Schalk Frank B, Guerrieri Davide, Poetzl Johann, van de Merbel Nico C
ICON Bioanalytical Laboratories, Assen, The Netherlands.
Clinical Development Biopharmaceuticals, Hexal (a Sandoz company), Holzkirchen, Germany.
Bioanalysis. 2025 Jan;17(2):87-98. doi: 10.1080/17576180.2024.2441058. Epub 2024 Dec 18.
Commonly, ligand-binding platforms are being used for immunogenicity assessment, but with the recent advent of liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) for protein quantification, this technology has become an alternative for the measurement of anti-drug antibodies (ADAs), when combined with an immunocapture step to extract them out of the biological sample.
The monoclonal antibody adalimumab was immobilized on magnetic beads to isolate ADAs against this drug from serum samples. Multiple repetitions of immunopurification were used to minimize nonspecific binding and improve drug tolerance while maintaining sufficient recovery. A subsequent tryptic digestion released peptides, from which unique peptide sequences, originating from the constant region of seven ADA subclasses, were selected. These were then analyzed by LC-MS/MS against (unextracted) subclass-specific reference standards for semi-quantification.
With two immunocapture and two immunopurification steps, the method simultaneously measures the ADA subclasses IgG1, IgG2, IgG3, IgG4, IgM, IgE and IgA within their relevant ranges, with good repeatability and drug tolerance, and limited interference of endogenous immunoglobulins. The method was successfully applied for the analysis of serum samples of subjects dosed with adalimumab.
Hybrid LBA-LC-MS/MS is a viable platform for measuring ADAs and adds value, especially when ADA isotyping is needed.
通常，配体结合平台用于免疫原性评估，但随着液相色谱-串联质谱联用技术（LC-MS/MS）用于蛋白质定量的出现，当与免疫捕获步骤结合以从生物样品中提取抗药物抗体（ADA）时，该技术已成为测量ADA的一种替代方法。
将单克隆抗体阿达木单抗固定在磁珠上，以从血清样品中分离针对该药物的ADA。多次重复免疫纯化以尽量减少非特异性结合并提高药物耐受性，同时保持足够的回收率。随后的胰蛋白酶消化释放出肽段，从中选择源自七种ADA亚类恒定区的独特肽序列。然后通过LC-MS/MS针对（未提取的）亚类特异性参考标准进行分析以进行半定量。
通过两步免疫捕获和两步免疫纯化步骤，该方法可在相关范围内同时测量ADA亚类IgG1、IgG2、IgG3、IgG4、IgM、IgE和IgA，具有良好的重复性和药物耐受性，且内源性免疫球蛋白的干扰有限。该方法已成功应用于阿达木单抗给药受试者血清样品的分析。
混合LBA-LC-MS/MS是测量ADA的可行平台，尤其在需要ADA分型时具有价值。
