Krishna Kunal, Sarin Deepika, Trivedi Drashti, Bhattacharya Sanghati, Refaat Hesham, Ahmad Aziz, Nejadnik Reza, Rathore Anurag S
School of Interdisciplinary Research, Indian Institute of Technology, Delhi, Hauz Khas, New Delhi, 110016, India.
Department of Chemical Engineering, Indian Institute of Technology, Delhi, Hauz Khas, New Delhi, 110016, India.
AAPS J. 2025 May 2;27(4):88. doi: 10.1208/s12248-025-01078-x.
Extensive analytical and functional characterization of a biotherapeutic product is a regulatory requirement, more so for biosimilar products where approval is contingent on the manufacturer's ability to demonstrate comparability of their product to the corresponding reference product. Typical biotherapeutic formulations contain multiple excipients that are meant to stabilize the product and these can impact certain analytical and functional techniques that are typically used in the above-mentioned characterization and comparability exercises. In this study, we elucidate this interference using Trastuzumab (Tmab) reference product, Herclon, and its commercially available biosimilars, Herzuma and Vivitra, as an example. Excipients were removed one at a time from the drug product and impact of this removal on a spectrum of analytical and functional tools was examined. Removal of certain excipients (Trehalose, L-histidine HCl and Polysorbate 20) was found to impact the results of charge variant analysis (cation exchange HPLC), secondary structure analysis (FTIR and far-UV CD spectroscopy), and tertiary structure analysis (near-UV CD and intrinsic FLR spectroscopy) For charge variants, differences up to 3.62% in basic species were observed, while FTIR spectra in the amide I region were significantly impacted. The intrinsic fluorescence spectra displayed major wavelength maxima shifts of up to 6 nm. In view of these results, it is recommended that biosimilar manufacturers consider the impact of differences in formulation in the samples that are being compared as well as the impact of excipient removal, if they are extracting the therapeutic moiety from the drug product for comparability analysis (routinely done by biosimilar manufacturers).
对生物治疗产品进行广泛的分析和功能特性表征是一项监管要求，对于生物类似药产品而言更是如此，其批准取决于制造商证明其产品与相应参比产品具有可比性的能力。典型的生物治疗制剂含有多种旨在稳定产品的辅料，而这些辅料可能会影响上述特性表征和可比性研究中通常使用的某些分析和功能技术。在本研究中，我们以曲妥珠单抗（Tmab）参比产品赫赛汀（Herclon）及其市售生物类似药赫赛玛（Herzuma）和维托珠单抗（Vivitra）为例，阐明这种干扰情况。每次从药品中去除一种辅料，并检查这种去除对一系列分析和功能工具的影响。发现去除某些辅料（海藻糖、L-组氨酸盐酸盐和聚山梨酯20）会影响电荷变体分析（阳离子交换高效液相色谱法）、二级结构分析（傅里叶变换红外光谱法和远紫外圆二色光谱法）以及三级结构分析（近紫外圆二色光谱法和内源荧光光谱法）的结果。对于电荷变体，观察到碱性物质的差异高达3.62%，而酰胺I区域的傅里叶变换红外光谱受到显著影响。内源荧光光谱显示最大波长位移高达6 nm。鉴于这些结果，建议生物类似药制造商在比较样品时考虑制剂差异的影响，以及如果他们从药品中提取治疗部分进行可比性分析（生物类似药制造商通常会这样做）时辅料去除的影响。
