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高通量檢測治療用抗體製程中的有害副產品 — 非天然抗體

Scientific Reports》論文選讀
高通量檢測治療用抗體製程中的有害副產品 — 非天然抗體

治療用 IgG 抗體在生產過程中會經受多種壓力干擾,例如 pH 變化、反覆冷凍再解凍 (Freeze-thaw)、震盪和攪拌等,這些壓力都會導致抗體藥物失去天然的三級結構,形成非天然的 IgG。非天然的 IgG 更容易形成聚集體 (Aggregates),這些聚集體的存在不僅會削減抗體藥物的療效,更讓抗體藥物具有潛在危險的免疫源性 (Immunogenicity)。因此在抗體藥物的生產製程中,監測有害副產品「非天然抗體」的含量,是管控抗體藥物品質極為關鍵的一環。

目前已知的非天然 IgG 檢測方式有大小排阻層析法 (Size exclusion chromatography, SEC)、動態光散射技術 (Dynamic light scattering, DLS)、場流分離技術 (Field flow fractionation) 等,這些方式皆有其侷限性,無法滿足抗體藥物產業製程的五大需求指標:(1) 足夠高的通量;(2) 檢測時間短;(3) 可以定量;(4) 可以檢測細胞培養上清液;(5) 高專一性。

日本產業技術綜合研究所 (AIST) Shinya Honda 研究團隊結合了「AF.2A1 人工合成蛋白」與「ALPHA 技術」兩者的優勢特點,開發出一個能夠完全滿足以上製藥產業需求的非天然 IgG 高通量檢測方法。

偵測原理與檢測步驟

AF.2A1Shinya Honda 團隊早先研發的一種人工合成蛋白,25 個胺基酸大小,具有與非天然 IgG Fc 區域專一性結合的特性,對天然抗體的 Fc 區域沒有結合活性,因此可以用於區分天然和非天然的 IgG

Principle of the AF.2A1-AlphaScreen technique. (A) Schematic illustration of AlphaScreen for detection of non-native IgG. The detection of non-native IgG is enabled by the sandwich structure involving a pair of AF.2A1 proteins. One is chemically conjugated to acceptor beads and the other is biotinylated, which is captured by streptavidin donor beads. The AlphaScreen signal is generated when donor beads are brought into close proximity with acceptor beads, allowing transfer of a singlet oxygen from the photo-excited donor to the acceptor. (B) Procedure of the AF.2A1-AlphaScreen technique. IMAGE © Sci Rep. 2017 Sep 29;7(1):12466, Figure 1.

IMAGE © Sci Rep. 2017 Sep 29;7(1):12466, Figure 1.

ALPHA 則是由 PerkinElmer 公司專利研發的生物分子檢測技術,具有高訊噪比、流程簡易、應用彈性、高通量等卓越性能。Shinya Honda 團隊所開發的非天然 IgG 檢測方法「AF.2A1-AlphaScreen」 ,其偵測原理與步驟可簡化描述為「只需混合無需洗滌的 ELISA」。當樣本中含有非天然 IgG 時,包覆有 AF.2A1 的受體微珠 (Acceptor bead) 與鍵結有 AF-2A1* 的供體微珠 (Donor bead) 即會被拉近。在 680 nm 雷射光激發下,供體微珠表面所帶有的光敏感物質即會催化周遭的氧分子形成活化態的單態氧,並進一步將能量傳遞給專一性結合的受體微珠,最終產生 520-620 nm 的訊號 (*AF-2A1 是藉由 Biotin 標誌鍵結於標誌有 Streptavidin 的供體微珠上 )。

實驗結果

使用 AF.2A1-AlphaScreen 方法檢測天然 IgG 與非天然 IgG,兩條曲線具有明顯差異。AF.2A1-AlphaScreen 方法對於非天然 IgG 的偵測極限 (Limit of detection, LOD) 及定量極限 Limit of quantification, LOQ) 分別是 8.5 μg/ml25.6 μg/ml

IMAGE © Sci Rep. 2017 Sep 29;7(1):12466, Figure 2A.

PBS 溶液中混合不同濃度的天然 IgG 與非天然 IgG,結果顯示天然 IgG 的濃度變化並不會影響 AF.2A1-AlphaScreen 方法對於非天然 IgG 的檢測能力。

IMAGE © Sci Rep. 2017 Sep 29;7(1):12466, Figure 2B and 2C.

進一步鑑定 AF.2A1-AlphaScreen 方法對不同抗體的適用性,三種不同的單株抗體經過壓力後,都可以通過 AF.2A1-AlphaScreen 方法進行檢測。

IMAGE © Sci Rep. 2017 Sep 29;7(1):12466, Table 1 and Figure 3.

評估 AF.2A1-AlphaScreen 方法對於不同壓力來源產生的非天然 IgG 的檢測能力,測試壓力條件包含:強酸 (pH 2.0)、高溫 (70 °C, 10 min)、攪拌 (25 °C, 200 rpm, 5 h) 和震盪 (4 °C, 200 rpm, 2 weeks)。結果顯示,除震盪外,其他壓力條件產生的非天然 IgG 均可以產生足夠的 ALPHA 信號。 *下圖 (A, C, E, G)AF.2A1-AlphaScreen 檢測結果,(B, D, F, H)DLS 檢測結果。

IMAGE © Sci Rep. 2017 Sep 29;7(1):12466, Figure 4.

檢視非天然 IgG 顆粒大小對於 AF.2A1-AlphaScreen 檢測結果的影響。透過和 DLS 檢測結果的比對,可以發現 AF.2A1-AlphaScreen 方法對於 20 nm ~ 500 nm 顆粒大小的非天然 IgG 具有較好的檢測效果。 *下圖 (A, C)AF.2A1-AlphaScreen 檢測結果,(B, D)DLS 檢測結果。

IMAGE © Sci Rep. 2017 Sep 29;7(1):12466, Figure 5.

檢測細胞培養上清液和細胞溶解物 (Cell lysates) 對於 AF.2A1-AlphaScreen 檢測結果的影響性。結果顯示無論在細胞培養上清液還是細胞溶解物樣本中,AF.2A1-AlphaScreen 方法均有相當的檢測效果。

Analysis of non-native IgG in cell culture supernatant and lysate using the AF.2A1-AlphaScreen technique. (A) Mixture of CHO cell culture supernatant and non-native IgG. Day 0 of culture (lane 1), day 7 of culture (lane 2), day 0 of culture with 500 ng acid-stressed IgG (lane 3), and day 7 of culture with 500 ng stressed IgG (lane 4) were subjected to 12% SDS-PAGE and stained with Coomassie brilliant blue. Full-length gel image is shown in Supplementary Figure 1. (B) Detection of non-native IgG in the supernatant of CHO cell culture. Acid-stressed IgG was added to PBS, F12K medium, and CHO cell culture supernatant. (C) Luminescence intensity of AlphaScreen signals of non-native IgG in the CHO and 293 T cell lysate. The measurement samples were prepared using acid-stressed IgG, which was added to the CHO and 293 T cell lysates. The stressed IgG concentration was 0.5 mg/ml. (D) Luminescence intensity of AlphaScreen signals of non-native IgG from the CHO and 293 T cell lysate, with an acid-stressed IgG concentration of 0.25 mg/ml. Otherwise conditions were as described in (C). Three independent experiments were performed, and the data were presented as mean ± SD. MAb_A was used in this experiment. IMAGE © Sci Rep. 2017 Sep 29;7(1):12466, Figure 6.

Analysis of non-native IgG in cell culture supernatant and lysate using the AF.2A1-AlphaScreen technique. (A) Mixture of CHO cell culture supernatant and non-native IgG. Day 0 of culture (lane 1), day 7 of culture (lane 2), day 0 of culture with 500 ng acid-stressed IgG (lane 3), and day 7 of culture with 500 ng stressed IgG (lane 4) were subjected to 12% SDS-PAGE and stained with Coomassie brilliant blue. Full-length gel image is shown in Supplementary Figure 1. (B) Detection of non-native IgG in the supernatant of CHO cell culture. Acid-stressed IgG was added to PBS, F12K medium, and CHO cell culture supernatant. (C) Luminescence intensity of AlphaScreen signals of non-native IgG in the CHO and 293 T cell lysate. The measurement samples were prepared using acid-stressed IgG, which was added to the CHO and 293 T cell lysates. The stressed IgG concentration was 0.5 mg/ml. (D) Luminescence intensity of AlphaScreen signals of non-native IgG from the CHO and 293 T cell lysate, with an acid-stressed IgG concentration of 0.25 mg/ml. Otherwise conditions were as described in (C). Three independent experiments were performed, and the data were presented as mean ± SD. MAb_A was used in this experiment. IMAGE © Sci Rep. 2017 Sep 29;7(1):12466, Figure 6.

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更多詳細說明歡迎瀏覽原文AlphaScreen-based homogeneous assay using a pair of 25-residue artificial proteins for high-throughput analysis of non-native IgG. Sci Rep. 2017 Sep 29;7(1):12466.。完整 ALPHA 產品資訊與技術應用指引,歡迎洽詢 PerkinElmer 台灣代理 — 伯森生技 (線上聯絡伯森業務專員 • 線上留言諮詢)。您可透過下方連結瀏覽更多相關訊息:

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