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Abstract：
BACKGROUND: Pegfilgrastim-cbqv/CHS-1701 (UDENYCA®) (hereafter referred to as pegfilgrastim-cbqv) was approved in 2018 by the US Food and Drug Administration as a biosimilar for pegfilgrastim (Neulasta®) (hereafter referred to as pegfilgrastim). Both pegfilgrastim-cbqv and pegfilgrastim are conjugates of recombinant human granulocyte colony stimulating factor (r-metHuG-CSF) with a 20 kDa polyethylene glycol (PEG) indicated to decrease the incidence of infection, as manifested by febrile neutropenia, in patients receiving myelosuppressive anticancer drugs. The demonstration of analytical similarity for PEG-protein conjugates presents unique challenges since both the protein and PEG attributes must be characterized.
OBJECTIVE: The current study demonstrates the analytical similarity of pegfilgrastim-cbqv and the reference product, pegfilgrastim. In addition to the physicochemical and functional characterization of the protein, the study assessed attributes specific to PEGylation including PEG size and polydispersity, site of attachment, linker composition, and PEGylation process-related variants.
METHODS: The structural, functional, and stability attributes of pegfilgrastim-cbqv and pegfilgrastim were compared using state-of-the-art analytical methods. For the protein, the primary structure, disulfide structure, and secondary and tertiary structures were assessed using traditional protein characterization techniques such as mass spectrometry (MS), circular dichroism (CD), intrinsic fluorescence, and differential scanning calorimetry (DSC), as well as more advanced techniques such as two-dimensional (2D) nuclear magnetic resonance (NMR) and hydrogen deuterium exchange (HDX). For the PEG moiety, the site of attachment, occupancy, linker composition, size and polydispersity were compared using mass spectrometry (both intact and after endoprotease digestion), multiangle light scattering detection (MALS), and Edman degradation. Purity assessments included the assessment of both protein variants and PEGylation variants using chromatographic and electrophoretic analytical separation techniques. The functional similarity between pegfilgrastim-cbqv and pegfilgrastim was compared using both a cell-based bioassay and surface plasmon resonance (SPR). The degradation rates and stability profiles were compared under accelerated and stressed conditions.
RESULTS: Biosimilarity was demonstrated by a thorough assessment of physiochemical and functional attributes, as well as comparative stability, of pegfilgrastim-cbqv relative to pegfilgrastim. These studies demonstrated identical primary structure and disulfide structure, highly similar secondary and tertiary structure, as well as functional similarity. The impurity profile of pegfilgrastim-cbqv was comparable to that of pegfilgrastim with only minor differences in PEGylation variants and a slight offset in the PEG molar mass. These differences were not clinically relevant. The degradation profiles were qualitatively and quantitatively similar under accelerated and stress conditions.
CONCLUSIONS: The structural, functional, and stability data demonstrate that pegfilgrastim-cbqv is highly similar to the reference product, pegfilgrastim.
OBJECTIVE: The current study demonstrates the analytical similarity of pegfilgrastim-cbqv and the reference product, pegfilgrastim. In addition to the physicochemical and functional characterization of the protein, the study assessed attributes specific to PEGylation including PEG size and polydispersity, site of attachment, linker composition, and PEGylation process-related variants.
METHODS: The structural, functional, and stability attributes of pegfilgrastim-cbqv and pegfilgrastim were compared using state-of-the-art analytical methods. For the protein, the primary structure, disulfide structure, and secondary and tertiary structures were assessed using traditional protein characterization techniques such as mass spectrometry (MS), circular dichroism (CD), intrinsic fluorescence, and differential scanning calorimetry (DSC), as well as more advanced techniques such as two-dimensional (2D) nuclear magnetic resonance (NMR) and hydrogen deuterium exchange (HDX). For the PEG moiety, the site of attachment, occupancy, linker composition, size and polydispersity were compared using mass spectrometry (both intact and after endoprotease digestion), multiangle light scattering detection (MALS), and Edman degradation. Purity assessments included the assessment of both protein variants and PEGylation variants using chromatographic and electrophoretic analytical separation techniques. The functional similarity between pegfilgrastim-cbqv and pegfilgrastim was compared using both a cell-based bioassay and surface plasmon resonance (SPR). The degradation rates and stability profiles were compared under accelerated and stressed conditions.
RESULTS: Biosimilarity was demonstrated by a thorough assessment of physiochemical and functional attributes, as well as comparative stability, of pegfilgrastim-cbqv relative to pegfilgrastim. These studies demonstrated identical primary structure and disulfide structure, highly similar secondary and tertiary structure, as well as functional similarity. The impurity profile of pegfilgrastim-cbqv was comparable to that of pegfilgrastim with only minor differences in PEGylation variants and a slight offset in the PEG molar mass. These differences were not clinically relevant. The degradation profiles were qualitatively and quantitatively similar under accelerated and stress conditions.
CONCLUSIONS: The structural, functional, and stability data demonstrate that pegfilgrastim-cbqv is highly similar to the reference product, pegfilgrastim.
摘要：
背景：Pegfilgrastim-cbqv/CHS-1701（UDENYCA®）（以下简称pegfilgrastim-cbqv）于2018年被美国食品和药物管理局批准为pegfilgrastim（Neulasta®）的生物仿制药（以下简称pegfilstim）。pegfilgrastim-cbqv和pegfilgrastim都是重组人粒细胞集落刺激因子（r-metHuG-CSF）与20kDa聚乙二醇（PEG）的结合物，可降低感染发生率。表现为发热性中性粒细胞减少症，在接受骨髓抑制抗癌药物的患者中。PEG-蛋白质缀合物的分析相似性的证明提出了独特的挑战,因为必须表征蛋白质和PEG属性两者。
目的：当前的研究证明了pegfilgrastim-cbqv与参考产品的分析相似性，Pegfilgrastim.除了蛋白质的物理化学和功能表征外，该研究评估了聚乙二醇化特有的属性，包括PEG大小和多分散性，附件的位置，连接体成分,和聚乙二醇化过程相关的变体。
方法：结构，功能，使用最先进的分析方法比较了pegfilgrastim-cbqv和pegfilgrastim的稳定性属性。对于蛋白质，一级结构，二硫化物结构，二级和三级结构使用传统的蛋白质表征技术，如质谱（MS），圆二色性（CD），固有荧光，和差示扫描量热法（DSC），以及更先进的技术，如二维（2D）核磁共振（NMR）和氢氘交换（HDX）。对于PEG部分，附件的位置，占用,连接体成分,使用质谱(完整和内切蛋白酶消化后)比较大小和多分散性，多角度光散射检测(MALS),和埃德曼退化。纯度评估包括使用色谱和电泳分析分离技术评估蛋白质变体和PEG化变体。使用基于细胞的生物测定法和表面等离子体共振（SPR）比较了pegfilgrastim-cbqv和pegfilgrastim之间的功能相似性。在加速和应力条件下比较了降解速率和稳定性曲线。
结果：通过对生理化学和功能属性的全面评估证明了生物相似性，以及相对稳定性，pegfilgrastim-cbqv相对于pegfilgrastim。这些研究证明了相同的一级结构和二硫化物结构，高度相似的二级和三级结构，以及功能相似性。pegfilgrastim-cbqv的杂质谱与pegfilgrastim的杂质谱相当，PEG化变体的差异很小，PEG摩尔质量略有偏移。这些差异没有临床相关性。在加速和胁迫条件下，降解曲线在质量和数量上相似。
结论：结构，功能，和稳定性数据表明pegfilgrastim-cbqv与参考产品高度相似，Pegfilgrastim.
目的：当前的研究证明了pegfilgrastim-cbqv与参考产品的分析相似性，Pegfilgrastim.除了蛋白质的物理化学和功能表征外，该研究评估了聚乙二醇化特有的属性，包括PEG大小和多分散性，附件的位置，连接体成分,和聚乙二醇化过程相关的变体。
方法：结构，功能，使用最先进的分析方法比较了pegfilgrastim-cbqv和pegfilgrastim的稳定性属性。对于蛋白质，一级结构，二硫化物结构，二级和三级结构使用传统的蛋白质表征技术，如质谱（MS），圆二色性（CD），固有荧光，和差示扫描量热法（DSC），以及更先进的技术，如二维（2D）核磁共振（NMR）和氢氘交换（HDX）。对于PEG部分，附件的位置，占用,连接体成分,使用质谱(完整和内切蛋白酶消化后)比较大小和多分散性，多角度光散射检测(MALS),和埃德曼退化。纯度评估包括使用色谱和电泳分析分离技术评估蛋白质变体和PEG化变体。使用基于细胞的生物测定法和表面等离子体共振（SPR）比较了pegfilgrastim-cbqv和pegfilgrastim之间的功能相似性。在加速和应力条件下比较了降解速率和稳定性曲线。
结果：通过对生理化学和功能属性的全面评估证明了生物相似性，以及相对稳定性，pegfilgrastim-cbqv相对于pegfilgrastim。这些研究证明了相同的一级结构和二硫化物结构，高度相似的二级和三级结构，以及功能相似性。pegfilgrastim-cbqv的杂质谱与pegfilgrastim的杂质谱相当，PEG化变体的差异很小，PEG摩尔质量略有偏移。这些差异没有临床相关性。在加速和胁迫条件下，降解曲线在质量和数量上相似。
结论：结构，功能，和稳定性数据表明pegfilgrastim-cbqv与参考产品高度相似，Pegfilgrastim.
