使用富集微球与有限消化相结合的宿主细胞蛋白分析
Summary
提出了一种从药物产品（DP）中富集宿主细胞蛋白（HCP）和使用蛋白质组富集珠检测肽的方案。该方法使用内部制造的单克隆抗体 （mAb） 原料药 （DS） 进行演示，该物质是一种表征良好的参考物质，用于评估和比较不同方法的性能。
Abstract
宿主细胞蛋白 （HCP） 是会对治疗性蛋白质产生不利影响的杂质，即使是少量的。为了评估与药品相关的潜在风险，已经开发了识别低丰度HCP的方法。开发灵敏的HCP检测方法的一个关键方法是利用液相色谱-质谱联用（LC-MS）在分析前富集HCP，同时去除单克隆抗体（mAb）。
该协议提供了使用市售蛋白质组富集珠富集宿主细胞蛋白的详细说明。这些磁珠包含对不同蛋白质具有特异性亲和力的多种六肽配体库。该方案还包括使用纳米LC-MS/MS进行有限的酶解和随后的肽检测。通过采用这些技术，低丰度的HCP可以富集超过7000倍，从而获得低至0.002 ppm的令人印象深刻的检测限。值得注意的是，该协议能够使用 NIST mAb 以高置信度检测 850 个 HCP。此外，它被设计为用户友好型，并包括一个视频演示，以协助其实施。通过遵循这些步骤，研究人员可以有效地富集和检测HCP，提高药品风险评估的灵敏度和准确性。
Introduction
宿主细胞蛋白 （HCP） 是从宿主生物体的细胞培养物中释放出来并与单克隆抗体 （mAb） 共纯化的杂质1,2,3,4。痕量HCPs会对药品的质量产生负面影响5,6,7,8,9,10,11,12,13,14,15，因此，需要一种灵敏的HCP分析方法来检测亚ppm至ppm水平的HCP。
正交方法可用于检测低丰度的 HCP。酶联免疫吸附测定 （ELISA） 通常用于定量整体 HCP，如果有相应的抗体，它也可以检测和定量单个 HCP16。然而，HCP特异性抗体的生产既费时又费力。相比之下，液相色谱-质谱联用（LC-MS）可以提供有关单克隆抗体药物产品中单个HCP的全面信息，并广泛用于HCP鉴定4,7,9,10,12,13,14,15,17,18,19,20， 21、22、23、24、25、26、27。
已经开发了几种使用 LC-MS/MS 检测 HCP 的方法，包括有限酶切20、过滤17、蛋白 A 缺失21、免疫沉淀 （IP） 和 ProteoMiner 富集 （PM）18。大多数方法旨在减少mAb的用量，并在LC-MS/MS分析之前富集HCP，从而减小mAb肽和HCP肽之间的动态范围。该协议提出了一种蛋白质组学样品富集方法，该方法结合了 ProteoMiner 技术和有限消化 （PMLD）28。ProteoMiner 富集原理涉及使用市售的蛋白质组富集微球，其中包含多种组合肽配体库。这些配体特异性地与抗体药物产品上的蛋白质结合，从而去除多余的分子，同时将低丰度宿主细胞蛋白 （HCP） 集中在它们各自的亲和配体上。另一方面，有限消化的原理涉及使用低浓度的胰蛋白酶。该浓度足以消化低丰度的HCP，但不足以消化所有抗体药物产品。这种方法能够从溶液中回收和富集消化的HCP肽。
与过滤方法相比，PMLD技术不受检测到的HCPs大小的限制17。蛋白 A 缺失方法特异性地检测与抗体相关的 HCP21，而免疫沉淀仅限于来自特定细胞系（例如中国仓鼠卵巢 （CHO） 细胞系）的预定义 HCP，其中产生抗 HCP 抗体4。相比之下，PMLD可用于检测来自任何药物模块的HCP，以及与来自不同细胞系的药物产品共同纯化的宿主细胞蛋白。此外，与上述方法相比，PMLD 表现出更好的灵敏度17、18、20、21、24。
这种方法可以将HCP浓度富集7000倍，并将检测限降低到0.002 ppm28。实验设置如图 1所示。
Protocol
协议中使用的缩写列于 补充表1中。 1. 溶液和缓冲液的制备 注：所有试剂的商业细节均列在 材料表中。 通过将 1 mL 的 1 M Tris-HCl（pH 8.0）加入玻璃小瓶中的 9 mL 去离子水中，制备 0.1 M Tris-HCl（pH 8.0）溶液，并通过涡旋充分混合。在4°C下储存长达3个月。 通过将 10 mg SDC 溶解在 1 mL 的 0.1 M Tris-HCl（pH 8.0）…
Representative Results
该协议提供了一种样品制备工作流程，称为蛋白质富集与有限消化（PMLD）相结合，用于分析单克隆抗体（mAb）样品中的宿主细胞蛋白（HCP）。图 1 说明了 PMLD 的分步过程。研究人员比较了使用直接消化（如图 2 顶部面板所示）和 PMLD（如图 2 底部面板所示）的 HCP 分析结果。总离子色谱图（TIC）图谱表明，PMLD显著减少或消除了主?…
Discussion
市售的蛋白质富集微球有两种版本：一种容量较小，另一种容量较大（参见 材料表）。两种版本的富集微球在包装中都含有十种制备物。制造商的说明表明，小容量试剂盒中的每个制备都可用于富集 10 mg 总蛋白。然而，为了实现 DS 宿主细胞蛋白 （HCP） 富集的最佳性能，每次制备都适合 5 个 DS 样品。因此，每个试剂盒可用于从 50 个样品中富集 HCP。对于大容量试剂盒的每种制备方法…
Divulgations
The authors have nothing to disclose.
Acknowledgements
没有。
Materials
| 16 G, Metal Hub Needle, 2 in, point style 3 | Hamilton | 91016 | |
| Acclaim PepMap 100 C18 trap column (20 cm × 0.075 mm) | Thermo Fisher | 164535 | |
| Acetonitrile | Fisher-Scientific | A955 | |
| Acetonitrile with 0.1% Formic Acid (v/v), Optima LC/MS Grade | Fisher-Scientific | LS120-4 | |
| Amicon Ultra-0.5 Centrifugal Filter Unit | Millipore Sigma | UFC5010 | |
| C18 analytical column (0.075 mm × 1.7 μm × 30 cm, 100 Å) | CoAnn Technologies | HEB07503001718I | |
| Centrifuge 5424 | Eppendorf | 5405000646 | |
| Dithiothreitol (DTT) | Thermo Fisher | A39255 | |
| Frit for SPE cartridges, 9.5 mm, 3 mL, 100/pk | Agilent | 12131020 | |
| GL-Tip GC | GL Sciences Inc | 7820-11201 | |
| in-house mAb | Regeneron | concentration 200 mg/mL | |
| Iodoacetamide (30 x 9.3 mg) | Thermo Fisher | A39271 | |
| Isopropanol | Fisher-Scientific | 149320025 | |
| L-Histidine | Sigma Aldrich | H6034 | |
| L-Histidine monohydrochloride monohydrate | Sigma Aldrich | 53370 | |
| Methanol | Fisher-Scientific | A456-4 | |
| Milli-Q | Millpore | 30035 | |
| NanoDrop 2000 | Thermo Scientific | ND-2000 | |
| Orbitrap Exploris 480 | Thermo Fisher | BRE725539 | |
| Protein LoBind Tube 0.5 mL | Eppendorf (VWR) | 22431064 | |
| Protein LoBind Tube 2.0 mL | Eppendorf (VWR) | 22431102 | |
| Proteome Discoverer software 2.4 | Thermo Scientific | ||
| ProteoMiner Protein Enrichment Large-Capacity Kit | Bio-Rad | 1633007 | |
| ProteoMiner Protein Enrichment Small-Capacity Kit | Bio-Rad | 1633006 | |
| Sodium deoxycholate (SDC) | Sigma Aldrich | D6750 | |
| Sodium lauroyl sarcosinate (SLS) | Sigma Aldrich | L5777 | |
| SpeedVac | Labconco | 7970010 | |
| Thermomixer R | Eppendorf | 22670107 | |
| Trifluoracetic acid (TFA) | Fisher-Scientific | 28904 | |
| Trypsin (Sequencing Grade Modified) (5 x 20 ug) | Promega | V5111 | |
| Tube Revolver Rotator | Thermo Fisher | 88881001 | |
| UltiMate 3000 RSLC nano system | Thermo Fisher | ULTIM3000RSLCNANO | |
| UltraPure 1 M Tris-HCl pH 8.0 | Thermo Fisher | 15568-025 | |
| Vortex Genie 2 | VWR | 102091-234 | |
| Water with 0.1% Formic Acid (v/v), Optima LC/MS Grade | Fisher-Scientific | LS118-4 |
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Citer Cet Article
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