CN103725645B - 一种去除新生牛血清内毒素的工艺 - Google Patents
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Abstract
本发明公开了一种去除新生牛血清内毒素的工艺，步骤如下：取新生牛血清，4000r/min离心10min，取上清，加入上清液质量0.2%的活性炭进行吸附，吸附30min；吸附后，4000r/min离心20min，取上清，依次用0.8μm滤膜、0.65μm滤膜、0.45μm滤膜、0.22μm滤膜、0.1μm滤膜过滤，即得到去除了内毒素的新生牛血清。本发明采用活性炭吸附及逐级过滤的方法，可显著降低以往在新生牛血清生产过程中作为废弃物的高内毒素含量牛血清中内毒素的含量，增加新生牛血清生产的效率和附加值。
Description
技术领域
本发明涉及一种去除新生牛血清内毒素的工艺。
背景技术
内毒素是革兰氏阴性菌的菌体中存在的毒性物质的总称，是多种革兰氏阴性菌的细胞壁成分，由菌体裂解后释出，又称之为“热原”。其化学成分有磷脂多糖-蛋白质复合物，其毒性成分主要为类脂质A。内毒素位于细胞壁的最外层、覆盖于细胞壁的黏肽上。各种细菌的内毒素大都具有一定的毒性，可引起发热、微循环障碍、内毒素休克及播散性血管内凝血等。因此，含有内毒素的血清不能用于疫苗的生产，只有去除内毒素、保留血清中的营养成分，才可以使这类血清用于疫苗生产，从而变废为宝，得到充分利用。
在现有新生牛血清生产过程中，由于采血、存放等诸多原因造成细菌污染，会产生大量内毒素超标的血清，因此，为了充分利用生物资源、减少资源浪费，设计一种高效去除新生牛血清内毒素的工艺是亟需解决的难题。
发明内容
针对上述现有技术，本发明提供了一种去除新生牛血清内毒素的工艺，本发明采用活性炭吸附及逐级过滤的方法，可显著降低以往在新生牛血清生产过程中作为废弃物的高内毒素含量牛血清中内毒素的含量，增加新生牛血清生产的效率和附加值。
本发明是通过以下技术方案实现的：
一种去除新生牛血清内毒素的工艺，步骤如下：取新生牛血清，4000r/min离心10min，取上清，加入上清液质量0.2%的活性炭进行吸附，吸附30min；吸附后，4000r/min离心20min，取上清，依次用0.8μm滤膜、0.65μm滤膜、0.45μm滤膜、0.22μm滤膜、0.1μm滤膜过滤，即得到去除了内毒素的新生牛血清。
本发明采用活性炭吸附及逐级过滤的方法，可显著降低以往在新生牛血清生产过程中作为废弃物的高内毒素含量牛血清中内毒素的含量，增加新生牛血清生产的效率和附加值。本发明的活性炭的加入量，以及过滤膜的选择，均是特定的，是通过优化实验得到的最佳方案，该最佳方案，可以去掉新生牛血清中的内毒素，且对血清的增殖效果、血清蛋白浓度的影响最低。
具体实施方式
下面结合实施例对本发明作进一步的说明。
实施例去除新生牛血清内毒素实验
（1）原料新生牛血清的准备
采用新生牛血清生产过程中内毒素含量较高的新生牛血清作为本发明的原料牛血清。
血清内毒素含量的检测使用湛江博康海洋生物有限公司鲎试剂内毒素检测试剂盒。
（2）去除牛血清中内毒素的工艺
将称量的不同投加量活性炭（0.1%、0.2%、0.3%、0.4%、0.5%、1%、2%、5%）缓慢撒进血清并柔和搅拌至均匀，持续柔和搅拌30min后，经逐级过滤去除活性炭并获得新生牛血清。过滤后的血清经无菌试验验证，达到无菌要求。
（3）内毒素含量的检测
将鲎试剂用无内毒素蒸馏水100μL溶解，然后分别加入稀释40倍的原料血清留样100μL与鲎试剂混合，同时设有阴性对照和阳性对照，37℃孵育1h后通过观察凝集情况判断标本中的细菌内毒素含量范围。
活性炭处理含内毒素新生牛血清的结果见表1，试验中≥0.5%活性炭投加量的血清标本均未发生凝集，表明血清中的细菌内毒素低于0.125EU/mL（试剂盒检测限量为0.125EU/mL），而0.1%、0.2%、0.3%、0.4%的活性炭投加量的血清内毒素含量也都符合国标规定标准（中国药典2010版要求≤10EU/mL）。
表1活性炭处理血清内毒素检测结果
（4）细胞增殖试验
将呈对数生长状态的瓶内MA104细胞用0.125%胰酶消化，用含10%受试血清（终浓度）—MEM分散细胞并连续传代6代，第7代时将细胞计数并接种于96孔板内，24h镜下观察结合中性红染色测定A540值计算细胞增殖率。阳性对照血清采用GIBICO产品血清。
以上述含10%受试血清（终浓度）—MEM培养液连续培养MA104细胞6代，细胞生长良好，与标准产品比较，0.1%、0.2%、0.3%细胞相对增值率在95%以上，细胞生长良好。投加不同浓度树脂去除内毒素后的血清细胞增殖试验结果见表2。
表2MA104细胞增殖试验结果n=3
（5）蛋白含量测定
将处理后血清用BCA法检测蛋白含量，结果发现处理后的新生牛血清蛋白含量也有不同程度的下降，详见表3。
表3活性炭处理血清的蛋白含量
结论：去除新生牛血清内毒素的工艺的确定
从上述试验结果可以看出，采用适量的活性炭吸附方法，可以去除血清中内毒素成分。细胞增殖试验和蛋白检测结果表明，活性炭浓度对血清增殖效果和蛋白浓度有影响，适度控制使用量是运行生产程序中的要点。
结合活性炭处理血清内毒素含量、细胞增殖率以及蛋白含量的结果，选取0.2%的活性炭投加量作为去除新生牛血清高含量内毒素的最佳投加量。
最终的去除新生牛血清内毒素的工艺如下：
Claims (1)
1.一种去除新生牛血清内毒素的工艺，其特征在于：步骤如下：取新生牛血清，4000r/min离心10min，取上清，加入上清液质量0.2％的活性炭进行吸附，吸附30min；吸附后，4000r/min离心20min，取上清，依次用0.8μm滤膜、0.65μm滤膜、0.45μm滤膜、0.22μm滤膜、0.1μm滤膜过滤，采用活性炭吸附及逐级过滤的方法，降低高内毒素含量牛血清中内毒素的含量，增加新生牛血清生产的效率和附加值，即得到去除了内毒素的新生牛血清。
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| CN109971698A (zh) * | 2019-03-25 | 2019-07-05 | 哈尔滨维科生物技术有限公司 | 一种去除猪血清对传代细胞生长毒性的方法 |
| CN112322574A (zh) * | 2020-10-09 | 2021-02-05 | 赛澳美细胞技术(北京)有限公司 | 一种去除血清中抗生素的方法及其应用 |
| CN112662610A (zh) * | 2020-12-14 | 2021-04-16 | 五河县鑫盛生物技术有限公司 | 一种去除新生牛血清内毒素的工艺 |
| CN114225542B (zh) * | 2021-12-17 | 2022-09-06 | 呼和浩特市草原绿野生物工程材料有限公司 | 一种高效去除新生牛血清内毒素的方法 |
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| CN1935122A (zh) * | 2006-10-16 | 2007-03-28 | 上海市血液中心 | 亚甲蓝冻干制剂及其制备方法和应用 |
| CN102178955A (zh) * | 2011-04-26 | 2011-09-14 | 北京新华联协和药业有限责任公司 | 一种变应原溶媒及其制备方法 |
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| CN1935122A (zh) * | 2006-10-16 | 2007-03-28 | 上海市血液中心 | 亚甲蓝冻干制剂及其制备方法和应用 |
| CN102178955A (zh) * | 2011-04-26 | 2011-09-14 | 北京新华联协和药业有限责任公司 | 一种变应原溶媒及其制备方法 |
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| "细菌内毒素定量法研究活性炭与超滤法热原去除工艺";李存玉 等;《医药导报》;20100228;第29卷(第2期);第142-144页 * |
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