抗体及抗体融合蛋白是临床常用的生物治疗剂，其药效、药代动力学特性、免疫应答及体内半衰期均高度依赖自身糖基化状态。细胞培养过程中的各类环境变量，易导致抗体糖基化程度改变、聚糖多样性增加，因此在生产、研发及质控环节，精准、快速监测抗体N-聚糖谱，是保障生物治疗剂产品质量的核心关键。
传统PNGase F酶解法可特异性去除糖蛋白N-连聚糖，但存在明显短板：常规酶解反应耗时长达数小时，且聚糖释放存在偏好性，易造成去糖基化不完全，导致检测结果偏差，无法真实反映抗体糖基化组成。
PNGase F (快速增强版) 为改良型重组酶试剂，可在数分钟内完成抗体、抗体融合蛋白的完全、无偏好N-聚糖释放，适配HPLC、质谱等下游检测手段。产品保留了传统PNGase F的核心酶学特性，可切割绝大多数N-糖链，且非还原酶切条件下可完整保留抗体二硫键，获得完整去糖基化多聚体蛋白，是治疗性抗体研发、质量控制及相关蛋白组学研究的理想工具。
本产品仅无法切割最内侧GlcNAc上含α-1,3连接核心岩藻糖的特殊N-糖链（多见于植物、昆虫来源糖蛋白），可精准切割高甘露糖、杂合、复合寡糖结构，在糖蛋白最内侧N-乙酰葡萄糖胺（GlcNAc）与天冬酰氨残基（Asn）之间特异性切割，释放完整寡糖链。
组分与储存
| 储运单元货号 | 储运单元名称 | 组分货号 | 组分名称 | 物理外观 | 30T | 储存 | | P1528045WT | PNGase F(快速增强版) (2-8℃组分) | P1528045A | PNGase F(快速增强版) | 液体 | 30 μL | 2-8℃ | | P1528045DT | PNGase F(快速增强版) (-20℃组分) | P1528045B | 10×糖苷酶切反应液 （非还原） | 液体 | 1ml | -20℃ |
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| P1528045C | 10×糖苷酶切反应液 （还原） | 液体 | 1ml | -20℃ |
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| P1528045D | PNGase F增强剂 | 固体 | 3×1vials | -20℃ |
注：发货时各组分将按照对应储存温度（2-8°C/-20°C）独立包装，采用差异化温控运输，保障产品最佳性能。若同一订单包含多种温区组分，包裹可能分批次送达，请以实际收货情况为准。收货后，请立即依据组分表标注的温度条件分类存放。所有组分在未拆封、运输及储存条件均符合要求的情况下，有效期为自生产之日起 24 个月。 |
1.产品应用
① 快速完成抗体、抗体融合蛋白的高效完全去糖基化（数分钟完成反应）；
② 应用于抗体及抗体融合蛋白生产过程的糖基化状态监测；
③ 用于抗体及抗体融合蛋白类生物治疗剂的成品质量控制；
④ 适配各类蛋白质组学相关实验与基础研究。
2.产品优势
① 高效无偏好：快速、均衡释放各类N-连聚糖，规避传统酶解的结果偏差；
② 高纯度无杂质：无蛋白酶、糖苷酶污染，产品纯度≥95%，避免样品降解干扰检测；
③ 适用性广泛：可作用于多种结构的抗体及各类糖蛋白底物；
④ 稳定性优异：每批次产品均经过严格质控，酶活及反应效果批次稳定；
⑤ 蛋白完整性保留：非还原反应条件下可完整保留抗体二硫键，获得完整去糖基化蛋白，降低蛋白表征误差。
此外，本产品快速酶解的特性，可大幅缩短反应时长，有效规避传统长时间酶解引发的蛋白氧化、脱酰胺等非特异性修饰及杂质生成问题，提升实验准确性。
3.操作方法
3.1试剂准备
① 优先选用0.2ml薄壁微管或1.5ml离心管进行反应，保证传热效率；
② 使用前将PNGase F (快速增强版)试剂、反应增强剂冻干粉10000rpm离心10秒，确保试剂全部归集于管底；
③ 取1支反应增强剂冻干粉，加入40μl 5×反应缓冲液-Plus（还原/非还原通用），充分复溶制备5×工作试剂，瞬离至管底备用。
3.2非还原条件操作流程（保留抗体二硫键，获取完整多聚体蛋白）
① 取10-100μg抗体样品，加纯化水定容至15μl；
② 加入4μl 5×工作试剂（非还原），轻柔混匀，总反应体系为19μl；
③ 90℃预热5分钟，取出冷却至室温；
④ 加入1μl PNGase F (快速增强版)试剂，轻柔混匀；
⑤ 50℃水浴条件下反应10分钟，完成酶解。
3.3 还原条件操作流程
① 取10-100μg抗体样品，加纯化水定容至15μl；
② 加入4μl 5×工作试剂（还原），轻柔混匀，总反应体系为19μl；
③ 90℃预热5分钟，取出冷却至室温；
④ 加入1μl PNGase F (快速增强版)试剂，轻柔混匀；
⑤ 50℃水浴条件下反应10分钟，完成酶解。
4.产物数据分析
① N-聚糖分析：对酶解释放的N-聚糖进行还原胺化等衍生化处理，适配后续HPLC、质谱等下游检测；
② 去糖基化糖蛋白分析：样品可直接用于SDS-PAGE检测；经微量透析或微过滤缓冲液置换后，可用于质谱表征分析。
5.操作注意事项
① 体系适配范围：20μl标准反应体系中，1μl PNGase F (快速增强版)可适配10μg含Fc、Fab糖基化抗体（西妥昔单抗等）、15μg小鼠IgG2a单抗、30μg常规简单抗体（利妥昔单抗等）的完全去糖基化，特殊底物可根据实验需求优化反应体系；
② 反应体系选择：IgA1、胎球蛋白、绒毛膜促性腺激素等特殊样品，仅还原条件下可实现完全去糖基化，此类样品需选用还原酶解体系；
③ 热变性要求：实验需严格遵循两步操作，加入酶试剂前必须完成90℃、5分钟热变性处理；部分难酶解样品需升温至近100℃以实现完全去糖基化，需实时观察样品状态，避免高温孵育产生蛋白沉淀；
④ 样品储存要求：样品储存体系不得含SDS（SDS会抑制酶活性）；Tween、Triton X-100、NP-40、辛基葡糖苷、磺基甜菜碱及高浓度有机溶剂，均会降低酶解效率，需提前规避；
⑤ 酶灭活条件：75℃水浴处理10分钟可完全灭活PNGase F (快速增强版)酶活性；
⑥ 适用范围：本产品仅供科研使用，不得用于人或动物的临床诊断、治疗场景。
Antibodies and antibody fusion proteins are widely used biotherapeutics in clinical practice. Their pharmacodynamic properties, pharmacokinetic profiles, immune responses and in vivo half-life are highly dependent on their glycosylation status. Various environmental variables during cell culture can easily alter antibody glycosylation levels and increase glycan heterogeneity. Therefore, accurate and rapid monitoring of antibody N-glycan profiles in production, R&D and quality control processes is critical to guarantee the quality of biotherapeutic products.
The conventional PNGase F enzymatic method can specifically remove N-linked glycans from glycoproteins, yet it has notable drawbacks: standard digestion reactions take several hours, and glycan release exhibits bias, which may lead to incomplete deglycosylation, biased detection results and failure to truly reflect antibody glycan composition.
Rapid Enhanced PNGase F is a modified recombinant enzyme reagent. It enables complete, unbiased N-glycan release from antibodies and antibody fusion proteins within minutes, compatible with downstream detection methods including HPLC and mass spectrometry. This product retains the core enzymatic properties of traditional PNGase F, cleaving most N-glycans. Under non-reducing digestion conditions, antibody disulfide bonds remain intact, yielding fully deglycosylated multimeric proteins. It serves as an ideal tool for therapeutic antibody R&D, quality control and related proteomics research.
This product cannot cleave a special subset of N-glycans carrying α-1,3 linked core fucose on the innermost GlcNAc residue (commonly found in plant and insect-derived glycoproteins). It specifically cleaves between the innermost N-acetylglucosamine (GlcNAc) and asparagine (Asn) residues of glycoproteins to release intact oligosaccharides, including high-mannose, hybrid and complex glycan structures.
Contents & Storage
| Storage Unit No. | Storage Unit Name | Component No. | Component Name | Appearance | 50T | Storage | | P1528045WT | PNGase F (Rapid Enhanced) (2-8℃ Components) | P1528045A | PNGase F (Rapid Enhanced) | Liquid | 30 μL | 2-8℃. | | P1528045DT | PNGase F (Rapid Enhanced) (-20℃ Components) | P1528045B | 10× Glycosidase Reaction Buffer(Non-reducing) | Liquid | 1ml | -20℃. |
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| P1528045C | 10× Glycosidase Reaction Buffer(Reducing) | Liquid | 1ml | -20℃. |
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| P1528045D | PNGase F Enhancer | Solid | 3×1vials | -20℃. |
Note: During shipment, each component is individually packaged and transported under temperaturecontrolled conditions corresponding to its storage requirement ( 2-8°C/-20°C ) to maintain optimal product performance. For a single order containing components requiring different storage temperatures, packages may be delivered in separate shipments, subject to actual receipt. Upon receipt, store all components immediately according to the temperature specifications listed in the component list. The shelflife of all unopened components is 24 months from the date of manufacture, provided that both shipping and storage conditions are strictly followed. |
1. Applications
① Rapid, efficient and complete deglycosylation of antibodies and antibody fusion proteins (reaction completed within minutes);
② Monitoring glycosylation status during the production of antibodies and antibody fusion proteins;
③ Quality control of finished biotherapeutic products based on antibodies and antibody fusion proteins;
④ Compatibility with various proteomics experiments and basic research.
2. Product Advantages
① Efficient and unbiased: fast and uniform release of all types of N-linked glycans, eliminating result bias caused by conventional digestion;
② High purity, free of contaminants: no protease or glycosidase contamination; product purity ≥95%, avoiding sample degradation that interferes with detection;
③ Broad applicability: acts on antibodies with diverse structures and various glycoprotein substrates;
④ Excellent stability: every batch undergoes strict QC, with consistent enzymatic activity and reaction performance across batches;
⑤ Preservation of protein integrity: antibody disulfide bonds stay intact under non-reducing conditions to obtain fully deglycosylated proteins, reducing errors in protein characterization.
In addition, the rapid digestion feature greatly shortens reaction time and effectively prevents non-specific modifications such as protein oxidation and deamidation, as well as impurity generation induced by prolonged incubation in traditional protocols, improving experimental accuracy.
3. Protocol
3.1 Reagent Preparation
① Use 0.2 mL thin-walled microtubes or 1.5 mL centrifuge tubes for reactions to ensure efficient heat transfer;
② Before use, centrifuge Rapid Enhanced PNGase F reagent and enhancer lyophilate at 10,000 rpm for 10 seconds to collect all reagents at the bottom of tubes;
③ Reconstitute one vial of enhancer lyophilate with 40 μL 5× Reaction Buffer-Plus (compatible for both reducing and non-reducing conditions) to prepare 5× working reagent, then perform a brief spin-down and set aside.
3.2 Non-Reducing Protocol (Preserve antibody disulfide bonds, obtain intact multimeric proteins)
① Take 10–100 μg antibody sample and add purified water to a final volume of 15 μL;
② Add 4 μL 5× non-reducing working reagent, mix gently; total reaction volume = 19 μL;
③ Heat at 90 °C for 5 min, then cool to room temperature;
④ Add 1 μL Rapid Enhanced PNGase F reagent and mix gently;
⑤ Incubate in a water bath at 50 °C for 10 min to complete digestion.
3.3 Reducing Protocol
① Take 10–100 μg antibody sample and add purified water to a final volume of 15 μL;
② Add 4 μL 5× reducing working reagent, mix gently; total reaction volume = 19 μL;
③ Heat at 90 °C for 5 min, then cool to room temperature;
④ Add 1 μL Rapid Enhanced PNGase F reagent and mix gently;
⑤ Incubate in a water bath at 50 °C for 10 min to complete digestion.
4. Post-reaction Sample Analysis
① N-glycan analysis: Derive released N-glycans via reductive amination or other derivatization methods for downstream HPLC, MS and other detection platforms;
② Deglycosylated glycoprotein analysis: Samples can be directly analyzed by SDS-PAGE. After buffer exchange by microdialysis or microfiltration, samples are suitable for mass spectrometry characterization.
5. Precautions
① Reaction compatibility: In a standard 20 μL reaction system, 1 μL Rapid Enhanced PNGase F achieves full deglycosylation for 10 μg Fc / Fab glycosylated antibodies (e.g., cetuximab), 15 μg mouse IgG2a mAb, and 30 μg conventional simple antibodies (e.g., rituximab). Reaction conditions may be optimized for special substrates according to experimental requirements;
② Buffer selection: For special samples such as IgA1, fetuin and human chorionic gonadotropin, complete deglycosylation can only be achieved under reducing conditions; use reducing digestion system for these samples;
③ Heat denaturation requirement: Strictly follow the two-step workflow. 90 °C, 5 min heat denaturation must be finished before enzyme addition. For some refractory samples, heating near 100 °C may be required for complete deglycosylation. Monitor sample status closely to avoid protein precipitation upon high-temperature incubation;
④ Sample matrix requirement: Sample storage solution must not contain SDS (SDS inhibits enzyme activity). Tween, Triton X-100, NP-40, octyl glucoside, sulfobetaine and high concentrations of organic solvents will reduce digestion efficiency and should be avoided in advance;
⑤ Enzyme inactivation: Incubation in a 75 °C water bath for 10 min fully inactivates Rapid Enhanced PNGase F;
⑥ Intended use: This product is for research use only. It shall not be used for clinical diagnosis or treatment in humans or animals.
危险品化学品经营许可证（带存储）