 105% and correlation coefficients (r 2) above 0.998 (Table ). These findings confirm the method’s high reproducibility and linearity across the validated concentration range.

2. The Reproducibility and Linearity of the Standard Curve.

accuracy (%) day 1 day 2 day 3 day 4 day 5 mean
S1 (0.2 μg/mL) 100.83 100.67 101.17 99.17 98.67 100.10
S2 (0.4 μg/mL) 99.17 98.50 98.08 102.25 103.08 100.22
S3 (2 μg/mL) 95.23 101.95 97.72 97.60 97.98 98.10
S4 (10 μg/mL) 111.98 92.52 100.08 99.92 93.08 99.52
S5 (50 μg/mL) 95.57 110.97 97.51 95.54 110.57 102.03
S6 (200 μg/mL) 97.39 95.39 105.44 105.57 96.52 100.06
r 2 0.9999 0.9982 0.9996 0.9994 0.9986 0.9991
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Injection of 5 μL of the prepared human serum sample (S1, 0.2 μg/mL) produced a mean S/N ratio of 19.8:1 (Figure C), with a coefficient of variation (CV) of 6.5% (N = 15). Accordingly, the LOQ and LOD of the LC–MS/MS method were calculated as 0.1 and 0.03 μg/mL based on the S/N relationship, respectively.

No significant interference was observed in hemolytic or icteric human serum samples, with measured concentrations showing a negligible average change of −0.6%. Although a positive bias was observed in lipemic samples, with concentrations increasing by an average of 11.2%, this deviation remained within the acceptance criteria. These results indicate that while lipids may introduce a slight upward shift in measured concentrations, the overall impact on donanemab quantification is acceptable.

Analytical Recovery and Imprecision

The analytical recoveries for the LC–MS/MS method in human and mouse serum are listed in Table . The mean analytical recoveries and CVs were 93.1% ± 4.1%, 99.8% ± 6.5%, 103.4% ± 6.6%, and 101.5% ± 4.2% for spiked serum samples with 2, 20, 50, and 100 μg/mL, respectively (Table ). The within-run, between-run, and total precision of the LC–MS/MS method were in the ranges of 2.83–4.50%, 2.71–5.17%, and 4.18–6.71%, respectively, at four levels (2–100 μg/mL, Table ). Overall, these findings indicate that the performance of the developed LC–MS/MS method meets established analytical criteria and is comparable to that reported for existing ELISA methods (Table ).

3. Analytical Percentage Recovery and the Precision LC–MS/MS Method for Both Mice and Human Serum .

recovery (%) low 2 μg/mL middle 1 (20 μg/mL) middle 2 (50 μg/mL) high (100 μg/mL)
mean (mice) 93.1 (96.8) 99.8 (97.2) 103.4 (98.4) 101.5 (94.9)
range 88.4–103.1 91.0–111.5 92.2–114.3 94.0–107.5
precision (%)        
within-run (mice) 3.62 (3.44) 4.50 (2.84) 2.83 (3.46) 3.19 (2.72)
between-run (mice) 2.75 (2.23) 4.97 (3.46) 5.17 (3.89) 2.71 (3.24)
total (mice) 4.54 (3.90) 5.17 (3.82) 5.90 (4.23) 4.18 (4.62)
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a

Note: Values in parentheses represent results obtained from mice serum.

4. The Comparison between Our LC–MS/MS and Published ELISA Method for Donanemab.

method LOQ detection range accuracy precision
ELISA 1 0.1 μg/mL 0.1–5 μg/mL –1.0%–5.8% ≤17.2%
ELISA 2 0.2 μg/mL 0.2–5 μg/mL –1.5–7.0% 4.0–9.7%
LC–MS/MS 0.1 μg/mL 0.2–200 μg/mL 93–101% (recovery %) 2.7–6.7%
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Method Application to Real Biological Samples

The developed LC–MS/MS method was further applied to real biological samples from an AD mouse model to evaluate its practical applicability. As shown in Figure , donanemab in mouse and AD patient serum samples was clearly detected. And mice serum collected at different time points exhibited a distinct time-dependent decline, which was consistent with the known pharmacokinetic characteristics of the drug in AD patients. And the CV% for each sample tested three times was less than 5%. On the other hand, the estimated AUC0–t was 11,432.2 μg·h/mL, and the AUC0–∞ was 11,966.5 μg·h/mL, with an extrapolated fraction of less than 5%, indicating adequate characterization of the terminal phase. The terminal elimination rate constant (λz) was 0.02096 h–1, corresponding to a terminal half-life (t 1/2) of 33.1 h. Total systemic clearance (CL_tot) was estimated to be 0.000836 L·h–1·kg–1, and the apparent volume of distribution (V_z) was 0.0399 L·kg–1.

2.

2

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The mean donanemab concentration in serum from AD Mice. Note: the exact duration of treatment for the AD patient was not available.

It should be noted that donanemab has only recently become available in China and remains extremely expensive (approximately $28,000 US dollar per treatment course). As a result, the number of patients currently receiving this therapy is very limited, and we obtained only one clinical serum sample at this stage. To overcome this limitation and to validate the method using real biological matrices, we therefore conducted a simple PK investigation in the AD model. Notably, this PK study was not intended to fully characterize donanemab pharmacokinetics, which has been well established, but rather to demonstrate the applicability of the LC–MS/MS method in real biological samples.

Pharmacokinetic Studies in Mice

Sample Preparation Interpretation

Tween-20 was used to enhance antigen–antibody binding efficiency during immunocapture, which can promote complex formation by facilitating molecular interactions and stabilizing donanemab in the mixed solution. Additionally, it effectively blocked unoccupied binding sites, thereby reducing the level of nonspecific binding.

When performing absolute quantification of therapeutic antibodies using LC–MS/MS, fully isotopically labeled antibodies are widely regarded as the gold standard because they can correct for recovery and matrix effects across the entire sample-preparation workflow. However, the synthesis of isotopically labeled full-length antibodies is technically demanding and cost prohibitive, which limits their routine use in many laboratories. As a result, numerous published LC–MS/MS methods for monoclonal antibody quantification have instead employed isotope-labeled signature peptides as internal standards. − These peptide standards provide excellent chromatographic and mass-spectrometric matching with the target analyte, but they do not participate in the digestion process and therefore cannot compensate for the variability introduced during enzymatic digestion or immunocapture. In light of these considerations, we employed analogue mAbs cetuximab as an IS due to its structural similarity to donanemab, as both are humanized IgG1 antibodies. And this “analogue protein” method was also used by many LC–MS/MS studies for mAbs quantification. ,, In this study, cetuximab IS was added at the beginning of the sample preparation process and underwent the entire immunocapture and enzymatic digestion procedure alongside donanemab, which can better compensate for errors arising during sample preparation, particularly during the digestion step. However, its ability to correct chromatographic separation, ionization variability, and matrix effects is somewhat inferior to that of isotope-labeled peptides. Despite these limitations, our validation results demonstrated that the method met all acceptance criteria for accuracy, precision, and matrix effect, supporting the adequacy of the analogue-protein internal standard for donanemab quantification in this study.

Conclusion

In conclusion, we have developed the first reported LC–MS/MS method for quantifying donanemab in serum. This novel approach demonstrated excellent accuracy, high sensitivity, and a rapid turnaround time. With the growing number of mAb therapeutics entering clinical development and practice, the technical framework established here may serve as a model for developing assays for other therapeutic antibodies, especially those with similar IgG1 structures.

Acknowledgments

The authors would like to thank the financial support from National Natural Science Foundation of China (Grant No. 82502779) and Shanghai Innovative Medical Device Demonstration Project (Grant No. 23SHS06200-01). The graphical abstract was created with BioRender.com.

§.

Y.D and F.X. contributed equally to this work. Y.H.D. performed the experiments, analyzed the results, wrote the main manuscript text, and edited the final draft. H.Q.J and M.G designed the study, supervised the work, interpreted the results, wrote the main manuscript text, and revised and edited the final draft. F.H.X., F.M.H, Y.J.G, and Q.T.Z did the experiment. W.Q.W. and D.W. consulted on the neurological part, revised, edited, and approved the final version. All authors have read and agreed to the published version of the manuscript.

The authors declare no competing financial interest.

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