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Anti-CD20rh MAb Quality Evaluation and Monoclonal Cell Line Screening
Hai-jiao JI,Wen-lei LI,Rui-jing Huang,Jian LI,Han-mei XU
China Biotechnology ›› 2018, Vol. 38 ›› Issue (8) : 34-40.
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Anti-CD20rh MAb Quality Evaluation and Monoclonal Cell Line Screening
({{custom_author.role_en}}), {{javascript:window.custom_author_en_index++;}}Objective: To screen high expression monoclonal cell lines and optimize the production and quality of Anti-CD20rh MAb. Method: Screen high expression monoclonal cell lines by finite dilution method, and evaluated the yield of monoclonal cell lines by double-sandwich ELISA. In order to screen the best cell culture program, two to three cells were picked based on the cells’ growth state, yield, viability, and so on. The result were evaluated by the sugar type, the isoelectric point, the purity and the distribution of the acid and alkali base peak. Results: The production of CHOS cells tripled after a series of optimization, increased from nearly 500mg/L to 2 290mg/L. After the optimization of culture program, the purity of the target protein reached up to 97.48%, and the distribution of the acid and alkali base peak looks more close to the ideal state. Conclusion: The production and quality of the target antibody were optimized by cloning and culture optimization, these results are of great significance to the later experimental research and industrial production.
Monoclonal / Medium optimization / Quality evaluation {{custom_keyword}} /
Fig.2 Growth status and antibody expression of monoclonal cell lines after optimization(a) Growth status of monoclonal cell lines after optimization (b) Antibody expression of monoclonal cell lines after optimization; A、B are different cells; 1~11 are different cell culture and flow plus scheme 图2 培养基优化后细胞株生长状态及蛋白表达量 |
Table 1 The concentration of ProteinA purified intermediate表1 ProteinA捕获中间体抗体蛋白浓度分析 |
| 样品编号 | 发酵液体积(ml) | UV(OD) | 浓度(mg/ml) | 捕获体积(ml) | 抗体蛋白产量(mg) |
|---|---|---|---|---|---|
| A1 | 35 | 0.6707 | 5.047 | 6.45 | 32.55 |
| A4 | 30 | 0.8178 | 6.224 | 5.7 | 35.48 |
| B1 | 35 | 1.2092 | 9.674 | 6.25 | 60.46 |
| B4 | 30 | 0.9489 | 7.273 | 5.00 | 36.37 |
| Note: A、B are different cells; 1、4 are different cell cultures and flow plus schemes |
Fig.4 The SEC-HPLC chromatograms(a) SEC-HPLC chromatograms of Sample A1 (b) SEC-HPLC chromatograms of Sample A4 (c) SEC-HPLC chromatograms of Sample B1 (d) SEC-HPLC chromatograms of Sample B4; A、B are different cells; 1、4 are different cell culture and flow plus schemes 图4 样品SEC-HPLC分析图谱 |
Table2 Results of CIEF表2 毛细管电泳分析结果 |
| 样品 | 酸峰 (%) | 主峰 (%) | 碱峰 (%) | 主峰+碱 峰(%) | 等电点 |
|---|---|---|---|---|---|
| A1 | 44.83 | 21.01 | 34.16 | 55.17 | 8.274 |
| A4 | 68.57 | 18.79 | 12.63 | 31.42 | 8.332 |
| B1 | 48.04 | 20.48 | 31.48 | 51.96 | 8.354 |
| B4 | 55.14 | 28.65 | 16.22 | 44.87 | 8.377 |
| Note: A、B are different cells; 1、4 are different cell cultures and flow plus schemes |
Table 3 Results of glycosylation analysis of HPLC-FLR表3 HPLC-FLR糖型分析结果(%) |
| 峰4 | 峰5 | 峰7 | 峰8 | 峰9, 10 | 峰17 | |
|---|---|---|---|---|---|---|
| 样品 | Man5 | G0 | G0F | G1 | G1F | G2F |
| A1 | 1.37 | 1.84 | 61.24 | 2.48 | 25.42 | 3.32 |
| A4 | 2.71 | 1.89 | 51.93 | 4.7 | 28.28 | 6.97 |
| B1 | 0.73 | 1.47 | 57.39 | 2.3 | 30.04 | 4.82 |
| B4 | 1.79 | 2.52 | 56.79 | 3.56 | 26.46 | 6.42 |
| Mean | 1.65 | 1.93 | 56.84 | 3.26 | 27.55 | 5.38 |
| Std.Dev | 0.83 | 0.44 | 3.82 | 1.11 | 2.04 | 1.65 |
| CV(%) | 50.32 | 22.57 | 6.72 | 34.04 | 7.40 | 30.65 |
| Note: A、B are different cells; 1、4 are different cell cultures and flow plus schemes |
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B cell antigen receptor(BCR) signaling initiates sustained cellular calcium influx necessary for the development, differentiation and activation of B-lymphocytes.CD20 is a B cell-restricted tetraspanning protein organized in the plasma membrane and involved in BCR-activated calcium entry.Recent studies provide here direct evidences of CD20 homo-oligomerization into tetramers.Anti-CD20 mAb-induced calcium signaling was also investigated, and it is reported that only type mAb are capable of inducing a calcium flux in B cells.Additionally, CD20 involves in signal transduction through store-operated calcium entry(SOCE).In this review, researches of CD20 forming homo-oligomer, associating with BCR, and participating in regulation of calcium flux in B lymphocytes were discussed.
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宋小红, 于婷, 李冰 , 等. 促进CHO 细胞生长及其产物hNGF 表达的培养条件的初步研究. 中国生物工程杂志, 2010,30(4):13-19.
<p>以稳定表达人神经生长因子（hNGF）的重组工程CHO细胞株为对象，采用无血清流加悬浮培养（Fedbatch culture）方式，考察使用基础培养基(无特殊添加物)，分别添加丁酸钠、DMSO、KH2PO4的培养基及不同培养温度（32℃和37℃）对细胞生长和重组蛋白表达的影响。每日取样检测细胞密度、细胞活率、葡萄糖浓度、重组蛋白浓度。结果表明细胞培养温度由37℃下降至32℃，细胞生长周期明显延长，重组蛋白产量增加。5mmol/L丁酸钠和2% DMSO的加入虽然提高了重组蛋白的表达量，但严重抑制细胞生长。最大的蛋白比生成速率（qNGF）出现在37℃培养且添加2% DMSO的培养条件下，而最高蛋白表达量则出现于32℃培养添加3.65mmol/L KH2PO4的培养条件下。研究表明，将培养温度设为32℃，在基础培养基中添加3.65mmol/L KH2PO4或1% DMSO是提高hNGF表达水平的有效方法。</p>
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Abstract Here we review the current state of the art in analytical and structural methods used to characterize therapeutic monoclonal antibodies (mAbs) and derivatives. First a general introductive background on immunoglobulins G (IgGs) structures and micro-heterogeneities is presented. Then the review focuses on analytical papers published mainly the last two years. As a result, a compendium of dozens of micro-variants that have been identified for mAbs both from a separation sciences and a mass spectrometry perspective, is proposed. These small structural differences with the main IgG isoform that are detailed, include glycoforms, charge, cysteine-related, oxidized, size and low level point mutation variants. In addition, the functional impact of these micro-variants is discussed, helping their classification as low, medium or high critical quality attributes (CQA) and contributing to the development of Quality by Design (QbD) paradigm.1 Then, analytical methods for higher order structures, mAb/antigens complexes and aggregates are discussed. Finally quantitative aspects are presented including mAbs quantification in biofluids and residual Host Cell Proteins (HCPs).
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衣常红, 高春芳 . IgG糖基化修饰及其意义研究进展. 中国免疫学杂志, 2010,26(11):1051-1056.
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The authors have declared that no competing interests exist.
作者已声明无竞争性利益关系。
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Fig.1 Growth status and antibody expression of monoclonal cell linesFig.2 Growth status and antibody expression of monoclonal cell lines after optimization
Table 1 The concentration of ProteinA purified intermediateFig.3 SDS-PAGE electrophoresis mapFig.4 The SEC-HPLC chromatogramsTable2 Results of CIEFFig.5 Chromatogram of glycosylation analysis of HPLC-FLRTable 3 Results of glycosylation analysis of HPLC-FLR/
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