工艺验证方案模板

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名称 Title Doc. No. 文件编号 Process Validation (PV) Protocol for XXX XXX工艺验证方案 Page 页码

Process Validation (PV) Protocol XXX XXX工艺验证方案 Written by 起草人 Reviewed by审核人 Approved by批准人 Written department 起草部门 Issued by: Written date 起草日期 Reviewed date 审核日期 Approved date 批准日期 Effective date 生效日期 Change record: Revision number 变更记载： Date of approval Effective date 修订号批准日期生效日期年月日年月日年月日年月日颁发部门： [ ] Copy number: 拷贝号： [ ] Change reason and goal: 变更原因及目的： Distribution department: 分发部门: 所长办公室[ ] 人力资源部[ ] 后勤保卫部[ ] 物资供应部[ ] 工程服务部[ ] 生产管理部[ ] 质量保证部[ ] 销售部[ ] 仓储部[ ] 疫苗一室[ ] 疫苗二室[ ] 疫苗三室[ ] 疫苗五室[ ] 疫苗六室[ ] 生化制品室[ ] 细胞因子室[ ] 血清制品室[ ] 实验动物室[ ] 分包装室[ ] 培养基室[ ]

Verification department (department): 验证科室（部门）： Verify completion date:

验证完成日期： Validity:

有效期：

名称 Title Doc. No. 文件编号 Process Validation (PV) Protocol for XXX XXX工艺验证方案 Page 页码

Index 目录

1. Purpose目的 ............................................................................................................. 5 2. Scope范围 ................................................................................................................ 5 3. Responsibility职责................................................................................................... 5

3.1 3.2

AAA responsibilities AAA的职责 .................................................................................... 5 XXX responsibilities XXX的职责 .................................................................................... 6

4. Regulation and Guidance 法规和指南 .................................................................... 8 5. Abbreviations缩略语................................................................................................ 9 6. Protocol Description方案说明 ............................................................................... 11 7. Product And Process Description产品和工艺描述 .............................................. 14

7.1 7.2 7.3 7.4 7.5 7.6 7.7 7.8 7.9

Product specification ......................................................................................................... 14 Drug approval No. ............................................................................................................. 14 Standard Followed: ........................................................................................................... 14 Raw Materials and Adjuvants原辅料情况......................................................................... 14 Process description: .......................................................................................................... 16 Process Recipe工艺处方： .............................................................................................. 21 List of Production Plants生产车间一览 ............................................................................. 26 List of Production Equipment 生产设备一览 .................................................................... 29 Process Flow Diagram 工艺流程图 .................................................................................. 33

7.10 CPP Identification CPP确认 .......................................................................................... 34 7.11 Validation Plan验证计划 ................................................................................................... 49

8. Validation prerequisite验证先决条件 .................................................................... 51

8.1 8.2 8.3 8.4 8.5 8.6

Personal Identification人员确认 ....................................................................................... 51 Process Documentation Verification 生产文件确认 ......................................................... 51 Training Verification 培训确认 .......................................................................................... 52 System and Equipment Instruments Calibration Verification 系统和设备仪表校验确认 .. 52 System and Equipment Instruments Calibration Verification 生产相关验证状态确认 ..... 53 Test Instruments Calibration Verification 测试仪器和仪表校验确认 ................................. 53

名称 Title Doc. No. 文件编号 Process Validation (PV) Protocol for XXX XXX工艺验证方案 Page 页码

8.7 8.8

Testing Method Validation Verification检验方法验证的确认 ............................................. 54 Raw Material and Excipient Verification原辅料确认 ........................................................ 54

9. Sampling Plan and Evaluation Criteria取样计划及评估标准 ............................... 56 10. Sample Coding Method样品编码原则 ................................................................... 70 11. Process Validation Test Execution 工艺验证执行 ............................................... 72

11.1 Liquid Preparation 液体配制 ............................................................................................. 72 11.2 Cell Resurrection and Passage细胞复苏及传代 .............................................................. 74 11.3 Harvest liquid test 收获液检测 .......................................................................................... 81 11.4 Clarification Filtration Efficacy Test 澄清过滤效果检测 .................................................... 83 11.5 UF Concentration 1 Efficacy Test超滤浓缩1效果检测 .................................................... 83 11.6 Ammonium sulfate precipitation efficacy test 硫酸铵沉淀效果检测 ............................... 84 11.7 Potassium bromide extraction efficacy test 溴化钾提取效果检测 .................................... 85 11.8 First density gradient centrifugation efficacy test 一次密度梯度离心效果检测 ................ 86 11.9 Second density gradient centrifugation efficacy test 二次密度梯度离心效果检测 ........... 87 11.10 UF Concentration 2 Efficacy Test 超滤浓缩2效果检测 ................................................... 88 11.11 Chromatography purification product test 层析纯化产物检测 .......................................... 90

12. Bulk Determination原液检验 ................................................................................. 92 13. Process Validation Summarization工艺验证总结 ................................................ 93 14. PV Report工艺验证报告 ......................................................................................... 94

14.1 Process Validation Report 工艺验证报告 ......................................................................... 94 14.2 Process Validation Analysis and Evaluation 工艺验证分析与评价 ................................... 94

15. Requirements on Execution Records 执行记录要求 ........................................... 95 16. Deviation Handling 偏差处理 ............................................................................... 95 17. Change Handling 变更处理 ................................................................................... 95 18. Test Form测试表 .................................................................................................... 96

表格 1 缩略语 ................................................................................................................................ 9 表格 2原辅料质量标准表 ............................................................................................................. 11 表格 3物料消耗表1 .................................................................................................................... 21

名称 Title Doc. No. 文件编号 Process Validation (PV) Protocol for XXX XXX工艺验证方案 Page 页码

表格 4物料消耗表2 .................................................................................................................... 21 表格 5所用液体配料表 ................................................................................................................ 22 表格 6生产车间一览表 ................................................................................................................ 26 表格 7主要生产设备一览表 ......................................................................................................... 29 表格 8工艺风险分析表 ................................................................................................................ 33 表格 9工艺时间表 ....................................................................................................................... 49 表格 10取样计划表 ..................................................................................................................... 56 表格 11样品编码原则 .................................................................................................................. 70 表格 12配液取样检测信息表 ....................................................................................................... 72 表格 13复苏和传代取样表 .......................................................................................................... 74 表格 14复苏和传代样品编码 ....................................................................................................... 80 表格 15收获液标准 ..................................................................................................................... 81 表格 16澄清过滤标准 .................................................................................................................. 83 表格 17超滤浓缩1标准 .............................................................................................................. 83 表格 18硫酸铵沉淀标准 .............................................................................................................. 85 表格 19溴化钾提取标准 .............................................................................................................. 86 表格 20一次密度梯度离心标准 ................................................................................................... 87 表格 21二次密度梯度离心标准 ................................................................................................... 88 表格 22超滤浓缩2标准 .............................................................................................................. 89 表格 23纯化产物标准 .................................................................................................................. 90 表格 24原液标准 ......................................................................................................................... 92 表格 25测试表列表 ..................................................................................................................... 96

名称 Title Doc. No. 文件编号 Process Validation (PV) Protocol for XXX XXX工艺验证方案 Page 页码

1. Purpose目的

This purpose of this process validation is to provide documented evidence to show that the combination of the personnel, materials, equipment, methods, environmental conditions and the other related utilities XXX Co., LTD (XXX) can ensure the consistent production of products conforming to the enterprise internal standards and the national standards, and the processes are reliable and conforming to the GMP requirements. The process validation and the test results are to be documented based on this validation protocol.

本工艺验证用于提供文件化的证据，证明XXX疫苗一室生产XXX原液的人员、材料、设备、方法、环境条件以及其它有关公用设施的组合可以始终如一的生产出符合企业内控标准及国家法定标准的产品，工艺稳定可靠，符合GMP要求，工艺验证的过程和检查的结果将按照该验证方案进行记录。

2. Scope范围

This protocol is applicable to the process validation of XXX produced by the equipment located XXX.

本方案适用于XXX原液生产的工艺验证。

由于工作细胞库制备的过程并非常规生产过程，因此，不在本次验证中进行考察。

The labeling and packaging processes are not within the scope of this document since these processes have been validated per PQ protocol (labeling) and PQ protocol (packaging), and these processes have minimal impact on finished product quality.

贴标签和包装也不在此文件范围内，因为这些工艺已经按照PQ方案（贴标签）和PQ方案(包装)验证过，并且这些工艺对产品质量极少有影响。

3. Responsibility职责

3.1 AAA responsibilities AAA的职责

 Compile Protocol

方案的编写

 Guide Protocol execution, data collection/ Provide test data for review upon request by

customer

指导方案的实施，数据的收集/在客户要求时提供测试数据供其审核  Final report Review/ Analyze and compile data into the summary report

最终报告的审核/对数据进行分析并将其编辑入总结报告之中

名称 Title Doc. No. 文件编号 Process Validation (PV) Protocol for XXX XXX工艺验证方案 Page 页码

3.2 XXX responsibilities XXX的职责

 Production department 生产部  Review and approve protocol

审核并批准验证方案

 Assure all the equipment used for validation can be maintained timely

保证验证用所有设备均能按SOP进行及时的维护和保养

 Assure all the raw materials used for validation can be supplied timely and comply with

domestic quality criteria of enterprise

保证验证用原辅包装材料能按时到货，并符合企业内控标准  Research and development department 研发部

 Provide product registration criteria and review whether recipe and process step

described in the protocol is complying with registered criteria

提供产品的注册工艺，审核方案中生产处方、工艺与注册资料的一致性

 Review and make assurance the compliance of raw material supplier with registered

supplier

审核原辅料供应商和注册资料的一致性

 Review relevant quality criteria including raw material, intermedia and finished product

审核原辅料标准、中控质量标准和成品的质量标准  Vaccine one Plant 疫苗一室

 Coordination and implementation of this validation protocol

协调、实施本验证方案

 Training of personnel based on this validation protocol

按验证方案对有关人员进行培训

 Collection, sorting and reviewing of validation data and drafting of validation reports

(including the deviation report, if there are any deviations)

收集、整理和审核验证数据，起草验证报告（如方案执行有偏差，要完成偏差报告）。  Utility support department 工程服务部

 Assistance in implementation of this validation protocol

协助本验证方案的实施

 Calibration of instruments related to this validation protocol and providing relevant

calibration certificates and quality certificates

负责校验和本验证方案有关的仪器、仪表，出具校验报告并贴合格证

名称 Title Doc. No. 文件编号 Process Validation (PV) Protocol for XXX XXX工艺验证方案 Page 页码

 Assure normal operation of the utility system

保证公用系统的正常运行  Final report Review

最终报告的审核

 Quality control of quality department 质量部质量控制  Determination of test methods used in this validation protocol

负责确定本验证方案的检验方法

 Validation of the necessary test methods for XXXXX involved in this validation protocol

负责本验证方案涉及的重组乙型肝炎疫苗（CHO细胞）原液生产的必要的检验方法的验证

 The quality inspections on XXXXX Tablet involved in this validation protocol, and

presenting full item certificates of analysis and quality inspection reports and records 负责本验证方案涉及的重组乙型肝炎疫苗（CHO细胞）原液生产的质量检验，并出具全项检验报告，提供质量检验报告书及检验记录

 Providing technical supports and guidance for tests at each of the process steps

负责各工序检验的技术支持和指导

 Coordinating the plant in the implementation of this validation protocol

协同车间实施本验证方案  Final report Review

最终报告的审核

 Quality assurance of quality department 质量部质量保证

 Supervision of validation process, review and approval of this protocol, and ensuring

the implementation following this protocol

负责验证过程监督，方案的审核与批准，确保按批准的方案执行  Sampling at each process steps during the process validation

负责工艺验证中各工序的取样

 Coordination among different departments involved in the validation activities

负责验证涉及到的各部门之间的协调

 Providing equipment validation reports related to this validation protocol

负责提供和本验证方案有关的设备验证报告  Identification, audit and assessment of suppliers

供应商的确认、审计及评价

名称 Title Doc. No. 文件编号 Process Validation (PV) Protocol for XXX XXX工艺验证方案 Page 页码

 Assessment and handling of changes and deviations occur during this process

validation

负责对工艺验证实施过程中出现的变更和偏差的评价和处理  Final report Review

最终报告的审核

4. Regulation and Guidance 法规和指南

 EudraLex Volume 4 EU Guidelines to Good Manufacturing Practice Medicinal Products for

Human and Veterinary Use, November, 2008

EudraLex 第四卷欧盟药品生产质量管理规范指南医药产品人用及兽用，2008年11月

 Final Version of Annex 15 to the EU Guide to Good Manufacturing Practice : Qualification

and Validation , September 2001

EudraLex 第四卷药品生产质量管理规范，附录15 验证与确认，2001年9月

 EMEA - Note for guidance on process validation, September 2001

欧洲药品管理局 – 工艺验证指南，2001年9月

 EMEA - Annex II to note for guidance on process validation, January 2005

欧洲药品管理局 – 附录II 工艺验证指南

 FDA 21 Code of Federal Regulations, Part 211, current Good Manufacturing Practice for

Finished Pharmaceuticals, April 2011

FDA联邦法典第21篇第211部分，现行药物制剂生产质量管理规范，2011年4月

 FDA Process Validation: General Principles and Practices , January 2011

FDA工艺验证：一般原则和实践，2011年01月

 SFDA Good Manufacturing Practice (2010 Revision), March, 2011

SFDA 药品生产质量管理规范（2010年修订），2011年03月

 PIC PIC/S Pharmaceutical Inspection Convention, Pharmaceutical Inspection Co-operation

Scheme, “Recommendations on Validation Master Plan, Installation and Operational Qualification, Non-sterile Process Validation, Cleaning Validation”, July 2004

PIC/S药品监管公约，药品监管合作计划，“有关验证主计划、安装和运行确认、非无菌工艺验证、清洁验证的建议”，2004年7月

 A WHO Guide to good manufacturing practice(GMP) requirements , Part 2 Validation , 1997

WHO 关于GMP要求的指南，第二部分：验证，1997年

 Chinese Pharmacopoeia (CP) 2010 Edition

中国药典（CP）2010年版

名称 Title Doc. No. 文件编号 Process Validation (PV) Protocol for XXX XXX工艺验证方案 Page 页码

 Process Validation of Protein Manufacturing (Technical Report No. 42 Supplement Vol. 59,

5. Abbreviations缩略语

The abbreviations which will be used in this protocol are listed in the following form. 在下面的表格中规定了本方案中使用的缩略语。

表格 1 缩略语

Abbreviations 缩略语 API CPP CQA Definition 定义 Active Pharmaceutical Ingredient (or “Drug Substance”) 原料药（或药品物质） Critical Process Parameter 关键工艺参数 Critical Quality attribute 关键质量属性 Cleaning Validation 清洗验证 Installation Qualification 安装确认 Media Fill Validation 培养基模拟灌装试验 Not applicable 不适用 Operation Qualification 运行确认 Programmable Logic Controller 可编程逻辑控制器 Performance Qualification 性能确认 Process Validation 工艺验证 Purified Water 纯化水 Quality Assurance 质量保证 Risk Assessment 风险分析 CV IQ MFV N/A OQ PLC PQ PV PW QA RA 名称 Title Doc. No. 文件编号 Process Validation (PV) Protocol for XXX XXX工艺验证方案 Page 页码

SOP URS VMP WFI Standard Operation Procedure 标准操作程序 User Requirement Specification 用户使用要求 Validation Master Plan 验证主计划 Water for Injection 注射用水名称 Title Doc. No. 文件编号 Process Validation (PV) Protocol for XXX XXX工艺验证方案 Page 页码

6. Protocol Description方案说明

 Using this defined process validation shall be performed for the results of the Process Risk

Assessment.

本方案实施的工艺验证，是基于工艺风险评估结果的。

 Using this defined process validation shall be performed for three consecutive successful

runs based on GMP requirements to demonstrate the process control reliability and the repeatability.

鉴于XXX的生产已经有近20年历史，本次验证仅是对生产地址发生变更后且主要设备未发生变化的再验证，考虑到本产品生产周期长（总生产周期为158天）的原因，本方案实施的工艺验证将生产1批成功的产品以证明其可靠性。

 The determination of the intermediate and final product will implement enterprise internal

standard but release finished product should be in accordance with criteria of the item “XXX” in Chinese Pharmacopoeia 2010 Edition.

 半成品、成品按企业内控标准检验，成品发放按中国药典2010版三部“XXX ”标准执行

Raw material internal standard原辅料内控标准：

表格 2原辅料质量标准表

Quality Standard Doc. 质量控制标准文件 Sodium Chloride (BP) Quality Specification 氯化钠（药用级）检验质量标准 Sodium Dihydrogen Phosphate (BP) Quality Specification 磷酸二氢钠（药用级）检验质量标准 Crystal Aluminum Chloride AR Quality Specification 结晶氯化铝AR检验质量标准 Glucose (BP) Quality Specification 葡萄糖（药用级）检验质量标准 Glutamine Quality Specification 谷氨酰胺质量标准 Potassium Bromide Quality Specification 溴化钾质量标准 Disodium Edetate Quality Specification 乙二胺四乙酸二钠质量标准 Sodium Hydroxide (BP) Quality Specification 氢氧化钠（药用级）质量标准 Disodium Hydrogen Phosphate (BP) Quality Specification 磷酸氢二钠(药用级)质量标准 Merthiolate (BP) Quality Specification 硫柳汞（药用）质量标准 Doc. Number 文件编号名称 Title Doc. No. 文件编号 Process Validation (PV) Protocol for XXX XXX工艺验证方案 Page 页码

Quality Standard Doc. Doc. Number 质量控制标准文件文件编号 Hydrochloric Acid (BP) Quality Specification 盐酸（药用级）检验质量标准 Proline (BP) Quality Specification 脯氨酸（药用）质量标准 Sodium Bicarbonate (BP) Quality Specification 碳酸氢钠（药用级）检验质量标准 DMEM Quality Specification DMEM质量标准 Formaldehyde Solution (BP) Quality Specification 甲醛溶液(药用)质量标准 Ammonium Sulfate AR Quality Specification 硫酸铵AR质量标准 Glycine (BP) Quality Specification 甘氨酸（药用）质量标准 Trypsin (BP) Quality Specification 胰蛋白酶(药用)质量标准 New Born Bovine Serum Quality Specification 新生牛血清质量标准 Methotrexate 氨甲喋呤 Dimethyl Sulphoxide 二甲基亚砜 Semi-product of XXX standard, include the Harvest liquid, the Purification liquid and the Bulk. XXX中间品质量标准，包括细胞收获液、纯化产物、原液（文件号：XXX） Final product of XXX standard XXX成品质量标准：（文件号：XXX）

 The validation procedures, items and contents are divided based on the system process

steps. The process steps, validation items, sampling methods, testing methods and acceptance criteria are described for each of the process steps.

验证的程序、项目和内容中以系统工序为单位，对各工序中的所涉及的工艺步骤、验证项目、取样方法、检验方法和接受标准进行了规定和描述。

 The process recipe of Bulk of XXX (Registration batch No.: for 20μg: GUOYAOZHUNZI XXX,

and for 10μg GUOYAOZHUNZI XXX) is briefly described in this protocol. The contents of the recipe are described and the process flow diagram is listed. The process procedure is conformed to the registration standard.

本方案对XXX原液的工艺处方（注册批号：20μg：国药准字 XXX、10μg：国药准字 XXX）进行了简介，对处方内容进行描述，并且列出直观化的工艺流程图。工艺规程与注册标准一致。  This protocol is used to specify the purpose and the scope of this validation and the

responsibilities of each of the departments during the implementation of this validation

名称 Title Doc. No. 文件编号 Process Validation (PV) Protocol for XXX XXX工艺验证方案 Page 页码

protocol.

本方案旨在指明验证的目的、范围，并规定了该验证方案在实施过程中的各部门的职责。  Summarize and evaluate the entire process validation based on the validation of each of the

process steps and record the evaluation results to the report.

在各工序验证的基础上，对整个工艺验证进行了总结评价，评价结果记录到验证报告中。

名称 Title Doc. No. 文件编号 Process Validation (PV) Protocol for XXX XXX工艺验证方案 Page 页码

7. Product And Process Description产品和工艺描述

7.1 Product specification

(1) Ampoule, one human dose of 1.0ml, containing 10μg of HBsAg (1) 安瓿瓶，每1次人用剂量为1.0ml，含HBsAg 10μg；

(2) Ampoule, one human dose of 1.0ml, containing 20μg of HBsAg (2) 安瓿瓶，每1次人用剂量为1.0ml，含HBsAg 20μg； (3) Vial, one human dose of 1.0ml, containing 20μg of HBsAg (3) 西林瓶，每1次人用剂量为1.0ml，含HBsAg 20μg； (4) Vial, one human dose of 0.5ml, containing 10μg of HBsAg (4) 西林瓶，每1次人用剂量为0.5ml，含HBsAg 10μg。 7.2 Drug approval No.

20μg：GUOYAOZHUNZI S10960047 20μg：国药准字 S10960047 10μg：GUOYAOZHUNZI S10960080 10μg：国药准字 S10960080

7.3 Standard Followed:

Chinese Pharmacopoeia 2010 Edition 执行标准：中国药典2010版

7.4 Raw Materials and Adjuvants原辅料情况

Table 3 Raw Materials and Adjuvants

表格 4原辅料信息表

Name Sodium Chloride Disodium hydrogen phosphate Crystal Aluminum Chloride Glucose Glutamine Potassium Bromide Disodium Edetate Specifications BP BP AP AP usp Grade AP AP Manufacturer Tianjin Haiguang Pharmaceutical Industrial Co., Ltd. Hunan Jiudian Pharmaceutical Co., Ltd. Beijing Yili Fine Chemical Co., Ltd. Baitaike Bioltechnology Co., Ltd. (filling) invitrogen corporation (Gibco) Beijing Yili Fine Chemical Co., Ltd. Sinopharm Chemical Reagent Co., Ltd Packaging Specifications 1000g/bag 0.5kg/bottle 500g/bottle 500g/bottle 1kg /drum 500g/bottle 250g/bottle 名称 Title Doc. No. 文件编号 Process Validation (PV) Protocol for XXX XXX工艺验证方案 Page 页码

Name Sodium Hydroxide Mono-water disodium hydrogen phosphate Merthiolate Hydrochloric acid Proline Sodium bicarbonate DMEM Formaldehyde Ammonium sulfate Glycine Pancreatin Methotrexate Dimethyl Sulphoxide New born bovine serum Specifications AP BP BP (USP/BP/EP) AP - AP - AP AP AP - ≥99.0% - Top grade - Manufacturer Beijing Chemical Factory Hunan Jiudian Pharmaceutical Co., Ltd. GIHON Beijing Chemical Factory Shanghai Xiehe Amino Acid Co., Ltd. Beijing Chemical Factory Gibco Sinopharm Chemical Reagent Co., Ltd Beijing Yili Fine Chemical Co., Ltd. Sinopharm Chemical Reagent Co., Ltd BD Gibco sigma-aldrich（fluka-analytical） SERVA Electrophoresis GmbH Wuhan Sanli Biotechnology Co., Ltd. Inner Mongolia Jinyuankang Biotechnology Co., Ltd. Packaging Specifications 500g/bottle 0.5kg/bottle 1kg /drum 2500ml/ bottle 500g/bag 500g/bottle 6687g/drum 500ml/ bottle 25kg /drum 100g/ bottle 500g/bottle 100g/ bottle 100mg/ tube 1L/ bottle 1000ml/ bottle 500ml/ bottle 原辅料名称氯化钠磷酸氢二钠结晶氯化铝葡萄糖谷氨酰胺溴化钾乙二胺四乙酸二钠氢氧化钠一水磷酸二氢钠硫柳汞盐酸规格药用级药用级分析纯分析纯 usp Grade 分析纯分析纯分析纯药用级药用级（USP/BP/EP）分析纯生产厂家天津海光药业有限公司湖南九典制药有限公司北京益利精细化学品有限公司宝泰克生物科技公司（分装） invitrogen corporation（Gibco）北京益利精细化学品有限公司国药集团化学试剂有限公司北京化工厂湖南九典制药有限公司 GIHON 北京化工厂包装规格 1000g/袋 0.5kg/瓶 500g/瓶 500g/瓶 1kg/桶 500g/瓶 250g/瓶 500g/瓶 0.5kg/瓶 1Kg/桶 2500ml/瓶名称 Title Doc. No. 文件编号 Process Validation (PV) Protocol for XXX XXX工艺验证方案 Page 页码

原辅料名称脯氨酸碳酸氢钠 DMEM 甲醛硫酸铵甘氨酸胰酶氨甲喋呤二甲基亚砜新生牛血清 - 内蒙古金源康生物工程有限公司 500ml/瓶规格 - 分析纯 - 分析纯分析纯分析纯 - Gibco ≥99.0% - 优级 sigma-aldrich（fluka-analytical） SERVA Electrophoresis GmbH 武汉三利生物技术有限公司 100g/瓶 100mg/管 1L/瓶 1000ml/瓶生产厂家上海协和氨基酸有限公司北京化工厂 Gibco 国药集团化学试剂有限公司北京益利精细化学品有限公司国药集团化学试剂有限公司 BD公司包装规格 500g/袋 500g/瓶 6687g/桶 500ml/瓶 25kg/桶 100g/瓶 500g/瓶 7.5 Process description:

工艺简述：

 Process Cycle 工艺周期

Table 5 Process Schedule

表格 6工艺时间表

8 C31 - first 15L bottle incubation C31-15L瓶第一次培养 C32 - second 15L bottle incubation C32-15L瓶第二次培养 20 consecutive harvest 连续20次收获液 Filtration for cell residue removal 去细胞残渣过滤 UF concentration 超滤浓缩 Ammonium sulfate precipitation 硫酸铵沉淀 Potassium bromide extraction 溴化钾提取 First density gradient centrifugation 一次密度梯度离心 Second density gradient centrifugation 二次密度梯度离心 UF concentration 超滤浓缩 Chromatography purification 层析纯化 Bulk solution combination 原液合并 5~7 9 10 11 12 13 14 15 5~7 40 0.5 0.5 4~5 1 1.5 16 17 18 19 1.5 1 1 0.5  Preparation of production cells生产用细胞制备 Take 1 to 2 tubes of cells from the working cell bank (of a same batch No.), resurrect, mix and inoculate them into a 100ml cell cultivation bottle in a ratio of 1:1. After the cells have grown full and adhering to the wall to a single layer, perform the 0.25% trypsin digestion and passage. And then inoculate them into a 100ml cell cultivation bottle in a ratio of 1:4. Passage and proliferate following the sequence of 100ml cell cultivation bottle, Kolle flask and the rotary bottle. The ratio is determined according to different cell cultivation bottle specifications. Two 100ml cell cultivation bottles are used for the passage to 1 Kolle flask. One Kolle flask is used for the passage to 1 3L rotary bottle. One 3L rotary bottle is used for the passage to 7 to 8 3L rotary bottles. One 3L rotary bottle is used for the passage to 1 15L rotary bottle. One 15L rotary bottle is used for the passage to 7 to 8 15L rotary bottles. Incubate them while the bottles are kept still or rotated at 36±1℃ (the 100ml cell cultivation bottles are kept still during incubation and rotary bottles are rotated). The duration of each expansion incubation step is 5 to 7 days. After proliferated to generation No. 32, the change solution is maintained. 取工作细胞库细胞1～2支（同一批号），复苏混合后按1：1比例接种100ml细胞培养瓶，待细胞贴壁长满至单层后，0.25%胰蛋白酶消化传代，按1：4比例接种100ml细胞培养瓶。按100ml细胞培养瓶→克氏瓶→转瓶传代扩增，比例根据不同规格细胞培养瓶而定，2个100ml

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细胞培养瓶传1个克氏瓶；1个克氏瓶传1个3L转瓶；一个3L转瓶传7～8个3L转瓶；一个3L转瓶传1个15L转瓶；1个15L转瓶传7～8个15L转瓶。置36±1℃条件下，采用静止或旋转培养（100ml细胞培养瓶和克氏瓶采用静止培养，转瓶采用旋转培养法）。每次扩增培养时间为5～7天，传代扩增至32代，开始维持换液。  Incubation media培养液

The growth media used for cell passage and proliferation is DMEM solution containing 9% to 10% inactivated new born bovine serum with a final concentration of methotrexate of 10-6mol/L and L-glutamine of 2mmol/L. The maintenance solution is DMEM solution containing 3% to 5% inactivated new born bovine serum.

细胞传代扩增用生长液为含有9%～10%灭能新生牛血清的DMEM液，其中含终浓度为10-6mol/L氨甲喋呤、2mmol/L L-谷氨酰胺。维持液为含有3%～5%灭能新生牛血清的DMEM液。

 Harvesting收获

After passage to the No. 32 generation and the cells have grown into a compact single layer adhering to the wall (Day 5 to 7), discard the growth liquor, add the maintenance solution, discard the incubation liquor 48 hours later and add the maintenance solution. Replace the maintenance solution every 48 hours thereafter and harvest the incubation liquor. Measure the HBsAg content in the harvested supernatant with ELISA (or reverse hemagglutination method), the result shall be above 1.0mg/L (RPHA titer 1:128). At the same time a sterility test shall be performed for the harvested solution of each bottle. The number of harvesting for a same batch of production cells shall be 19 to 21 (20±1) and the harvest solution of 1 to 2 batches can be combined for purification.

传代扩增至32代的细胞贴壁长成致密单层后（5～7天），弃去生长液，加入维持液，48小时后弃去培养液，加入维持液。此后，每48小时更换一次维持液，并收获培养液。收获的培养上清液采用ELISA法(或反向血凝法)检测HBsAg含量，应在1.0mg/L(RPHA滴度1：128)以上，同时每瓶细胞培养收获液应进行无菌检查，同一批生产细胞收液次数应达到19～21（20±1）次，每1～2批收获液可合并纯化。  Purification纯化

 Filtration过滤

The harvested culture media go first through a 0.8μm filter and then a 0.2μm filter to remove any cell residues.

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收获的培养液通过0.8μm，然后通过0.2μm滤柱，过滤去除细胞碎片。

The filtrate goes through a filter that can retain molecules of 100KD and above. Then it is concentrated 3 to 5 times through UF.

滤液通过截留分子量100KD的滤柱，超滤浓缩至3～5倍。

 Ammonium sulfate precipitation 硫酸铵沉淀

After UF concentration, 312g of ammonium sulfate is added to each liter of the obtained culture media. It is then allowed to stand at room temperature for 4 to 5 days after stirring to allow HBsAg to precipitate.

超滤浓缩后的培养液每升加入312g硫酸铵，搅拌均匀后，室温放置4～5天，使HBsAg沉淀。

 Potassium bromide extraction溴化钾提取

The precipitation solution is centrifuged for 30min at 4000r/min. The supernatant is discarded. The precipitates are sufficiently dissolved after adding potassium bromide powder (4.5 to 5.5g of potassium bromide is added to each liter of the harvest solution). Then 50% potassium bromide is used for the extraction of HBsAg.

沉淀液4000r/min离心30min，弃上清，沉淀中加入溴化钾固体粉末充分溶解（每升收获液加4.5～5.5g溴化钾），再用50%溴化钾溶液提取HBsAg。

 Potassium bromide density gradient centrifugation (twice) 溴化钾密度梯度离心（两

次）

HBsAg is separated with two potassium bromide density gradient centrifugations. The density ratios of the two density gradient centrifugation were both:

采用两次溴化钾密度梯度离心HBsAg，两次离心时密度梯度液的比重均为： Solution A:1.04g/ml sodium chloride solution A液:1.04g/ml的氯化钠溶液；

Solution B: 1.28g/ml sodium bromide solution B液:1.28g/ml的溴化钾溶液； Solution C: 1.32g/ml test substance C液:1.32g/ml的供试品；

Solution D: 1.34g/ml sodium bromide solution

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D液:1.34g/ml的溴化钾溶液。

Inject the samples following the sequence of solution A, solution B, solution C and solution D. The speed of the centrifuge is 2000rpm both at the time of sample injection and at the time of sample collection. The centrifugation is performed at a speed o 25000rpm for 22 hours. HBsAg peaks are collected according to the UV absorbance value at 280nm. The HBsAg peak is peak B. The same method is used for the collection of HBsAg for the two potassium bromide density gradient centrifugation.

按A液、B液、C液、D液的顺序依次上样，上样和收样时离心机的转速均为每分钟2000转。以每分钟25000转的转速离心22小时，根据波长为280nm的紫外吸光度值收集HBsAg峰。HBsAg峰为B峰。两次溴化钾密度梯度离心收集HBsAg的方法相同。

 Column chromatography 柱色谱

The samples collected through the two density gradient centrifugation are filtered through a UF membrane that can retain molecules of 100KD to concentrate it (for 4 to 5 times). And 20mmol/L phosphate buffer solution with a pH value of 7.0 is used for washing. Sepharose 4 FF gel filtration and chromatography are performed for the concentrated samples. The sample injection amount is 4% to 6% of the column volume. The mobile phase is 20mmol/L phosphate buffer solution with a pH value of 7.0, the elution speed is 480ml/h and HBsAg peaks are collected according to a UV absorbance value at 280nm. The HBsAg peak is peak B. The liquid collected after sterile filtration is the purified product.

二次密度梯度离心收集样品通过截留分子量100KD的超滤膜超滤浓缩（4～5倍），并用pH 7.0的20mmol/L 磷酸盐缓冲液进行冲洗。浓缩样品进行Sepharose 4 FF凝胶过滤层析，上样量为柱体积的4%～6%，流动相为pH 7.0的20mmol/L 磷酸盐缓冲液，洗脱流速为480ml/h，根据波长为280nm的紫外吸光度值收集HBsAg峰。HBsAg峰为B峰。经除菌过滤后收集的液体即为纯化产物。

 Formaldehyde treatment 甲醛处理

Add 5% formaldehyde solution to the HBsAg purified product. The final concentration of formaldehyde is 200μg/ml. Maintain the temperature at 36℃±1℃ for 72 hours.

向HBsAg纯化产物中加入5%甲醛溶液，甲醛终浓度为200μg/ml，置36℃±1℃保温72小时。  Sterilization of Purified Product 纯化产物除菌

After acceptable results are obtained, HBsAg purified products from a same cell digestion batch are combined. And the bulk is obtained after UF, concentration and 0.2μm sterile

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filtration.

同一细胞消化批来源的HBsAg纯化产物检定合格后合并，经超滤、浓缩、0.2μm除菌过滤后即为原液。

7.6 Process Recipe工艺处方： Major Recipe主处方：

Materials consumed for the production of 1000 units of vaccines (1.0ml/vial): 按生产1000支疫苗(1.0ml/瓶) 计算，所消耗物料量为：

Table 7 Material Consumption Table 1

表格 8物料消耗表1

Material Name Material Code Consumption 物料名称物料代码用量 DMEM HP0010-2 636.9g New born bovine serum HP0002-2 1429ml～2381ml 新生牛血清 Ammonium sulfate H01020-1 14857g 硫酸铵 Potassium Bromide H02132-1 238g 溴化钾 Sodium Chloride H01002 9350mg 氯化钠 Aluminum Chloride H01018 2.68g 氯化铝 Merthiolate HW0011 33000μg 硫柳汞 Materials consumed for the production of 1000 units of vaccines (0.5.0ml/vial): 按生产1000支疫苗(0.5ml/瓶) 计算，所消耗物料量为：

Table 9 Material Consumption Table 2

表格 10物料消耗表2

Material Name 物料名称 DMEM New born bovine serum 新生牛血清 Ammonium sulfate 硫酸铵 Potassium Bromide 溴化钾 Material Code 物料代码 HP0010-2 HP0002-2 Consumption 用量 318.5g 715ml～1191ml 7429g 119g H01020-1 H02132-1 名称 Title Doc. No. 文件编号 Process Validation (PV) Protocol for XXX XXX工艺验证方案 Page 页码

Sodium Chloride H01002 氯化钠 Aluminum Chloride H01018 氯化铝 Merthiolate HW0011 硫柳汞 The HBsAg contained in a 10μg/unit vaccine is 13200μg. 规格为10μg/支的疫苗含乙型肝炎病毒表面抗原为13200μg； The HBsAg contained in a 20μg/unit vaccine is 24200μg. 规格为20μg/支的疫苗含乙型肝炎病毒表面抗原为24200μg。

5100mg 1.46g 18000μg The contents of merthiolate and residual bovine serum protein conform to requirements in Volume III of CP 2010.

硫柳汞含量、牛血清白蛋白残留量等符合《中华人民共和国药典》2010版三部要求。

For the consumption of DMEM, 1337.5g DMEM is used for the preparation of each 1000L DMEM culture media.

DMEM用量折算：每配制1000L DMEM培养液需用1337.5g DMEM。

For the consumption of aluminum chloride, it = aluminum hydroxide consumption ×241.43÷78. 氯化铝用量折算：氯化铝用量=氢氧化铝用量×241.43÷78

Table 11 Liquid Material Consumption Table

表格 12所用液体配料表

3％DMEM culture media 3％DMEM培养液 DMEM powder 133.75g DMEM粉末 Glycine 0.1g 甘氨酸 L-Proline 0.35g L-脯氨酸

Sodium bicarbonate 37g powder

碳酸氢钠粉末

Water For Injection 至10L 注射用水

New born bovine 3％ serum 新生牛血清 Total 󰀀10L 总计

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4％DMEM culture media 4％DMEM培养液 DMEM powder 133.75g DMEM粉末 Glycine 0.1g 甘氨酸 L-Proline 0.35g L-脯氨酸

Sodium bicarbonate 37g powder

碳酸氢钠粉末

Water For Injection 至10󰀀 注射用水

New born bovine 4％ serum 新生牛血清 Total 10L 总计

5％DMEM culture media 5％DMEM培养液 DMEM powder 133.75g DMEM粉末 Glycine 0.1g 甘氨酸 L-Proline 0.35g L-脯氨酸

Sodium bicarbonate 3󰀀g powder

碳酸氢钠粉末

Water For Injection 至10L 注射用水

New born bovine 5％ serum 新生牛血清 Total 10L 总计

10％DMEM culture media 10％DMEM培养液 DMEM powder 133.75g DMEM粉末 Glycine 0.1g 甘氨酸 L-Proline 0.35g L-脯氨酸

Sodium bicarbonate 37󰀀 powder

碳酸氢钠粉末

Water For Injection 至10L

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注射用水

New born bovine serum 新生牛血清 Total 总计

10％ 10L

Solution A for ultra-speed centrifugation 超离所需液体A Sodium Chloride 500g 氯化钠 Edetate 3󰀀2g 乙二胺四乙酸

Water For Injection 至8L 注射用水 Total 8L 总计

Solution B for ultra-speed centrifugation 超离所需液体B Sodium Chloride 500g 氯化钠 Edetate 3.2g 乙二胺四乙酸

Potassium bromide 3500g 溴化钾

Water For Injection 至8.󰀀L 注射用水 Total 8.5L 总计

Solution D for ultra-speed centrifugation 超离所需液体D

Potassium bromide 5000g 溴化钾

Water For Injection 至8.󰀀L 注射用水 Total 8.5L 总计

0.25 trypsin solution 0.25胰蛋白酶溶液 Sodium Chloride 85g 氯化钠 Typsin 25g 蛋白胰酶

Water For Injection 至10L 注射用水 Total 10L 总计

75% ammonium sulfate solution

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75%硫酸铵溶液 Ammonium sulfate 硫酸铵

Water For Injection 注射用水 Total 总计

7.5kg 至10L 10L

0.2mol/L glutamine 0.2mol/L谷氨酰胺

Glutamine 292.3󰀀 谷氨酰胺

10g Glucose 葡萄糖

Water For Injection 至10L 注射用󰀀 Total 10L 总计

MTX solution MTX溶液

Methotrexate 454mg 甲氨蝶呤

Sodium hydroxide 20g 氢氧化钠

Water For Injection 至10L 注射用水 Total 10L 总󰀀

8% sodium bicarbonate 8%碳酸氢钠

Sodium bicarbonate 800g 碳酸氢钠

Water For Injection 至10L 注射用水 Total 󰀀0L 总计

20mmol/L phosphate buffer solution (pH 7.0) 20mmol/L磷酸盐缓冲液（PH值7.0） Disodium hydrogen 17 phosphate 磷酸氢二钠

Sodium dihydrogen 10.2g phosphate 磷酸二氢钠

Water For Injection 至10L 注射用水 Total 10L 总计

5% formaldehyde solution 5%甲醛溶液

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Formaldehyde 500ml 甲醛

Water For Injection 至10L 注射用水 Total 10L 总计

1mol/L hydrochloric acid solution 1mol/L盐酸溶液 Hydrochloric acid 1L 盐酸

Water For Injection 至10L 注射用水 Total 1󰀀L 总计

0.85% sodium chloride solution 0.85%氯化钠溶液 Sodium chloride 85g 氯化钠

Water For Injection 至10L 注射用水 Total 10L 总计

0.1mol/L sodium hydroxide solution 0.1mol/L氢氧化钠溶液 Sodium hydroxide氢氧化钠

Water For Injection 注射用水 Total 总计

40g 至10L 10L

7.7 List of Production Plants生产车间一览

Table 13 List of Production Plants

表格 14生产车间一览表

Group 组别 Operation Room Name 操作间名称 Operation Room (5)-1 Cell 细胞 Operation Room (5)-2 16-XB-C-04 A/C 操作间(5)-1 No. 编号 Classification 洁净级别 Temperature and Humidity Requirements 温湿度要求 Temperature: 18 to 26℃ and relative humidity 45% to 65% 温度18～26℃，相对湿度45％～65％ Temperature: 18 to 26℃ and relative humidity 45% to 65% 温度18～26℃，相对湿度45％～16-XB-C-03 C 名称 Title Doc. No. 文件编号 Process Validation (PV) Protocol for XXX XXX工艺验证方案 Page 页码操作间(5)-2 Thermostatic room (2) 恒温室(2) Operation Room (1) 操作间(1) Operation Room (2) 操作间(2) Operation Room Production (3) 生产操作间(3) Thermostatic room (1)-1 恒温室(1)-1 Thermostatic room (1)-2 恒温室(1)-2 Rough extraction room 粗提间 Centrifugation room Purification 离心间纯化 Chromatography room 层析间 Sterilization room 除菌间 16-CH-C-03 C 65％ Temperature: 18 to 26℃ and relative humidity 45% to 65% 温度35～37℃，相对湿度45％～65％ Temperature: 18 to 26℃ and relative humidity 45% to 65% 温度18～26℃，相对湿度45％～65％ Temperature: 18 to 26℃ and relative humidity 45% to 65% 温度18～26℃，相对湿度45％～65％ Temperature: 18 to 26℃ and relative humidity 45% to 65% 温度18～26℃，相对湿度45％～65％ Temperature: 18 to 26℃ and relative humidity 45% to 65% 温度35～37℃，相对湿度45％～65％ Temperature: 18 to 26℃ and relative humidity 45% to 65% 温度35～37℃，相对湿度45％～65％ Temperature: 18 to 26℃ and relative humidity 45% to 65% 温度18～26℃，相对湿度45％～65％ Temperature: 18 to 26℃ and relative humidity 45% to 65% 温度18～26℃，相对湿度45％～65％ Temperature: 18 to 26℃ and relative humidity 45% to 65% 温度18～26℃，相对湿度45％～65％ Temperature: 18 to 26℃ and relative humidity 45% to 65% 温度18～26℃，相对湿度45％～16-XB-C-05 C 16-SC-A/C-01 A/C 16-SC-A/C-02 A/C 16-SC-A/C-03 A/C 16-SC-C-04 C 16-SC-C-05 C 16-CH-C-01 C 16-CH-C-02 C 16-CH-A/C-04 A/C 名称 Title Doc. No. 文件编号 Process Validation (PV) Protocol for XXX XXX工艺验证方案 Page 页码 65％ Aluminum preparation room 制铝间 Combination room 稀合间 Temperature: 18 to 26℃ and relative humidity 45% to 65% 温度18～26℃，相对湿度45％～65％ Temperature: 18 to 26℃ and relative humidity 45% to 65% 温度18～26℃，相对湿度45％～65％ 16-CH-C-08 C 16-CH-A/B-12 A/B 名称 Title Process Validation (PV) Protocol for XXX XXX工艺验证方案 Page 页码 Doc. No. 文件编号

7.8 List of Production Equipment 生产设备一览

Table 15 List of Major Production Equipment

表格 16主要生产设备一览表

Model and Specifications 规格型号 FFS02/GB-P0617D QuanProduction Capacity Equipment No. tity 数生产能力设备编号量 150-121 -- 1 Temperature control range 5℃ to 50℃. 控温范围5～50℃ JRSH-2.4型 Capacity 2.4 m3 容积2.4 m3 Capacity 2.8 m3 容积2.8 m3 Capacity 2.4 m3 容积2.4 m3 -- 002-210、 002-211 048-546、048-547 2 094-177 1 094-176 1 001-231 1 Name 名称 Integrity tester 完整性测试仪 Incubator 孵箱 Class 100 dry heat sterilization oven 百级净化干热灭菌烘箱 Class 100 dry heat sterilization oven 百级净化干热灭菌烘箱 Pure steam autoclave 纯蒸汽压力蒸汽灭菌器 Disk filter 圆盘过滤器 Manufacturer 厂家 PALL Sanyo of Japan 日本三洋 Nanjing Kangda Bioengineering Equipment Co., Ltd. 南京康达生物工程设备厂 Nanjing Kangda Bioengineering Equipment Co., Ltd. 南京康达生物工程设备厂 Changchun Huari Medical Appliances Co., Ltd. 长春市华日医疗器械有限公司 MILLIPORE MCO-20AIC JRSH-2.8型 YXQ-2.4Q2 293mm 2 名称 Title Process Validation (PV) Protocol for XXX XXX工艺验证方案 Page 页码 Doc. No. 文件编号

Name 名称 Column filter 柱式滤器 Cell incubation rotary bottle machine 细胞培养转瓶机 25 bottles 蠕动泵 Chromatograph column 层析柱 Stainless steel tank 不锈钢罐 Stainless steel tank 不锈钢罐 Peristaltic pump 蠕动泵 Manufacturer 厂家 PALL Nanjing Kangda Bioengineering Equipment Co., Ltd. 南京康达生物工程设备厂 Baoding Lange Constant Flow Pump Co., Ltd. 保定兰格恒流泵有限公司 Pharmacia Changchun First Liquid Equipment Manufacturing Co., Ltd. 长春富士特流体设备制造有限公司 Changchun Huari Medical Appliances Co., Ltd. 长春市华日医疗器械有限公司 Baoding Lange Constant Flow Pump Co., Ltd. 保定兰格恒流泵有限公司 Model and Specifications 规格型号 30英寸 QuanProduction Capacity Equipment No. tity 数生产能力设备编号量 048-551～-- 10 048-559 25 bottles 25瓶位 Rotation speed: 0 to 99rpm 转速0～99rpm Volume: 10L 容积10L Volume: 1000L 222-117～222-124 259-002、259-003 061-383、061-383 052-774～052-833 XP-3 60 BT00-100M 7 10×100cm 2 1000L 容积1000L 2 600L Volume: 600L 容积600L Rotation speed: 0 to 99rpm 转速0～99rpm 061-344～061-348 222-179、222-180 5 YT600-1J 2 名称 Title Process Validation (PV) Protocol for XXX XXX工艺验证方案 Page 页码 Doc. No. 文件编号

Name 名称 Roll UF equipment 卷式超滤设备 Cam transfer pump 凸轮形输送泵 Thermostatic incubation oscillator 恒温培养振荡器 Ultra-speed centrifuge 超速离心机 Low speed centrifuge 低速离心机 Microscope 显微镜 Supported laminar flow hood 支架式层流罩 Manufacturer 厂家 Beijing Shengwanquan Xinli Membrane Technology Co., Ltd. 北京圣万泉新立膜科技有限公司 Shanghai Nuoni Machinery Co., Ltd. 上海诺尼轻工机械有限公司 Shanghai Zhicheng Analytical Instrument Manufacturing Co., Ltd. 上海智诚分析仪器制造有限公司 Model and Specifications 规格型号 40m2 QuanProduction Capacity Equipment No. tity 数生产能力设备编号量 048-488、Filtration area: 40m2 048-489 2 过滤面积40m2 Stepless variable speed 无级变速 Temperature control range: 5℃ to 300℃. 控温范围5～300℃ 178-088 1 057-586 1 TR-1-2-50 ZHWY-100B Beckman optimaTM L-90K Rotation speed: 0 to 30000rpm 转速0～30000rpm 025-538～025-540 3 Thermo scientific Chongqing Guangdian 重庆光电 Suzhou Kangda Bioengineering Equipment Co., Ltd. 苏州康达生物工程设备厂 RC 3Bp+ Rotation speed: 0 to 5000rpm 转速0～5000rpm 025-541、025-542 2 XDS-1B -- Air flow: 0.36 to 0.54 m/s 风速0.36～0.54 m/s 019-225、019-226 158-362 2 C10090155T 1 名称 Title Process Validation (PV) Protocol for XXX XXX工艺验证方案 Page 页码 Doc. No. 文件编号

名称 Title Process Validation (PV) Protocol for XXX XXX工艺验证方案 Page 页码 Doc. No. 文件编号 7.9 Process Flow Diagram 工艺流程图

图表 1Process Flow Diagram工艺流程图

名称 Title Process Validation (PV) Protocol for XXX XXX工艺验证方案 Page 页码 Doc. No. 文件编号 7.10 CPP Identification CPP确认

The critical process parameters (CPP) are identified according to the process risk assessment detailed in the table above and the qualification plan is established accordingly. Please see the table below.

根据上表的工艺风险分析，确认关键工艺参数，并制定确认计划，见下表

Table 17 CPP Identification Table

表格 18CCP确认表

No. 序号 CPP Process Steps 工艺步骤 Taking out working cells from liquid nitrogen tank 从液氮罐中取出工作细胞 Operations 操作 Operation Requirements 操作要求 Working cell bank name, batch No., generation No., date, operator and checker There shall be no damages of the freezing tube upon visual inspection. 工作细胞库名称，批号，代次，日期，操作人，复核人冻存管外观应无破损 The odor of the frozen cell suspension changes and the suspension becomes clear without floating ice 细胞冻存液变味澄清液体，无浮冰。 Identification Steps 确认步骤 Remarks 备注 1 Taking out 取出 2 Thawing 解冻 Putting the cells into water bath for heating after taking them out from liquid nitrogen tank 从液氮罐取出后放入水浴加热 3 4 5 Mix the frozen cell suspension and the culture solution homogeneously and evenly place the resulted suspension at the bottom of the square bottle 细胞冻存液与培养液混合均匀，平铺于100ml方瓶的底部 Mix the frozen cell suspension and the culture solution Transfer and homogeneously and evenly addition of Mix place the resulted suspension culture solution homogenously at the bottom of the square 转移及添加培养混匀 bottle 液细胞冻存液与培养液混合均匀，平铺于100ml方瓶的底部 Mix the frozen cell suspension and the culture solution Transfer and homogeneously and evenly Stoppering and addition of place the resulted suspension wrapping with culture solution parchment paper at the bottom of the square 转移及添加培养加塞及包裹硫酸纸 bottle 液细胞冻存液与培养液混合均匀，平铺于100ml方瓶的底部 Transfer the cell suspension to a Transfer and 100ml square addition of culture solution bottle 转移及添加培养在超净台下吸取细胞悬液至100ml方液瓶中名称 Title Process Validation (PV) Protocol for XXX XXX工艺验证方案 Page 页码 Doc. No. 文件编号 Place the square bottles in a CO2 incubator and C25 square incubate for 5 to 7 bottle cultivation days 方瓶C25培养方瓶静置在CO2培养箱中培养5~7天 The cell growth is satisfactory during days 5 to 7. Discard the incubation liquid. Add 0.25% Pancreatin pancreatin and digestion shake to allow the 胰酶消化 cells drop down. 5~7天后细胞生长良好，则弃去培养液，加入0.25%胰酶，摇动，使细胞脱落 Add DMEM incubation liquid Transfer to 5 square bottles and mix well 转移至5个方瓶加入DMEM培养液，混匀 Stoppering and Transfer to 9 wrapping with square bottles parchment paper 转移至9个方瓶加塞及包裹硫酸纸 Place the square bottles in a CO2 incubator and C26 square incubate for 5 to 7 bottle cultivation days 方瓶C26培养方瓶静置在CO2培养箱中培养5~7天 The cell growth is satisfactory during days 5 to 7. Discard the incubation liquid. Add 0.25% Pancreatin pancreatin and digestion shake to allow the 胰酶消化 cells drop down. 5~7天后细胞生长良好，则弃去培养液，加入0.25%胰酶，摇动，使细胞脱落 Add DMEM Transfer to 2 incubation liquid Kolle flask and mix well 转移至2个克氏加入DMEM培养瓶液，混匀 Stoppering Transfer to 2 Kolle flask 加塞 The cells grow into a compact single layer. The growth is satisfactory and the growth liquor is orange in color (pH about 7.2). 细胞长成致密单层，生长状态良好，生长液橙色（pH值7.2左右） 6 7 The cells shall return to the round shape and drop down into the square bottles. A homogenous liquid shall be obtained. 细胞应恢复圆形，从方瓶中脱落，形成均匀的液体 8 The cells are suspended in the incubation liquid homogenously. 细胞均匀悬浮于培养液中 The stopper is placed closely. 加塞严密 The cells grow into a compact single layer. The growth is satisfactory and the growth liquor is orange in color (pH about 7.2). 细胞长成致密单层，生长状态良好，生长液橙色（pH值7.2左右） 9 10 11 The cells shall return to the round shape and drop down into the square bottles. A homogenous liquid shall be obtained. 细胞应恢复圆形，从方瓶中脱落，形成均匀的液体 12 The cells are suspended in the incubation liquid homogenously. 细胞均匀悬浮于培养液中 The stopper is placed closely. 加塞严密 13 名称 Title Process Validation (PV) Protocol for XXX XXX工艺验证方案 Page 页码 Doc. No. 文件编号转移至2个克氏瓶 Place the Kolle flasks in a CO2 incubator and C27 Kolle flask incubate for 5 to 7 incubation days 克氏瓶C27培养克氏瓶静置在CO2培养箱中培养5~7天 The cell growth is satisfactory during days 5 to 7. Discard the incubation liquid. Add 0.25% Pancreatin pancreatin and digestion shake to allow the 胰酶消化 cells drop down. 5~7天后细胞生长良好，则弃去培养液，加入0.25%胰酶，摇动，使细胞脱落 Add DMEM Transfer to 2 4L incubation liquid rotary bottles and mix well 转移至2个4L加入DMEM培养转瓶液，混匀 Place the 3L rotary bottles on a rotary bottle machine and incubate for 5 to 7 C28 3L rotary days under a bottle constant incubation temperature of 3L转瓶C28培36±1℃ 养 3L转瓶在转瓶机上，转瓶恒温36±1℃培养5~7天 The cell growth is satisfactory during days 5 to 7. Discard the incubation liquid. Add 0.25% Pancreatin pancreatin and digestion shake to allow the 胰酶消化 cells drop down. 5~7天后细胞生长良好，则弃去培养液，加入0.25%胰酶，摇动，使细胞脱落 Respectively add Combination DMEM into each 合并 of the two 3L rotary bottles. The cells grow into a compact single layer. The growth is satisfactory and the growth liquor is orange in color (pH about 7.2). 细胞长成致密单层，生长状态良好，生长液橙色（pH值7.2左右） 14 15 The cells shall return to the round shape and drop down into the square bottles. A homogenous liquid shall be obtained. 细胞应恢复圆形，从方瓶中脱落，形成均匀的液体 16 The cells are suspended in the incubation liquid homogenously. 细胞均匀悬浮于培养液中 17 The cells grow into a compact single layer. The growth is satisfactory and the growth liquor is orange in color (pH about 7.2). 细胞长成致密单层，生长状态良好，生长液橙色（pH值7.2左右） 18 The cells shall return to the round shape and drop down into the square bottles. A homogenous liquid shall be obtained. 细胞应恢复圆形，从方瓶中脱落，形成均匀的液体 19 The cells are suspended in the incubation liquid homogenously. 细胞均匀悬浮于培养液中名称 Title Process Validation (PV) Protocol for XXX XXX工艺验证方案 Page 页码 Doc. No. 文件编号分别在两个3L转瓶中加入DMEM液 Combine the cell suspension from the 2 rotary bottles. 将2个转瓶中的细胞悬液合并 The cell suspensions are completely transferred. 细胞悬液完全转移 20 Combination 合并 21 22 23 24 25 26 Transfer to 16 4L rotary bottles Stoppering 转移至16个4L加塞转瓶 Place the 3L rotary bottles on a rotary bottle machine and incubate for 5 to 7 C29 3L rotary days under a bottle constant incubation temperature of 3L转瓶C29培36±1℃ 养 3L转瓶在转瓶机上，转瓶恒温36±1℃培养5~7天 The cell growth is satisfactory during days 5 to 7. Discard the incubation liquid. Add 0.25% Pancreatin pancreatin and digestion shake to allow the 胰酶消化 cells drop down. 5~7天后细胞生长良好，则弃去培养液，加入0.25%胰酶，摇动，使细胞脱落 Respectively add DMEM into each of the 16 3L rotary Combination bottles. 合并分别在16个3L转瓶中加入DMEM液 Combine the cell suspension from the 16 rotary Combination bottles. 合并将16个转瓶中的细胞悬液合并 Transfer to 128 4L rotary bottles Stoppering 转移至128个4L加塞转瓶 The stopper is placed closely. 加塞严密 The cells grow into a compact single layer. The growth is satisfactory and the growth liquor is orange in color (pH about 7.2). 细胞长成致密单层，生长状态良好，生长液橙色（pH值7.2左右） The cells shall return to the round shape and drop down into the square bottles. A homogenous liquid shall be obtained. 细胞应恢复圆形，从方瓶中脱落，形成均匀的液体 The cells are suspended in the incubation liquid homogenously. 细胞均匀悬浮于培养液中 The cell suspensions are completely transferred. 细胞悬液完全转移 The stopper is placed closely. 加塞严密名称 Title Process Validation (PV) Protocol for XXX XXX工艺验证方案 Page 页码 Doc. No. 文件编号 Place the 3L rotary bottles on a rotary bottle machine and incubate for 5 to 7 days under a constant temperature of 36±1℃ 3L转瓶在转瓶机上，转瓶恒温36±1℃培养5~7天 The cell growth is satisfactory during days 5 to 7. Discard the incubation liquid. Add 0.25% pancreatin and shake to allow the cells drop down. 5~7天后细胞生长良好，则弃去培养液，加入0.25%胰酶，摇动，使细胞脱落 Respectively add DMEM into each of the 128 3L rotary bottles. 分别在128个3L转瓶中加入DMEM液 Combine the cell suspension from the 128 rotary bottles. 将128个转瓶中的细胞悬液合并 Stoppering 加塞 Place the 15L rotary bottles on a rotary bottle machine and incubate for 5 to 7 days under a constant temperature of 36±1℃ 15L转瓶在转瓶机上，转瓶恒温36±1℃培养5~7天 The cell growth is satisfactory during days 5 to 7. Discard the incubation liquid. The cells grow into a compact single layer. The growth is satisfactory and the growth liquor is orange in color (pH about 7.2). 细胞长成致密单层，生长状态良好，生长液橙色（pH值7.2左右） 27 C30 3L rotary bottle incubation 3L转瓶C30培养 28 Pancreatin digestion 胰酶消化 The cells shall return to the round shape and drop down into the square bottles. A homogenous liquid shall be obtained. 细胞应恢复圆形，从方瓶中脱落，形成均匀的液体 The cells are suspended in the incubation liquid homogenously. 细胞均匀悬浮于培养液中 29 Combination 合并 The cell suspensions are completely transferred. 细胞悬液完全转移 The stopper is placed closely. 加塞严密 30 Combination 合并 31 Combination 合并 32 C31 15L rotary bottle incubation 15L转瓶C31培养 The cells grow into a compact single layer. The growth is satisfactory and the growth liquor is orange in color (pH about 7.2). 细胞长成致密单层，生长状态良好，生长液橙色（pH值7.2左右） 33 Pancreatin digestion 胰酶消化 The cells shall return to the round shape and drop down into the square bottles. A homogenous liquid shall be obtained. 名称 Title Process Validation (PV) Protocol for XXX XXX工艺验证方案 Page 页码 Doc. No. 文件编号 Add 0.25% pancreatin and shake to allow the cells drop down. 5~7天后细胞生长良好，则弃去培养液，加入0.25%胰酶，摇动，使细胞脱落 Respectively add DMEM into each of the 128 15L rotary bottles. 分别在128个15L转瓶中加入DMEM液 Combine the cell suspension from the 128 rotary bottles. 将128个转瓶中的细胞悬液合并 Divide averagely into 1024 15L rotary bottles. 均分至1024个15L转瓶中 Stoppering 加塞 Place the 15L rotary bottles on a rotary bottle machine and incubate for 5 to 7 days under a constant temperature of 36±1℃ 15L转瓶在转瓶机上，转瓶恒温36±1℃培养5~7天 After secured the rotary bottle machine, connect the power and allow it to run continuously. 转瓶机停稳后通上电源，使转瓶机继续运转细胞应恢复圆形，从方瓶中脱落，形成均匀的液体 The cells are suspended in the incubation liquid homogenously. 细胞均匀悬浮于培养液中 34 Combination 合并 The cell suspensions are completely transferred. 细胞悬液完全转移 35 Combination 合并 36 37 Transfer to 1024 15L rotary bottles 转移至1024个15L转瓶 Transfer to 1024 15L rotary bottles 转移至1024个15L转瓶 The amount of cell suspension in each of the 2 flasks is about the same. 两个中的细胞悬液量基本一致 The stopper is placed closely. 加塞严密 38 C32 15L rotary bottle incubation 15L转瓶C32培养 The cells grow into a compact single layer. The growth is satisfactory and the growth liquor is orange in color (pH about 7.2). 细胞长成致密单层，生长状态良好，生长液橙色（pH值7.2左右） 39 Perform 20 consecutive runs and harvest the culture liquid every 2 days. 连续20次，每2天一次收获培养液 The power cut period shall not exceed 20 minutes. 断电时间不应超过20分钟名称 Title Process Validation (PV) Protocol for XXX XXX工艺验证方案 Page 页码 Doc. No. 文件编号 Take a 15L rotary bottle, open the stopper under laminar flow protection, and pour the culture liquid into a collection drum. Repeat the operations for another 15L bottle until all the culture liquid collection operations are completed. 取下一个15L转瓶，在层流保护下打开胶塞，将培养液倒入收集桶中，完成一个后，进行下一个，至所有15L瓶中的培养液全部收集完毕 Put the prepared DMEM to the emptied rotary bottles. 将配制好的DMEM液加入已倒出收获液的转瓶中 40 Perform 20 consecutive runs and harvest the culture liquid every 2 days. 连续20次，每2天一次收获培养液 Pour all the culture media into the collection drum through aseptic operations. 在无菌操作下，将所有培养基全部倒入收集桶中 41 42 Perform 20 consecutive runs and harvest the culture liquid every 2 days. 连续20次，每2天一次收获培养液 Perform 20 consecutive runs and harvest the culture liquid every 2 days. 连续20次，每2天一次收获培养液 Add 1.5L of DMEM. 倒入1.5LDMEM液 Stoppering 加塞 The stopper is placed closely. 加塞严密 43 Perform 20 consecutive runs and harvest the culture liquid every 2 days. 连续20次，每2天一次收获培养液 After stoppering, the rotary bottles are returned to the rotary bottle machine. Cut the power to the rotary bottle machine after the operations on all the rotary bottles are completed. Return the rotary bottle machine to the thermostatic room. Connect the power and start the machine after it is well positioned. 加完塞的转瓶返回 Handle with care when placing the rotary bottles back. The rotary bottles shall be held stably when moving them. The machine stop period shall not be more than 20 minutes. 转瓶放回时应轻拿轻放，移动时转瓶机时应平稳，停机时间不超过20分钟名称 Title Process Validation (PV) Protocol for XXX XXX工艺验证方案 Page 页码 Doc. No. 文件编号转瓶机，待所有转瓶均完成操作后，断开转瓶机电源，将转瓶机移回恒温室，就位后，通电、开机运转 Suck the harvest liquid in the collection drum to Filtration for cell a 500L buffer tank residue removal under negative 去细胞残渣过滤 pressure. 将收集桶中的收获液用负压抽至500L缓冲罐中 Transfer to column filter for filtration through an impeller pump and remove cell Filtration for cell residues through residue removal 0.8+0.2um 去细胞残渣过滤 filtration 用叶轮泵送至柱式过滤器过滤，通过0.8+0.2um的过滤，去除细胞残渣 The filtrated harvest liquid is collected in a Filtration for cell 500L stainless residue removal steel tank. 去细胞残渣过滤过滤后收获液收集在500L不锈钢罐中 Transfer the harvest liquid to the UF UF concentration concentration tank through the 超滤浓缩 peristaltic pump 使用蠕动泵将收获液进行超滤浓缩 The concentrate is collected in a UF 300L stainless concentration steel drum. 超滤浓缩浓缩液收集在300L不锈钢桶中 Add 75% ammonium sulfate solution equals to the Ammonium amount of the sulfate harvest liquid and precipitation stir for 30 minutes 硫酸铵沉淀加入与收获液等量75%硫酸铵溶液使用搅拌器搅拌30分钟 The vacuum pump can suck the harvest liquid to the buffer tank. The buffer tank is used as a buffering device. 负压真空泵能将收获液抽至缓冲罐，缓冲罐起缓冲作用， 44 45 All the live/dead cells and cell residues shall be removed. 应去除所有死/活细胞和细胞碎片 The quantity shall be determined. 应进行定量 46 47 The UF system can retain proteins above 100kd to concentrate the harvest liquid to less than 240L. 超滤系统能截留100kd以上蛋白，使收获液体积浓缩至240L以下 It shall not be more than 240L. 应不大于240L 48 49 It shall be sufficiently homogenous. 应充分混匀名称 Title Process Validation (PV) Protocol for XXX XXX工艺验证方案 Page 页码 Doc. No. 文件编号 50 Ammonium sulfate precipitation 硫酸铵沉淀 Allow to stand for 4 to 5 days at room temperature 室温下静置4~5天 Remove the floating foams on day 2 of standing and break the suspended proteins. 静置第二天撇去浮沫，打散悬浮蛋白 After standing, siphon and discard the supernatant. 静置完成后虹吸法弃去上清液， Transfer the precipitation to the low speed centrifuge. 沉淀液转移至低速离心机离心杯中， The centrifuge is set to 4℃ and the temperature dropping period is 15 minutes. 离心机设置为4℃，降温15分钟 4000rpm low speed centrifugation for 30 min to separate the supernatant and the precipitates 4000rpm低速离心30min，分离上清液和沉淀 Dry heat sterilize the potassium bromide powder at 180℃ for 2 hours. 溴化钾粉末经180℃干热灭菌2小时 4000rpm low speed centrifugation for 10min 4000rpm低速离心10min， Combine the all the supernatants. 合并历次离心的上清液 The complex of proteins and ammonium sulfate has been precipitated. 蛋白与硫酸铵的复合物已沉淀 51 Ammonium sulfate precipitation 硫酸铵沉淀 Allow all the suspended proteins to participate in precipitation. 使悬浮蛋白全部能够参与沉淀 The supernatant shall be siphoned completely as much as possible. 上清液尽量吸取彻底 52 Ammonium sulfate precipitation 硫酸铵沉淀 Equal amount of precipitate shall be added into each cup. 每杯中应加等量沉淀液 53 Low speed centrifugation 低速离心 The temperature in the centrifugation chamber shall drop to 4℃. 离心机腔室应降为4℃ 54 Low speed centrifugation 低速离心 The complex is separated into the supernatant and a kind of paste like precipitate. 复合物分离为上清液和糊状沉淀 55 Low speed centrifugation 低速离心 The sterilized materials shall be sterile. 灭菌后物料应为无菌 56 Potassium bromide extraction 溴化钾提取 The mixture shall be sufficiently separated. 混合液应充分分离 57 Potassium bromide extraction 溴化钾提取 Potassium bromide extraction 溴化钾提取 58 The quantity shall be determined. 应定量名称 Title Process Validation (PV) Protocol for XXX XXX工艺验证方案 Page 页码 Doc. No. 文件编号 First density gradient centrifugation 一次密度梯度离心 Set the centrifugation program and start the centrifuge 设定离心程序，开启离心机 When the rotation speed reaches 2000rpm, add 500ml solution A, 200ml solution B and 1000ml solution C one after another. 当转速达到2000rpm后，依次加入A液500ml、B液200ml、C液1000ml Set the rotation speed to 25000rpm and the temperature to 20℃. Centrifuge for 20 hours. 设定转速为25000rpm，温度设定20℃，离心20小时 After the set time has elapsed, set the centrifuge rotation speed to 2000rpm and add 30ml of solution D. 到达设定时间后，设置离心机转速为2000rpm，加入D液30ml With the addition of solution D, the original liquid flows out of the centrifuge. Monitor with a UV light at 280nm. Collect the antigen solution. 随着D液加入，之前的液体流出离心机，用280nm波长紫外灯监测，收集抗原溶液 Set the centrifugation program and start the centrifuge 设定离心程序，开启离心机 Set the rotation speed to 2000rpm 设定转速2000rpm 59 60 First density gradient centrifugation 一次密度梯度离心 Solutions A, B and C shall be added one after another. A、B、C液应顺序全部加入 61 First density gradient centrifugation 一次密度梯度离心 The equipment shall run stably within this 20 hours. 20小时中设备应平稳运行 62 First density gradient centrifugation 一次密度梯度离心 Solution D shall be added completely. D液应全部加入 63 First density gradient centrifugation 一次密度梯度离心 Collect all the antigen containing liquid. 将含抗原的部分的液体全部收集 64 Second density gradient centrifugation 二次密度梯度离心 Set the rotation speed to 2000rpm 设定转速2000rpm 名称 Title Process Validation (PV) Protocol for XXX XXX工艺验证方案 Page 页码 Doc. No. 文件编号 When the rotation speed reaches 2000rpm, add e00ml solution A, 700ml solution B and 700ml solution C one after another. 当转速达到2000rpm后，依次加入A液300ml、B液700ml、C液700ml Set the rotation speed to 25000rpm and the temperature to 20℃. Centrifuge for 20 hours. 设定转速为25000rpm，温度设定20℃，离心22小时 After the set time has elapsed, set the centrifuge rotation speed to 2000rpm and add 30ml of solution D. 到达设定时间后，设置离心机转速为2000rpm，加入D液30ml With the addition of solution D, the original liquid flows out of the centrifuge. Monitor with a UV light at 280nm. Collect the antigen solution. 随着D液加入，之前的液体流出离心机，用280nm波长紫外灯监测，收集抗原溶液 Transfer the antigen solution to the UF concentration tank through the peristaltic pump 使用蠕动泵将抗原溶液进行超滤浓缩 The concentrate is collected in a 300L stainless steel drum. 65 Second density gradient centrifugation 二次密度梯度离心 Solutions A, B and C shall be added one after another. A、B、C液应顺序全部加入 66 Second density gradient centrifugation 二次密度梯度离心 The equipment shall run stably within this 22 hours. 22小时中设备应平稳运行 67 Second density gradient centrifugation 二次密度梯度离心 Solution D shall be added completely. D液应全部加入 68 Second density gradient centrifugation 二次密度梯度离心 Collect all the antigen containing liquid. 将含抗原的部分的液体全部收集 69 UF concentration 超滤浓缩 The UF system can retain proteins above 100kd to concentrate the harvest liquid to less than 240L. 超滤系统能截留100kd以上蛋白，使收获液体积浓缩至240L以下 70 UF concentration 超滤浓缩 It shall not be more than 240L. 应不大于240L 名称 Title Process Validation (PV) Protocol for XXX XXX工艺验证方案 Page 页码 Doc. No. 文件编号浓缩液收集在300L不锈钢桶中 Transfer the precipitation to the High speed low speed centrifugation centrifuge. 高速离心沉淀液转移至低速离心机离心杯中， 8000rpm low speed centrifugation for 35 min to High speed separate the centrifugation supernatant and 高速离心 the precipitates 8000rpm低速离心35min，分离上清液和沉淀 Elute with 20mmol/L Gel phosphate buffer chromatography solution. 凝胶层析用20mmol/L的磷酸缓冲液洗脱 Use a UV analyzer to indicate the Gel chromatography absorption peak solution 凝胶层析用紫外检测仪显示吸收峰型收液， Add formaldehyde into the solution obtained from the previous step to a Formaldehyde concentration of treatment 1/2000 甲醛处理在上步收液中加入甲醛至浓度为1/2000， Standing at 37℃ Formaldehyde incubator for 72 treatment hours 甲醛处理 37℃孵箱静置72小时 Use a 0.22um sterile filter for the sterile filtration of the product. A peristaltic pump is Sterile filtration used to providing 过滤除菌 the driving force. 用0.22um除菌过滤器对产品进行除菌过滤，以蠕动泵为动力。 Potassium Dry heat sterilize bromide the potassium extraction bromide powder 溴化钾提取 at 180℃ for 2 Equal amount of precipitate shall be added into each cup. 每杯中应加等量沉淀液 71 72 The complex is separated into the supernatant and a kind of precipitate. 复合物分离为上清液和沉淀 The antigen solution is effectively separated. 抗原溶液有效分离 73 Starting from the start to the end of the second peak. 从第二峰起峰至峰底 74 75 The formaldehyde concentration shall be 1/2000 甲醛浓度应为1/2000 The temperature is 37℃ and the time is 72 hours. 温度37℃，时间为72小时 76 77 The product shall be sterile. 产品应无菌 78 The sterilized materials shall be sterile. 灭菌后物料应为无菌名称 Title Process Validation (PV) Protocol for XXX XXX工艺验证方案 Page 页码 Doc. No. 文件编号 hours. 溴化钾粉末经180℃干热灭菌2小时 4000rpm low speed centrifugation for 10min 4000rpm低速离心10min， Combine the all the supernatants. 合并历次离心的上清液 Set the centrifugation program and start the centrifuge 设定离心程序，开启离心机 When the rotation speed reaches 2000rpm, add 500ml solution A, 200ml solution B and 1000ml solution C one after another. 当转速达到2000rpm后，依次加入A液500ml、B液200ml、C液1000ml Set the rotation speed to 25000rpm and the temperature to 20℃. Centrifuge for 20 hours. 设定转速为25000rpm，温度设定20℃，离心20小时 After the set time has elapsed, set the centrifuge rotation speed to 2000rpm and add 30ml of solution D. 到达设定时间后，设置离心机转速为2000rpm，加入D液30ml With the addition of solution D, the original liquid flows out of the centrifuge. Monitor with a UV The mixture shall be sufficiently separated. 混合液应充分分离 79 Potassium bromide extraction 溴化钾提取 Potassium bromide extraction 溴化钾提取 First density gradient centrifugation 一次密度梯度离心 80 The quantity shall be determined. 应定量 81 Set the rotation speed to 2000rpm 设定转速2000rpm 82 First density gradient centrifugation 一次密度梯度离心 Solutions A, B and C shall be added one after another. A、B、C液应顺序全部加入 83 First density gradient centrifugation 一次密度梯度离心 The equipment shall run stably within this 20 hours. 20小时中设备应平稳运行 84 First density gradient centrifugation 一次密度梯度离心 Solution D shall be added completely. D液应全部加入 85 First density gradient centrifugation 一次密度梯度离心 Collect all the antigen containing liquid. 将含抗原的部分的液体全部收集名称 Title Process Validation (PV) Protocol for XXX XXX工艺验证方案 Page 页码 Doc. No. 文件编号 light at 280nm. Collect the antigen solution. 随着D液加入，之前的液体流出离心机，用280nm波长紫外灯监测，收集抗原溶液 Set the centrifugation program and start the centrifuge 设定离心程序，开启离心机 When the rotation speed reaches 2000rpm, add e00ml solution A, 700ml solution B and 700ml solution C one after another. 当转速达到2000rpm后，依次加入A液300ml、B液700ml、C液700ml Set the rotation speed to 25000rpm and the temperature to 20℃. Centrifuge for 20 hours. 设定转速为25000rpm，温度设定20℃，离心22小时 After the set time has elapsed, set the centrifuge rotation speed to 2000rpm and add 30ml of solution D. 到达设定时间后，设置离心机转速为2000rpm，加入D液30ml With the addition of solution D, the original liquid flows out of the centrifuge. Monitor with a UV light at 280nm. Collect the antigen solution. 随着D液加入，之前的液体流出离心机，用280nm波长86 Second density gradient centrifugation 二次密度梯度离心 Set the rotation speed to 2000rpm 设定转速2000rpm 87 Second density gradient centrifugation 二次密度梯度离心 Solutions A, B and C shall be added one after another. A、B、C液应顺序全部加入 88 Second density gradient centrifugation 二次密度梯度离心 The equipment shall run stably within this 22 hours. 22小时中设备应平稳运行 89 Second density gradient centrifugation 二次密度梯度离心 Solution D shall be added completely. D液应全部加入 90 Second density gradient centrifugation 二次密度梯度离心 Collect all the antigen containing liquid. 将含抗原的部分的液体全部收集名称 Title Process Validation (PV) Protocol for XXX XXX工艺验证方案 Page 页码 Doc. No. 文件编号紫外灯监测，收集抗原溶液 91 92 93 94 95 96 97 98 Transfer the antigen solution to the UF UF concentration concentration tank through the 超滤浓缩 peristaltic pump 使用蠕动泵将抗原溶液进行超滤浓缩 The concentrate is collected in a UF 300L stainless concentration steel drum. 超滤浓缩浓缩液收集在300L不锈钢桶中 Transfer the precipitation to the High speed low speed centrifugation centrifuge. 高速离心沉淀液转移至低速离心机离心杯中， 8000rpm low speed centrifugation for 35 min to High speed separate the centrifugation supernatant and 高速离心 the precipitates 8000rpm低速离心35min，分离上清液和沉淀 Elute with 20mmol/L Gel phosphate buffer chromatography solution. 凝胶层析用20mmol/L的磷酸缓冲液洗脱 Use a UV analyzer to indicate the Gel chromatography absorption peak solution 凝胶层析用紫外检测仪显示吸收峰型收液， Add formaldehyde into the solution obtained from the previous step to a Formaldehyde concentration of treatment 1/2000 甲醛处理在上步收液中加入甲醛至浓度为1/2000， Formaldehyde Standing at 37℃ treatment incubator for 72 甲醛处理 hours The UF system can retain proteins above 100kd to concentrate the harvest liquid to less than 240L. 超滤系统能截留100kd以上蛋白，使收获液体积浓缩至240L以下 It shall not be more than 240L. 应不大于240L Equal amount of precipitate shall be added into each cup. 每杯中应加等量沉淀液 The complex is separated into the supernatant and a kind of precipitate. 复合物分离为上清液和沉淀 The antigen solution is effectively separated. 抗原溶液有效分离 Starting from the start to the end of the second peak. 从第二峰起峰至峰底 The formaldehyde concentration shall be 1/2000 甲醛浓度应为1/2000 The temperature is 37℃ and the time is 72 hours. 温度37℃，时间为72小时名称 Title Process Validation (PV) Protocol for XXX XXX工艺验证方案 Page 页码 Doc. No. 文件编号 37℃孵箱静置72小时 Use a 0.22um sterile filter for the sterile filtration of the product. A peristaltic pump is used to providing the driving force. 用0.22um除菌过滤器对产品进行除菌过滤，以蠕动泵为动力。 99 Sterile filtration 过滤除菌 The product shall be sterile. 产品应无菌 7.11 Validation Plan验证计划

One production batch of XXX is to be used for this validation. The table below lists the relevant process times.

本验证采用生产1批XXX进行验证，工艺时间表如下：

Table 19 Process Times 表格 20工艺时间表

No. 序号 1 2 Process Steps 工艺步骤 Resurrection 复苏 C25 - First square bottle incubation C25-方瓶第一次培养 C26 - second square bottle incubation C26-方瓶第二次培养 C27 - Kolle flask incubation C27-克氏瓶培养 C28 - First 3L rotary bottle incubation C28-3L转瓶第一次培养 C29 - second 3L rotary bottle incubation C29-3L转瓶第二次培养 C30 - third 3L rotary bottle incubation C30-3L转瓶第三次培养 C31 - first 15L bottle incubation C31-15L瓶第一次培养 C32 - second 15L bottle incubation C32-15L瓶第二次培养 20 consecutive harvest 连续20次收获液 Filtration for cell residue removal 去细胞残渣过滤 Time (Day) 时间（天） 1 5~7 5~7 5~7 5~7 5~7 5~7 5~7 5~7 40 0.5 3 4 5 6 7 8 9 10 11 名称 Title Process Validation (PV) Protocol for XXX XXX工艺验证方案 Page 页码 Doc. No. 文件编号 12 13 14 15 UF concentration 超滤浓缩 Ammonium sulfate precipitation 硫酸铵沉淀 Potassium bromide extraction 溴化钾提取 First density gradient centrifugation 一次密度梯度离心 Second density gradient centrifugation 二次密度梯度离心 UF concentration 超滤浓缩 Chromatography purification 层析纯化 Bulk solution combination 原液合并 0.5 4~5 1 1.5 1.5 1 1 0.5 16 17 18 19

名称 Title Process Validation (PV) Protocol for XXX XXX工艺验证方案 Page 页码 Doc. No. 文件编号 8. Validation prerequisite验证先决条件

To insure the process can be implemented in accordance with the approved protocol and the result date is authenticity and reliability, the following item should be checked and qualified. 为确保工艺验证能够按既定的方案进行，验证数据真实可靠，应在工艺验证实施之前进行下列项目的检查。

8.1 Personal Identification人员确认  Purpose目的

To document the signatures of those persons who executed and reviewed the protocol. 记录执行和检查方案的人员的签名。  Procedure 步骤

List and identify all personnel involved in the execution of the present protocol (Name, Company/Department, Title and Signature).

列出所有执行本方案的人员（姓名、公司/部门、职务和签名）。  Acceptance Criteria可接受标准

All the operators are recorded by Name, Company/Department, Title and Signature. 所有执行本方案的人员（姓名、签名、公司/部门和职务）已记录。  Test Report测试报告

Document Signatures of individuals carrying out the execution of this protocol on Test Report 1, Personnel Verification.

每个执行该验证方案的人员需要在测试报告1，人员确认中签名记录 8.2 Process Documentation Verification 生产文件确认  Purpose目的

Verify whether the SOPs are accurate in their operations. 确认在操作过程中按照工艺规程和岗位SOP准确操作。  Procedure 步骤

Verify the availability of process documents listed in the test report and record title, date of issued, revision and location.

核实在测试报告中所列的生产文件是可用的，并记录文件名称、发布日期、版本号和存放位置。  Acceptance Criteria可接受标准

All required process documents must been available.

名称 Title Process Validation (PV) Protocol for XXX XXX工艺验证方案 Page 页码 Doc. No. 文件编号所有必需的生产文件必须是可用的。  Test Report测试报告

Document Signatures of individuals carrying out the execution of this protocol on Test Report 1, Personnel Verification.

记录填入测试报告2，生产文件确认。 8.3 Training Verification 培训确认  Purpose目的

All personnel involved in the execution of the present protocol should be appropriately trained in order to execute the verifications and testing in the protocols. 所有参与方案实施的人员应得到适当的培训，以实施本方案中的检查和测试。  Procedure 步骤

All the operators have been trained.

以岗位对方案实施人员的培训档案进行检查，并记录培训过的文件名称、发布日期、版本号。  Acceptance Criteria可接受标准

All the operators have been trained. 所有执行本方案人员已得到培训。  Test Report测试报告

Document Signatures of individuals carrying out the execution of this protocol on Test Report 1, Personnel Verification. 记录填入测试报告3，培训确认。

8.4 System and Equipment Instruments Calibration Verification 系统和设备仪表校验确认  Purpose目的

Verify that the system and equipment instruments have been calibrated. 确认用于工艺验证系统和设备的仪表已经校准。  Procedure 步骤

All the operators have been trained.

检查车间校验计划，并记录系统/设备名称、仪表名称、仪表编号、校验证书编号、校准日期、有效期。

 Acceptance Criteria可接受标准

Verify that the system and equipment instruments have been calibrated. 确认用于工艺验证系统和设备的仪表已经校准。

名称 Title Process Validation (PV) Protocol for XXX XXX工艺验证方案 Page 页码 Doc. No. 文件编号 Each instrument is within its calibration period during the execution of the protocol. 在方案执行期间，所有系统和设备的仪表都在其校验有效期内。  Test Report测试报告

Document Signatures of individuals carrying out the execution of this protocol on Test Report 1, Personnel Verification.

记录填写在测试报告4，系统和设备仪表校验确认。

8.5 System and Equipment Instruments Calibration Verification 生产相关验证状态确认  Purpose目的

Verify that the system and equipment instruments have been calibrated. 确认用于生产相关系统、设备和活动已经按照车间验证矩阵进行了确认和验证。  Procedure 步骤

All the operators have been trained.

检查车间验证矩阵，并记录系统、设备和活动的名称及其确认报告名称、编号、状态和位置。  Acceptance Criteria可接受标准

Verify that the system and equipment instruments have been calibrated. 确认用于生产相关系统、设备和活动已经按照车间验证矩阵要求进行了确认。  Test Report测试报告

Document Signatures of individuals carrying out the execution of this protocol on Test Report 1, Personnel Verification.

记录填写在测试报告5，生产相关验证状态确认。

8.6 Test Instruments Calibration Verification 测试仪器和仪表校验确认  Purpose目的

Verify that the system and equipment instruments have been calibrated. 确认用于测试的仪器和仪表已经校准。  Procedure 步骤

All the operators have been trained.

检查QC校验计划和测试仪器清单，并记录仪器名称、仪表名称、仪表编号、校验证书编号、校准日期、有效期。

 Acceptance Criteria可接受标准

Verify that the system and equipment instruments have been calibrated. 确认用于测试用仪器和仪表已经校准。

名称 Title Process Validation (PV) Protocol for XXX XXX工艺验证方案 Page 页码 Doc. No. 文件编号 Each instrument is within its calibration period during the execution of the protocol. 在方案执行期间，所有测试用仪器和仪表都在其校验有效期内。  Test Report测试报告

Document Signatures of individuals carrying out the execution of this protocol on Test Report 1, Personnel Verification.

记录填写在测试报告6，测试用仪器和仪表校验确认。 8.7 Testing Method Validation Verification检验方法验证的确认  Purpose目的

The necessary relevant testing methods specified in the documents have been effectively validated to ensure that they can effectively ensure the production need. 文件中必要的相关检验方法已得到有效验证，能够有效的满足生产需要。  Qualification Procedure确认步骤

Prior to starting validation, confirm that the validation of the analytical methods to be used during this process validation has been completed and relevant persons shall check the names of the analytical methods, the validation document numbers and whether these documents have been approved or not.

在验证开始前，确认在本次工艺验证中将要用到的检验方法的验证已完成，相关人员需对检验方法的名称、验证文件号及是否批准进行确认。  Acceptance Criteria可接受标准

Record the results in Test 8 “Testing Method Validation Verification“. 检验结果记录到测试7 “检验方法验证确认表”。 8.8 Raw Material and Excipient Verification原辅料确认  Purpose目的

The suppliers of the raw materials, excipients and packaging materials used for the production of the three batches of finished products during the process validation have been effectively audited, the full item tests have been performed according to the enterprise internal standard, and the test results conform to the enterprise internal standards.

名称 Title Process Validation (PV) Protocol for XXX XXX工艺验证方案 Page 页码 Doc. No. 文件编号工艺验证生产的所用的原辅料及包装材料厂家的供应商已得到有效的确认，且已根据企业内控标准全项检验，检验结果符合企业内控标准。  Qualification Procedure确认步骤

After the raw materials and the excipients for the production of the validation batches have been delivered to the plant, operators of the relevant posts need to check the name, the batch number, the manufacturer, the supplier qualification, and the certificate of analysis for the factory acceptance test of each of them to verify that the raw materials and excipients used are conforming to the requirements.

验证批次生产所需的原辅料到达车间后，相关岗位操作人员对原辅料的名称、批号、生产厂家、供应商资质确认、入厂检验报告等进行确认，确认使用的原辅料符合要求。  Acceptance Criteria可接受标准

工艺验证生产的所用的原辅料及包装材料厂家的供应商已得到有效的确认，且已根据企业内控标准全项检验，检验结果符合企业内控标准。  Test Report测试报告

Record the results in Test 9“Raw Material and Excipient Verification“. 检查结果记录在测试9“原辅料确认表”中。

名称 Title Process Validation (PV) Protocol for XXX XXX工艺验证方案 Page 页码 Doc. No. 文件编号 9. Sampling Plan and Evaluation Criteria取样计划及评估标准

According process steep and product quality criteria perform sample plan including appropriate and necessary additional tests as well as the acceptance criteria and the corresponding analytical validation (if necessary) to make assure that the process parameter is reasonable and process control is engaged in stability.

根据工艺过程及产品质量标准，确定取样计划，包括适宜的必要的附加测试和可接受标准，及相应分析验证（如果需要），用充足的符合数据来确定工艺参数制定的合理性，工艺过程控制的稳定性。

Table 21 Sampling Schedule

表格 22取样计划表

No 编号 Evaluation Items 评估项目 Sterility test 无菌检查 Process step 工艺步骤 0.2mol/L glutamine 0.2mol/L谷氨酰胺 0.85% sodium chloride solution 0.85%氯化钠溶液 0.25 trypsin solution 0.25胰蛋白酶溶液 1mol/L hydrochloric acid solution 1mol/L盐酸溶液 Evaluation criteria 评估标准 Conforming to the specifications 应符合规定 Conforming to the specifications 应符合规定 Conforming to the specifications 应符合规定 Conforming to the specifications 应符合规定 7.0 Conforming to the specifications 应符合规定 Conforming to the specifications 应符合规定 Conforming to the specifications 应符合规定 Conforming to the specifications 应符合规定 Conforming to the specifications 应符合规定 Sampling Plan 取样计划 Take samples after finishing preparation 配制完成后取样 Take samples after finishing preparation 配制完成后取样 Take samples after finishing preparation 配制完成后取样 Take samples after finishing preparation 配制完成后取样 Take samples after finishing preparation 配制完成后取样 Take samples after finishing preparation 配制完成后取样 Take samples after finishing preparation 配制完成后取样 Take samples after finishing preparation 配制完成后取样 Take samples after finishing preparation 配制完成后取样 Amount 取样量 Once 20ml 20ml/次 Once 20ml 20ml/次 Once 20ml 20ml/次 Once 20ml 20ml/次 Once 10ml 10ml/次 Once 20ml 20ml/次 Once 20ml 20ml/次 Once 20ml 20ml/次 Once 20ml 20ml/次 Once 20ml 20ml/次 1. 2. Sterility test 无菌检查 3. Sterility test 无菌检查 4. Sterility test 无菌检查 5. Solution preparation 液体配制 20mmol/L pH phosphate buffer solution (pH 7.0) 20mmol/L磷酸盐Sterility test 缓冲液（PH值7.0）无菌检查 10％DMEM culture media 10％DMEM培养液 3％DMEM culture media 3％DMEM培养液 4％DMEM culture media 4％DMEM培养液 5％DMEM culture media 5％DMEM培养液 Sterility test 无菌检查 6. 7. Sterility test 无菌检查 8. Sterility test 无菌检查 9. Sterility test 无菌检查名称 Title Process Validation (PV) Protocol for XXX XXX工艺验证方案 Page 页码 Doc. No. 文件编号 No 编号 Process step 工艺步骤 5% formaldehyde solution 5%甲醛溶液 50% potassium bromide solution 50％溴化钾溶液 75% ammonium sulfate solution 75%硫酸铵溶液 8% sodium bicarbonate 8%碳酸氢钠 MTX solution MTX溶液 Evaluation Items 评估项目 Sterility test 无菌检查 Evaluation criteria 评估标准 Conforming to the specifications 应符合规定 Conforming to the specifications 应符合规定 Conforming to the specifications 应符合规定 Conforming to the specifications 应符合规定 Conforming to the specifications 应符合规定 Conforming to the specifications 应符合规定 1.040g/ml Conforming to the specifications 应符合规定 1.280g/ml Conforming to the specifications 应符合规定 1.340g/ml It shall be a kind of clear liquid. 应为澄清液体 The cell grow is good. 细胞生长状态良好 The cell grow is good. 细胞生长状态良好 The cell grow is good. 细胞生长状态良好 The cell grow is good. 细胞生长状态良好 Sampling Plan 取样计划 Take samples after finishing preparation 配制完成后取样 Take samples after finishing preparation 配制完成后取样 Take samples after finishing preparation 配制完成后取样 Take samples after finishing preparation 配制完成后取样 Take samples after finishing preparation 配制完成后取样 Take samples after finishing preparation 配制完成后取样 Amount 取样量 Once 20ml 20ml/次 Once 20ml 20ml/次 Once 20ml 20ml/次 Once 20ml 20ml/次 Once 20ml 20ml/次 Once 20ml 20ml/次 Once 100ml 100ml/次 Once 20ml 20ml/次 Once 100ml 100ml/次 Once 20ml 20ml/次 Once 100ml 100ml/次 1 unit 1支 1 bottle 1瓶 10. 11. Sterility test 无菌检查 12. Sterility test 无菌检查 13. Sterility test 无菌检查 14. Sterility test 无菌检查 Sterility test 无菌检查 Density 密度 Sterility test 无菌检查 Density 密度 Sterility test 无菌检查 Density 密度 Visual inspection 目测 Microscopic inspection 显微观察 Microscopic inspection 显微观察 Microscopic inspection 显微观察 Microscopic inspection 显微观察 15. Solution A for ultra-speed centrifugation 超离所需液体A 16. Solution B for ultra-speed centrifugation 超离所需液体B Take samples after finishing preparation 配制完成后取样 17. Solution D for ultra-speed centrifugation 超离所需液体D Working cell bank thawing 工作细胞库解冻 Day 1 第1天 Take samples after finishing preparation 配制完成后取样 Observation at site 现场观察 Observation at site in square bottles 方瓶中现场观察 Observation at site in square bottles 方瓶中现场观察 Observation at site in square bottles 方瓶中现场观察 Observation at site in square bottles Resurrection 18. 复苏 C25 - First square bottle 19. incubation C25-方瓶第一次培养 Day 2 第2天 1 bottle 1瓶 Day 3 第3天 Day 4 第4天 1 bottle 1瓶 1 bottle 1瓶名称 Title Process Validation (PV) Protocol for XXX XXX工艺验证方案 Page 页码 Doc. No. 文件编号 No 编号 Process step 工艺步骤 Evaluation Items 评估项目 Evaluation criteria 评估标准 Sampling Plan 取样计划方瓶中现场观察 Under microscope observation the cells have grown into a compact single layer, the cells are Microscopic inspection shuttle-shaped and the growth liquor is 显微观察 orange in color. 显微镜下观察培养瓶长成致密单层，细胞呈梭形，生长液为橙色 Under microscope observation the cells have grown into a compact single layer, the cells are Microscopic shuttle-shaped and inspection the growth liquor is 显微观察 orange in color. 显微镜下观察培养瓶长成致密单层，细胞呈梭形，生长液为橙色 Under microscope observation the cells have grown into a compact single layer, the cells are Microscopic inspection shuttle-shaped and the growth liquor is 显微观察 orange in color. 显微镜下观察培养瓶长成致密单层，细胞呈梭形，生长液为橙色 Microscopic inspection 显微观察 Microscopic inspection 显微观察 Microscopic inspection 显微观察 Microscopic inspection 显微观察 The cell grow is good. 细胞生长状态良好 The cell grow is good. 细胞生长状态良好 The cell grow is good. 细胞生长状态良好 The cell grow is good. 细胞生长状态良好 Amount 取样量 Day 5 第5天 Observation at site in square bottles 方瓶中现场观察 1 bottle 1瓶 Day 6 (if necessary) 第6天（如需） Observation at site in square bottles 方瓶中现场观察 1 bottle 1瓶 Day 7 (if necessary) 第7天（如需） Observation at site in square bottles 方瓶中现场观察 1 bottle 1瓶 Day 1 第1天 C26 - second square bottle 20. incubation C26-方瓶第二次培养 Day 2 第2天 Day 3 第3天 Day 4 第4天 Observation at site in square bottles 方瓶中现场观察 Observation at site in square bottles 方瓶中现场观察 Observation at site in square bottles 方瓶中现场观察 Observation at site in square bottles 方瓶中现场观察 4 bottles 4瓶 4 bottles 4瓶 4 bottles 4瓶 4 bottles 4瓶名称 Title Process Validation (PV) Protocol for XXX XXX工艺验证方案 Page 页码 Doc. No. 文件编号 No 编号 Process step 工艺步骤 Evaluation Items 评估项目 Evaluation criteria 评估标准 Sampling Plan 取样计划 Amount 取样量 Day 5 第5天 Day 6 (if necessary) 第6天（如需） Day 7 (if necessary) 第7天（如需） Under microscope observation the cells have grown into a compact single layer, the cells are Microscopic inspection shuttle-shaped and the growth liquor is 显微观察 orange in color. 显微镜下观察培养瓶长成致密单层，细胞呈梭形，生长液为橙色 Under microscope observation the cells have grown into a compact single layer, the cells are Microscopic inspection shuttle-shaped and the growth liquor is 显微观察 orange in color. 显微镜下观察培养瓶长成致密单层，细胞呈梭形，生长液为橙色 Under microscope observation the cells have grown into a compact single layer, the cells are Microscopic inspection shuttle-shaped and the growth liquor is 显微观察 orange in color. 显微镜下观察培养瓶长成致密单层，细胞呈梭形，生长液为橙色 Microscopic inspection 显微观察 The cell grow is good. 细胞生长状态良好 Observation at site in square bottles 方瓶中现场观察 4 bottles 4瓶 Observation at site in square bottles 方瓶中现场观察 4 bottles 4瓶 Observation at site in square bottles 方瓶中现场观察 4 bottles 4瓶 Day 1 第1天 C27 - Kolle flask 21. incubation C27-克氏瓶培养 Day 2 第2天 Microscopic inspection 显微观察 The cell grow is good. 细胞生长状态良好 Day 3 第3天 Day 4 第4天 Microscopic inspection 显微观察 Microscopic inspection The cell grow is good. 细胞生长状态良好 The cell grow is good. Observation at site in Kolle bottles 克氏瓶中现场观察 Observation at site in Kolle bottles 克氏瓶中现场观察 Observation at site in Kolle bottles 克氏瓶中现场观察 Observation at site in Kolle 2 bottles 2瓶 2 bottles 2瓶 2 bottles 2瓶 2 bottles 2瓶名称 Title Process Validation (PV) Protocol for XXX XXX工艺验证方案 Page 页码 Doc. No. 文件编号 No 编号 Process step 工艺步骤 Evaluation Items 评估项目显微观察 Evaluation criteria 评估标准细胞生长状态良好 Sampling Plan 取样计划 bottles 克氏瓶中现场观察 Amount 取样量 Day 5 第5天 Microscopic inspection 显微观察 Day 6 (if necessary) 第6天（如需） Microscopic inspection 显微观察 Day 7 (if necessary) 第7天（如需） Microscopic inspection 显微观察 Day 1 第1天 Day 2 第2天 Day 3 第3天 Day 4 第4天 Day 5 第5天 C28 - First 3L rotary bottle 22. incubation C28-3L转瓶第一次培养 Microscopic inspection 显微观察 Microscopic inspection 显微观察 Microscopic inspection 显微观察 Microscopic inspection 显微观察 Microscopic inspection Under microscope observation the cells have grown into a compact single layer, the cells are shuttle-shaped and the growth liquor is orange in color. 显微镜下观察培养瓶长成致密单层，细胞呈梭形，生长液为橙色 Under microscope observation the cells have grown into a compact single layer, the cells are shuttle-shaped and the growth liquor is orange in color. 显微镜下观察培养瓶长成致密单层，细胞呈梭形，生长液为橙色 Under microscope observation the cells have grown into a compact single layer, the cells are shuttle-shaped and the growth liquor is orange in color. 显微镜下观察培养瓶长成致密单层，细胞呈梭形，生长液为橙色 The cell grow is good. 细胞生长状态良好 The cell grow is good. 细胞生长状态良好 The cell grow is good. 细胞生长状态良好 The cell grow is good. 细胞生长状态良好 Under microscope observation the Observation at site in Kolle bottles 克氏瓶中现场观察 2 bottles 2瓶 Observation at site in Kolle bottles 克氏瓶中现场观察 2 bottles 2瓶 Observation at site in Kolle bottles 克氏瓶中现场观察 2 bottles 2瓶 Observation at site in 3L bottles 3L瓶中现场观察 Observation at site in 3L bottles 3L瓶中现场观察 Observation at site in 3L bottles 3L瓶中现场观察 Observation at site in 3L bottles 3L瓶中现场观察 Observation at site in 3L bottles 2 bottles 2瓶 2 bottles 2瓶 2 bottles 2瓶 2 bottles 2瓶 2 bottles 2瓶名称 Title Process Validation (PV) Protocol for XXX XXX工艺验证方案 Page 页码 Doc. No. 文件编号 No 编号 Process step 工艺步骤 Evaluation Items 评估项目显微观察 Evaluation criteria 评估标准 cells have grown into a compact single layer, the cells are shuttle-shaped and the growth liquor is orange in color. 显微镜下观察培养瓶长成致密单层，细胞呈梭形，生长液为橙色 Under microscope observation the cells have grown into a compact single layer, the cells are shuttle-shaped and the growth liquor is orange in color. 显微镜下观察培养瓶长成致密单层，细胞呈梭形，生长液为橙色 Under microscope observation the cells have grown into a compact single layer, the cells are shuttle-shaped and the growth liquor is orange in color. 显微镜下观察培养瓶长成致密单层，细胞呈梭形，生长液为橙色 The cell grow is good. 细胞生长状态良好 The cell grow is good. 细胞生长状态良好 The cell grow is good. 细胞生长状态良好 The cell grow is good. 细胞生长状态良好 Under microscope observation the cells have grown into a compact single layer, the cells are shuttle-shaped and Sampling Plan 取样计划 3L瓶中现场观察 Amount 取样量 Day 6 (if necessary) 第6天（如需） Microscopic inspection 显微观察 Observation at 2 bottles site in 3L bottles 2瓶 3L瓶中现场观察 Day 7 (if necessary) 第7天（如需） Microscopic inspection 显微观察 Observation at 2 bottles site in 3L bottles 2瓶 3L瓶中现场观察 Day 1 第1天 Day 2 第2天 C29 - second 3L rotary bottle 23. incubation C29-3L转瓶第二次培养 Day 3 第3天 Day 4 第4天 Microscopic inspection 显微观察 Microscopic inspection 显微观察 Microscopic inspection 显微观察 Microscopic inspection 显微观察 Observation at site in 3L bottles 3L瓶中现场观察 Observation at site in 3L bottles 3L瓶中现场观察 Observation at site in 3L bottles 3L瓶中现场观察 Observation at site in 3L bottles 3L瓶中现场观察 Observation at site in 3L bottles 3L瓶中现场观察 16 bottles 16瓶 16 bottles 16瓶 16 bottles 16瓶 16 bottles 16瓶 16 bottles 16瓶 Day 5 第5天 Microscopic inspection 显微观察名称 Title Process Validation (PV) Protocol for XXX XXX工艺验证方案 Page 页码 Doc. No. 文件编号 No 编号 Process step 工艺步骤 Evaluation Items 评估项目 Evaluation criteria 评估标准 the growth liquor is orange in color. 显微镜下观察培养瓶长成致密单层，细胞呈梭形，生长液为橙色 Under microscope observation the cells have grown into a compact single layer, the cells are shuttle-shaped and the growth liquor is orange in color. 显微镜下观察培养瓶长成致密单层，细胞呈梭形，生长液为橙色 Under microscope observation the cells have grown into a compact single layer, the cells are shuttle-shaped and the growth liquor is orange in color. 显微镜下观察培养瓶长成致密单层，细胞呈梭形，生长液为橙色 The cell grow is good. 细胞生长状态良好 The cell grow is good. 细胞生长状态良好 The cell grow is good. 细胞生长状态良好 The cell grow is good. 细胞生长状态良好 Under microscope observation the cells have grown into a compact single layer, the cells are shuttle-shaped and the growth liquor is orange in color. 显微镜下观察培养瓶长成致密单层，细胞呈梭形，生长液为Sampling Plan 取样计划 Amount 取样量 Day 6 (if necessary) 第6天（如需） Microscopic inspection 显微观察 Observation at site in 3L bottles 3L瓶中现场观察 16 bottles 16瓶 Day 7 (if necessary) 第7天（如需） Microscopic inspection 显微观察 Observation at site in 3L bottles 3L瓶中现场观察 16 bottles 16瓶 Day 1 第1天 Day 2 第2天 Day 3 第3天 C30 - third 3L rotary bottle 24. incubation C30-3L转瓶第三次培养 Day 4 第4天 Microscopic inspection 显微观察 Microscopic inspection 显微观察 Microscopic inspection 显微观察 Microscopic inspection 显微观察 Observation at site in 3L bottles 3L瓶中现场观察 Observation at site in 3L bottles 3L瓶中现场观察 Observation at site in 3L bottles 3L瓶中现场观察 Observation at site in 3L bottles 3L瓶中现场观察 20 bottles 20瓶 20 bottles 20瓶 20 bottles 20瓶 20 bottles 20瓶 Day 5 第5天 Microscopic inspection 显微观察 Observation at site in 3L bottles 3L瓶中现场观察 20 bottles 20瓶名称 Title Process Validation (PV) Protocol for XXX XXX工艺验证方案 Page 页码 Doc. No. 文件编号 No 编号 Process step 工艺步骤 Evaluation Items 评估项目 Evaluation criteria 评估标准橙色 Under microscope observation the cells have grown into a compact single layer, the cells are Microscopic inspection shuttle-shaped and the growth liquor is 显微观察 orange in color. 显微镜下观察培养瓶长成致密单层，细胞呈梭形，生长液为橙色 Under microscope observation the cells have grown into a compact single layer, the cells are Microscopic inspection shuttle-shaped and the growth liquor is 显微观察 orange in color. 显微镜下观察培养瓶长成致密单层，细胞呈梭形，生长液为橙色 Microscopic inspection 显微观察 The cell grow is good. 细胞生长状态良好 Sampling Plan 取样计划 Amount 取样量 Day 6 (if necessary) 第6天（如需） Observation at site in 3L bottles 3L瓶中现场观察 20 bottles 20瓶 Day 7 (if necessary) 第7天（如需） Observation at site in 3L bottles 3L瓶中现场观察 20 bottles 20瓶 Day 1 第1天 Day 2 第2天 Microscopic inspection 显微观察 The cell grow is good. 细胞生长状态良好 C31 - first 15L bottle incubation 25. C31-15L瓶第一次培养 Day 3 第3天 Microscopic inspection 显微观察 The cell grow is good. 细胞生长状态良好 Day 4 第4天 Microscopic inspection 显微观察 The cell grow is good. 细胞生长状态良好 Under microscope observation the cells have grown into a compact single layer, the cells are shuttle-shaped and the growth liquor is Observation at site in 15L bottles 15L瓶中现场观察 Observation at site in 15L bottles 15L瓶中现场观察 Observation at site in 15L bottles 15L瓶中现场观察 Observation at site in 15L bottles 15L瓶中现场观察 Observation at site in 15L bottles 15L瓶中现场观察 20 bottles 20瓶 20 bottles 20瓶 20 bottles 20瓶 20 bottles 20瓶 Day 5 第5天 Microscopic inspection 显微观察 20 bottles 20瓶名称 Title Process Validation (PV) Protocol for XXX XXX工艺验证方案 Page 页码 Doc. No. 文件编号 No 编号 Process step 工艺步骤 Evaluation Items 评估项目 Evaluation criteria 评估标准 Sampling Plan 取样计划 Amount 取样量 Day 6 (if necessary) 第6天（如需） Day 7 (if necessary) 第7天（如需） orange in color. 显微镜下观察培养瓶长成致密单层，细胞呈梭形，生长液为橙色 Under microscope observation the cells have grown into a compact single layer, the cells are Microscopic inspection shuttle-shaped and the growth liquor is 显微观察 orange in color. 显微镜下观察培养瓶长成致密单层，细胞呈梭形，生长液为橙色 Under microscope observation the cells have grown into a compact single layer, the cells are Microscopic inspection shuttle-shaped and the growth liquor is 显微观察 orange in color. 显微镜下观察培养瓶长成致密单层，细胞呈梭形，生长液为橙色 Microscopic inspection 显微观察 The cell grow is good. 细胞生长状态良好 Observation at site in 15L bottles 15L瓶中现场观察 20 bottles 20瓶 Observation at site in 15L bottles 15L瓶中现场观察 20 bottles 20瓶 Day 1 第1天 Day 2 第2天 C32 - second 15L bottle 26. incubation C32-15L瓶第二次培养 Microscopic inspection 显微观察 The cell grow is good. 细胞生长状态良好 Day 3 第3天 Microscopic inspection 显微观察 The cell grow is good. 细胞生长状态良好 Day 4 第4天 Microscopic inspection 显微观察 The cell grow is good. 细胞生长状态良好 Under microscope observation the cells have grown into a compact single layer, the Observation at site in 15L bottles 15L瓶中现场观察 Observation at site in 15L bottles 15L瓶中现场观察 Observation at site in 15L bottles 15L瓶中现场观察 Observation at site in 15L bottles 15L瓶中现场观察 Observation at site in 15L bottles 15L瓶中现场观20 bottles 20瓶 20 bottles 20瓶 20 bottles 20瓶 20 bottles 20瓶 Day 5 第5天 Microscopic inspection 显微观察 20 bottles 20瓶名称 Title Process Validation (PV) Protocol for XXX XXX工艺验证方案 Page 页码 Doc. No. 文件编号 No 编号 Process step 工艺步骤 Evaluation Items 评估项目 Evaluation criteria 评估标准 Sampling Plan 取样计划察 Amount 取样量 Day 6 (if necessary) 第6天（如需） Day 7 (if necessary) 第7天（如需） cells are shuttle-shaped and the growth liquor is orange in color. 显微镜下观察培养瓶长成致密单层，细胞呈梭形，生长液为橙色 Under microscope observation the cells have grown into a compact single layer, the cells are Microscopic inspection shuttle-shaped and the growth liquor is 显微观察 orange in color. 显微镜下观察培养瓶长成致密单层，细胞呈梭形，生长液为橙色 Under microscope observation the cells have grown into a compact single layer, the cells are Microscopic shuttle-shaped and inspection the growth liquor is 显微观察 orange in color. 显微镜下观察培养瓶长成致密单层，细胞呈梭形，生长液为橙色 Conforming to the Sterility test specifications 无菌检查应符合规定 HBsAg expression level HBsAg表达量 ELISA test, no less than 1.0mg/L ELISA法检测，应不低于1.0mg/L Observation at site in 15L bottles 15L瓶中现场观察 20 bottles 20瓶 Observation at site in 15L bottles 15L瓶中现场观察 20 bottles 20瓶 27. 20 consecutive harvest 连续20次收获液 28. Clarification filtration 澄清过滤 It shall be a kind of clear liquid without Visual or cell residues or microscopic dead/live cells. inspection 应为澄清液体，无细目测或镜检胞残渣或死/活细胞存在 Take altogether 20 samples, with one sample taken from the 500L buffer tank at each harvest 每次收获时，在500L缓冲罐中取样，共20次 Take altogether 20 samples, with one sample taken from the 500L buffer tank at each harvest after cell residues are removed. 每次收获后，去细胞残渣过滤后，在500L缓冲罐中取样，共20次 Once 20ml 20ml/次 Once 20ml 20ml/次 Once 50ml 50ml/次名称 Title Process Validation (PV) Protocol for XXX XXX工艺验证方案 Page 页码 Doc. No. 文件编号 No 编号 Process step 工艺步骤 Evaluation Items 评估项目 Evaluation criteria 评估标准 Sampling Plan 取样计划 Take altogether 20 samples from the concentrate, one sample after each UF concentration. 每次超滤浓缩后取浓缩液，共20次 Take altogether 20 samples from the membrane filtrate, one sample after each UF concentration. 每次超滤浓缩后取超滤膜下液，共20次 Take altogether 20 samples from the supernatant obtained after the finishing low speed centrifugation, one sample after each ammonium sulfate precipitation end point 取每次硫酸铵沉淀终点时，低速离心结束后的上清液，共20次 Take altogether 20 samples from the combined supernatant of low speed centrifugation 低速离心的上清液合并后取样，共20次 Take altogether 20 samples from the serum solution at the centrifugation end point 取离心终点的血清溶液，共20次 Take altogether 20 samples from the serum solution at the Amount 取样量 29. Concentrate 浓缩液 Protein content test 蛋白含量检测 Record the actual results. 记录实际结果 Once 10ml 10ml/次 UF concentration 1 超滤浓缩1 Membrane filtrate 膜下液 Protein content test 蛋白含量检测 Record the actual results. 记录实际结果 30. Once 10ml 10ml/次 Ammonium sulfate precipitation 31. 硫酸铵沉淀 Protein content test 蛋白含量检测 Record the actual results. 记录实际结果 Once 10ml 10ml/次 32. Potassium bromide extraction 溴化钾提取 Protein content test 蛋白含量检测 Record the actual results. 记录实际结果 Once 10ml 10ml/次 33. First density gradient centrifugation 一次密度梯度离心 Protein content test 蛋白含量检测 Record the actual results. 记录实际结果 Once 10ml 10ml/次 34. Second density gradient centrifugation 二次密度梯度离心 Protein content test 蛋白含量检测 Record the actual results. 记录实际结果 Once 10ml 10ml/次名称 Title Process Validation (PV) Protocol for XXX XXX工艺验证方案 Page 页码 Doc. No. 文件编号 No 编号 Process step 工艺步骤 Evaluation Items 评估项目 Evaluation criteria 评估标准 Sampling Plan 取样计划 centrifugation end point 取离心终点的血清溶液，共20次 Take altogether 20 samples from the concentrate, one sample after each UF concentration. 每次超滤浓缩后取浓缩液，共20次 Take altogether 20 samples from the membrane filtrate, one sample after each UF concentration. 每次超滤浓缩后取超滤膜下液，共20次 Amount 取样量 35. Concentrate 浓缩液 Protein content test 蛋白含量检测 Record the actual results. 记录实际结果 Once 10ml 10ml/次 UF concentration 2 超滤浓缩2 Membrane filtrate 膜下液 Protein content test 蛋白含量检测 Record the actual results. 记录实际结果 36. Once 10ml 10ml/次 37. Chromatography purification product 层析纯化产物 It shall be 100 to 200μg/m (Method Protein II of Appendix VI B) content 应为100～200μ蛋白质含量 g/ml（附录VI B 第二法） Use the reducing SDS polyacrylamide gel electrophoresis (Appendix IV C). There shall be belts of proteins with molecular weights of 23kD and 27kD. There can be belts Specific of proteins with a protein belt molecular weight of 特异蛋白带 30kD and HBsAg polymer proteins. 采用还原型SDS聚丙烯酰胺凝胶电泳法（附录IV C），在分离胶中应有分子量23kD、27kD蛋白带，可有30kD蛋白带及HBsAg多聚体蛋白带 Use the SEC-HPLC Purity method and the 纯度 result shall be no less than 95.5%. Take altogether 20 samples from the purification product after each gel column chromatography 每次凝胶柱层析结束后，取纯化产物检测，共20次 Once 210ml 210ml/次名称 Title Process Validation (PV) Protocol for XXX XXX工艺验证方案 Page 页码 Doc. No. 文件编号 No 编号 Process step 工艺步骤 Evaluation Items 评估项目 Evaluation criteria 评估标准 Sampling Plan 取样计划 Amount 取样量 38. Bulk Solution 原液 SEC-HPLC法，应不低于95.5% It shall be less than 10EU per 10μg Bacterial endotoxin protein (Appendix test XII E gel limit test) 细菌内毒素每10μg蛋白质应检查小于10EU（附录Ⅻ E凝胶限度试验） Conforming to the Sterility test specifications 无菌检查应符合规定 Mycoplasma Conforming to the test specifications 支原体检查应符合规定 It shall be 100 to 200μg/m (Method Protein II of Appendix VI B) content 应为100～200μ蛋白质含量 g/ml（附录VI B 第二法） Use the reducing SDS polyacrylamide gel electrophoresis (Appendix IV C). There shall be belts of proteins with molecular weights The bulk is of 23kD and 27kD. obtained after There can be belts combination and Specific of proteins with a sterile filtration. protein belt molecular weight of 在完成合并、除菌30kD and HBsAg 特异蛋白带 polymer proteins. 过滤后的产品为原液，采用还原型SDS聚丙烯酰胺凝胶电泳法（附录IV C），在分离胶中应有分子量23kD、27kD蛋白带，可有30kD蛋白带及HBsAg多聚体蛋白带 Bovine serum It shall be no more albumin than 45ng/dose residue 应不高于45ng/剂牛血清白蛋（附录ⅧⅠ）白残留量 Use the SEC-HPLC method and the result shall be no Purity less than 95.5%. 纯度 SEC-HPLC法，应不低于95.5% Once 230ml 230ml 名称 Title Process Validation (PV) Protocol for XXX XXX工艺验证方案 Page 页码 Doc. No. 文件编号 No 编号 Process step 工艺步骤 Evaluation Items 评估项目 Evaluation criteria 评估标准 Perform the test as specified in Method I in Appendix IX B of CP. The results shall be no more than 10pg/dose. 按现行《中国药典》三部“附录IX B第一法”进行。检测结果应不高于10pg/剂 The CHO cell protein residue shall be no more than 0.05% of the total protein content. CHO细胞蛋白质残留量应不高于总蛋白质含量的0.05% It shall be less than 10EU per 10μg protein (Appendix XII E gel limit test) 每10μg蛋白质应小于10EU（附录Ⅻ E凝胶限度试验） Sampling Plan 取样计划 Amount 取样量 CHO cell DNA residue CHO细胞DNA残留量 CHO cell protein residue CHO细胞蛋白质残留量 Bacterial endotoxin test 细菌内毒素检查

名称 Title Process Validation (PV) Protocol for XXX XXX工艺验证方案 Page 页码 Doc. No. 文件编号 10. Sample Coding Method样品编码原则

Table 23 Sample Coding Method

表格 24样品编码原则

No 序号 1 2 3 4 5 6 7 8 3 4 5 6 7 8 3 4 5 6 7 8 No 序号 1 2 Process step 工艺步骤 Solution preparation 液体配制 Resurrection 复苏 C25 - First square bottle incubation C25-方瓶第一次培养 C26 - second square bottle incubation C26-方瓶第二次培养 C27 - Kolle flask incubation C27-克氏瓶培养 C28 - First 3L rotary bottle incubation C28-3L转瓶第一次培养 C29 - second 3L rotary bottle incubation C29-3L转瓶第二次培养 C30 - third 3L rotary bottle incubation C30-3L转瓶第三次培养 C31 - first 15L bottle incubation C31-15L瓶第一次培养 C32 - second 15L bottle incubation C32-15L瓶第二次培养 20 consecutive harvest 连续20次收获液 Clarification filtration 澄清过滤 UF concentration 1 超滤浓缩1 Ammonium sulfate precipitation 硫酸铵沉淀 Potassium bromide extraction 溴化钾提取 First density gradient centrifugation 一次密度梯度离心 Second density gradient centrifugation 二次密度梯度离心 UF concentration 2 超滤浓缩2 Chromatography purification product 层析纯化产物 Bulk Solution 原液 Test Item 检验项目 Sterility 无菌 Visual inspection 目测 Code 代码 LP CR C25 C26 C27 C28 C29 C30 C31 C32 CL CF UF1 ASP KBr 1DGC 2DGC UF2 CH Bulk Code 代码 S IN 名称 Title Process Validation (PV) Protocol for XXX XXX工艺验证方案 Page 页码 Doc. No. 文件编号 Protein content test PD 蛋白含量检测 Endotoxin Test 4 ET 内毒素检测 Purity 5 PT 纯度 Specific protein belt 6 SPT 特异蛋白带 Density 7 DE 密度 Microscopic inspection 8 ME 显微观察 Notes: 1. The test items can be marked on the bulk samples. 2. When preparing liquids, the abbreviation of the major ingredient need to be put into the codes for different liquid samples. 注：1.原液中的样品直接标注检测项目即可 2.液体配制时，不同液体样品需用主料名称缩写加入编码中。 3 For example: LP-GLN-S-01 LP stands for liquid preparation. GLN stands for glutamine S stands for sterility test

01 stands for the first preparation

例如： LP-GLN-S-01

LP代表液体配制 GLN代表谷氨酰胺， S代表无菌检测 01代表第一次配制

名称 Title Process Validation (PV) Protocol for XXX XXX工艺验证方案 Page 页码 Doc. No. 文件编号 11. Process Validation Test Execution 工艺验证执行

11.1 Liquid Preparation 液体配制  Purpose 目的

Check that the various kinds of liquids prepared following the relevant preparation SOPs are conforming to their respective specifications.

检查生产中所需各种液体按照配制标准操作规程进行配制后，应能符合相应的质量标准要求。  Acceptance Criteria可接受标准

Table 25 Solution Preparation, Sampling and Testing Information

表格 26配液取样检测信息表

Solution Name 溶液名称 0.2mol/L glutamine 0.2mol/L谷氨酰胺 0.85% sodium chloride solution 0.85%氯化钠溶液 0.25 trypsin solution 0.25胰蛋白酶溶液 1mol/L hydrochloric acid solution 1mol/L盐酸溶液 20mmol/L phosphate buffer solution (pH 7.0) 20mmol/L磷酸盐缓冲液（PH值7.0） 10％DMEM culture media 10％DMEM培养液 3％DMEM culture media 3％DMEM培养液 4％DMEM culture Test Items 检测项目 Sterility test 无菌检查 Criteria 标准 Conforming to the specifications 应符合规定 Conforming to the specifications 应符合规定 Conforming to the specifications 应符合规定 Conforming to the specifications 应符合规定 7.0 Conforming to the specifications 应符合规定 Conforming to the specifications 应符合规定 Conforming to the specifications 应符合规定 Conforming to the Operation Procedures 操作规程 SOP for Preparation of 0.2mol/L Glutamine Solution 0.2mol/L谷氨酰胺溶液配制标准操作规程 SOP for Preparation of 0.85% Sodium Chloride Solution 0.85%氯化钠溶液配制标准规程 SOP for Preparation of 0.25 Trypsin Solution 0.25%胰蛋白酶配制标准操作规程 SOP for Preparation of 1mol/L Hydrochloric Acid Solution 1mol/L盐酸溶液配制标准操作规程 SOP for Preparation of 20mmol/L Phosphate Buffer Solution 20mmol/L磷酸盐缓冲液配制标准操作规程 SOP for Preparation of DMEM Solution DMEM溶液配制标准操作规程 SOP for Preparation of DMEM Solution DMEM溶液配制标准操作规程 SOP for Preparation of DMEM Solution SOP No. 规程编号 Sample No. 样品编号 1303.015-16-02 LP-GLN-S-XX Sterility test 无菌检查 1303.019-16-02 LP-NaCl-S-XX Sterility test 无菌检查 1303.014-16-02 LP-TP-S-XX Sterility test 无菌检查 pH Sterility test 无菌检查 1303.018-16-02 LP-HCl-S-XX LP-PB-pH-XX 1303.011-16-02 LP-PB-S-XX Sterility test 无菌检查 Sterility test 无菌检查 Sterility test 1303.013-16-02 LP-10%DMEM-S-XX 1303.013-16-02 LP-3%DMEM-S-XX 1303.013-16-02 LP-4%DMEM-S-XX 名称 Title Process Validation (PV) Protocol for XXX XXX工艺验证方案 Page 页码 Doc. No. 文件编号 specifications DMEM溶液配制标准应符合规定操作规程 Conforming SOP for Preparation 5％DMEM culture Sterility of DMEM Solution to the test media specifications DMEM溶液配制标准5％DMEM培养液无菌检查应符合规定操作规程 SOP for Preparation Conforming of 5% Formaldehyde 5% formaldehyde Sterility to the Solution solution test specifications 5%甲醛溶液配制标5%甲醛溶液无菌检查应符合规定准规程 SOP for Preparation Conforming of 50% Potassium 50% potassium Sterility to the Bromide Solution bromide solution test specifications 50%溴化钾溶液配制50％溴化钾溶液无菌检查应符合规定标准操作规程 SOP for Preparation Conforming of 75% Ammonium 75% ammonium Sterility to the Sulfate Solution sulfate solution test specifications 75%硫酸铵溶液配制75%硫酸铵溶液无菌检查应符合规定标准操作规程 SOP for Preparation Conforming of 8% Sodium 8% sodium Sterility to the Bicarbonate Solution bicarbonate test specifications 8%碳酸氢钠溶液配8%碳酸氢钠无菌检查应符合规定制标准操作规程 SOP for Preparation Conforming of 10﹣4mol/L MTX Sterility MTX solution to the test Solution specifications MTX溶液 ﹣4无菌检查 10mol/L MTX溶液应符合规定配制标准操作规程 Conforming SOP for Preparation Sterility to the of Solutions for Solution A for test specifications Ultra-speed ultra-speed 无菌检查应符合规定 Centrifugation centrifugation 超速离心所需溶液配超离所需液体A Density 1.040g/ml 制标准操作规程密度 Conforming SOP for Preparation Sterility to the of Solutions for Solution B for test specifications Ultra-speed ultra-speed 无菌检查应符合规定 Centrifugation centrifugation 超速离心所需溶液配超离所需液体B Density 1.280g/ml 制标准操作规程密度 Conforming SOP for Preparation Sterility to the of Solutions for Solution D for test specifications Ultra-speed ultra-speed 无菌检查应符合规定 Centrifugation centrifugation 超速离心所需溶液配超离所需液体D Density 1.340g/ml 制标准操作规程密度 Note: XX stands for the serial number of sampling. 注：XX代表取样流水号。无菌检查 media 4％DMEM培养液 1303.013-16-02 LP-5%DMEM-S-XX 1303.020-16-02 LP-HCHO-S-XX 1303.009-16-02 LP-KBr-S-XX 1303.008-16-02 LP-AS-S-XX 1303.017-16-02 LP-SHC-S-XX 1303.016-16-02 LP-MTX-S-XX LP-UCA-S-XX 1303.010-16-02 LP-UCA-D-XX LP-UCB-S-XX 1303.010-16-02 LP-UCB-D-XX LP-UCD-S-XX 1303.010-16-02 LP-UCD-D-XX  Sampling and testing method取样及检测方法 When taking the above samples, all samples for sterility test shall be taken and tested following the SOP for Sterility Test (No.: 1302.001-16-02).

以上样品取样时，凡无菌检测的，按照无菌检查标准操作规程（编号：1302.001-16-02）的要

名称 Title Process Validation (PV) Protocol for XXX XXX工艺验证方案 Page 页码 Doc. No. 文件编号求进行取样和检验。  Test Records 检测记录

When taking the above samples, a Sterility Test Record (Membrane Filtration Method) (Document No.: 2102.001-16-02) or a Sterility Test Record (Direct Inoculation Method) (Document No.: 2102.002-16-02) shall be filled for each of the sample for sterility test. 以上样品取样时，凡无菌检测的，记录填写在无菌检查（薄膜过滤法）记录（文件编号：2102.001-16-02）或无菌检查(直接接种法)记录（文件编号：2102.002-16-02）。

The test results are to be summarized in the Attachment 9 – List Preparation Test Record Summarization Table. The original test records shall be attached to this protocol. 检测结果统计在附表9-液体配制检测记录统计表，原始检测记录作为附件附在本方案后。 11.2 Cell Resurrection and Passage细胞复苏及传代  Purpose 目的

The growth of the cells during the resurrection and passage process is to be observed through microscopic or visual inspections to verify that the cells can grow normally under the set growing environment.

通过显微观察、目测等方式，观察细胞在复苏、传代过程中的生长状态，以确认在设定的生长环境中，细胞能够正常生长。  Acceptance Criteria可接受标准

Table 27 Sampling Table for Resurrection and Passage

表格 28复苏和传代取样表

Process Step 工艺过程 Ressurrection 复苏 Working cell bank thawing 工作细胞库解冻 Day 1 第1天 C25 - First square bottle incubation C25-方瓶第一次培养 Testing Method 检测方法 Visual inspection 目测 Microscopic inspection 显微观察 Microscopic inspection 显微观察 Microscopic inspection 显微观察 Microscopic inspection Acceptance Criteria 合格标准 It shall be a kind of clear liquid 应为澄清液体 The cell grow is good. 细胞生长状态良好 Sampling Method 取样方式 Site inspection 现场观察 Observation at site in square bottles 方瓶中现场观察 Observation at site in square bottles 方瓶中现场观察 Observation at site in square bottles 方瓶中现场观察 Observation at site in square Amount 取样量 1 unit 1支 1 bottle 1瓶 Day 2 第2天 The cell grow is good. 细胞生长状态良好 1 bottle 1瓶 Day 3 第3天 Day 4 The cell grow is good. 细胞生长状态良好 The cell grow is good. 1 bottle 1瓶 1 bottle 名称 Title Process Validation (PV) Protocol for XXX XXX工艺验证方案 Page 页码 Doc. No. 文件编号第4天显微观察细胞生长状态良好 Under microscope observation the cells have grown into a compact single layer, the cells are shuttle-shaped and the growth liquor is orange in color. 显微镜下观察培养瓶长成致密单层，细胞呈梭形，生长液为橙色 Under microscope observation the cells have grown into a compact single layer, the cells are shuttle-shaped and the growth liquor is orange in color. 显微镜下观察培养瓶长成致密单层，细胞呈梭形，生长液为橙色 Under microscope observation the cells have grown into a compact single layer, the cells are shuttle-shaped and the growth liquor is orange in color. 显微镜下观察培养瓶长成致密单层，细胞呈梭形，生长液为橙色 The cell grow is good. 细胞生长状态良好 bottles 方瓶中现场观察 1瓶 Day 5 第5天 Microscopic inspection 显微观察 Observation at site in square bottles 方瓶中现场观察 1 bottle 1瓶 Day 6 (if necessary) 第6天（如需） Microscopic inspection 显微观察 Observation at site in square bottles 方瓶中现场观察 1 bottle 1瓶 Day 7 (if necessary) 第7天（如需） Microscopic inspection 显微观察 Observation at site in square bottles 方瓶中现场观察 1 bottle 1瓶 Day 1 第1天 Microscopic inspection 显微观察 Microscopic inspection 显微观察 Microscopic inspection 显微观察 Microscopic inspection 显微观察 Day 2 第2天 The cell grow is good. 细胞生长状态良好 Day 3 第3天 The cell grow is good. 细胞生长状态良好 C26-方瓶第二次培养 Day 4 第4天 The cell grow is good. 细胞生长状态良好 Under microscope observation the cells have grown into a compact single layer, the cells are shuttle-shaped and the growth liquor is orange in color. 显微镜下观察培养瓶长成致密单层，细胞呈梭形，生长液为橙色 Under microscope observation the cells have grown into a compact single Observation at site in square bottles 方瓶中现场观察 Observation at site in square bottles 方瓶中现场观察 Observation at site in square bottles 方瓶中现场观察 Observation at site in square bottles 方瓶中现场观察 4 bottles 4瓶 4 bottles 4瓶 4 bottles 4瓶 4 bottles 4瓶 Day 5 第5天 Microscopic inspection 显微观察 Observation at site in square bottles 方瓶中现场观察 4 bottles 4瓶 Day 6 (if necessary) Microscopic inspection Observation at site in square bottles 4 bottles 4瓶名称 Title Process Validation (PV) Protocol for XXX XXX工艺验证方案 Page 页码 Doc. No. 文件编号第6天（如需） layer, the cells are shuttle-shaped and the growth liquor is orange in color. 显微镜下观察培养瓶长成致密单层，细胞呈梭形，生长液为橙色 Under microscope observation the cells have grown into a compact single layer, the cells are Microscopic shuttle-shaped and the inspection growth liquor is orange in 显微观察 color. 显微镜下观察培养瓶长成致密单层，细胞呈梭形，生长液为橙色显微观察 Microscopic inspection 显微观察 The cell grow is good. 细胞生长状态良好方瓶中现场观察 Day 7 (if necessary) 第7天（如需） Observation at site in square bottles 方瓶中现场观察 4 bottles 4瓶 Day 1 第1天 Day 2 第2天 Microscopic inspection 显微观察 The cell grow is good. 细胞生长状态良好 Day 3 第3天 Microscopic inspection 显微观察 The cell grow is good. 细胞生长状态良好 Day 4 第4天 C27-克氏瓶培养 Day 5 第5天 Microscopic inspection 显微观察 The cell grow is good. 细胞生长状态良好 Under microscope observation the cells have grown into a compact single layer, the cells are shuttle-shaped and the growth liquor is orange in color. 显微镜下观察培养瓶长成致密单层，细胞呈梭形，生长液为橙色 Under microscope observation the cells have grown into a compact single layer, the cells are shuttle-shaped and the growth liquor is orange in color. 显微镜下观察培养瓶长成致密单层，细胞呈梭形，生长液为橙色 Under microscope observation the cells have grown into a compact single Observation at site in Kolle bottles 克氏瓶中现场观察 Observation at site in Kolle bottles 克氏瓶中现场观察 Observation at site in Kolle bottles 克氏瓶中现场观察 Observation at site in Kolle bottles 克氏瓶中现场观察 2 bottles 2瓶 2 bottles 2瓶 2 bottles 2瓶 2 bottles 2瓶 Microscopic inspection 显微观察 Observation at site in Kolle bottles 克氏瓶中现场观察 2 bottles 2瓶 Day 6 (if necessary) 第6天（如需） Microscopic inspection 显微观察 Observation at site in Kolle bottles 克氏瓶中现场观察 2 bottles 2瓶 Day 7 (if necessary) 第7天（如需） Microscopic inspection 显微观察 Observation at site in Kolle bottles 2 bottles 2瓶名称 Title Process Validation (PV) Protocol for XXX XXX工艺验证方案 Page 页码 Doc. No. 文件编号 layer, the cells are shuttle-shaped and the growth liquor is orange in color. 显微镜下观察培养瓶长成致密单层，细胞呈梭形，生长液为橙色 Day 1 第1天 Day 2 第2天 Day 3 第3天 Day 4 第4天 Microscopic inspection 显微观察显微观察 Microscopic inspection 显微观察 Microscopic inspection 显微观察 Microscopic inspection 显微观察 The cell grow is good. 细胞生长状态良好 The cell grow is good. 细胞生长状态良好 The cell grow is good. 细胞生长状态良好 The cell grow is good. 细胞生长状态良好 Under microscope observation the cells have grown into a compact single layer, the cells are shuttle-shaped and the growth liquor is orange in color. 显微镜下观察培养瓶长成致密单层，细胞呈梭形，生长液为橙色 Under microscope observation the cells have grown into a compact single layer, the cells are shuttle-shaped and the growth liquor is orange in color. 显微镜下观察培养瓶长成致密单层，细胞呈梭形，生长液为橙色 Under microscope observation the cells have grown into a compact single layer, the cells are shuttle-shaped and the growth liquor is orange in color. 显微镜下观察培养瓶长成致密单层，细胞呈梭形，生长液为橙色 The cell grow is good. 细胞生长状态良好 The cell grow is good. 细胞生长状态良好 The cell grow is good. 细胞生长状态良好克氏瓶中现场观察 Observation at site in 3L bottles 3L瓶中现场观察 Observation at site in 3L bottles 3L瓶中现场观察 Observation at site in 3L bottles 3L瓶中现场观察 Observation at site in 3L bottles 3L瓶中现场观察 2 bottles 2瓶 2 bottles 2瓶 2 bottles 2瓶 2 bottles 2瓶 Day 5 第5天 C28-3L转瓶第一次培养 Microscopic inspection 显微观察 Observation at site in 3L bottles 3L瓶中现场观察 2 bottles 2瓶 Day 6 (if necessary) 第6天（如需） Microscopic inspection 显微观察 Observation at site in 3L bottles 3L瓶中现场观察 2 bottles 2瓶 Day 7 (if necessary) 第7天（如需） Microscopic inspection 显微观察 Observation at site in 3L bottles 3L瓶中现场观察 2 bottles 2瓶 Day 1 第1天 C29-3L转瓶第二次培养 Day 2 第2天 Day 3 第3天 Microscopic inspection 显微观察 Microscopic inspection 显微观察 Microscopic inspection 显微观察 Observation at 16 bottles site in 3L bottles 16瓶 3L瓶中现场观察 Observation at 16 bottles site in 3L bottles 16瓶 3L瓶中现场观察 Observation at site in 3L bottles 16 bottles 3L瓶中现场观察16瓶 3L瓶中现场观察名称 Title Process Validation (PV) Protocol for XXX XXX工艺验证方案 Page 页码 Doc. No. 文件编号 Day 4 第4天 Microscopic inspection 显微观察 The cell grow is good. 细胞生长状态良好 Under microscope observation the cells have grown into a compact single layer, the cells are shuttle-shaped and the growth liquor is orange in color. 显微镜下观察培养瓶长成致密单层，细胞呈梭形，生长液为橙色 Under microscope observation the cells have grown into a compact single layer, the cells are shuttle-shaped and the growth liquor is orange in color. 显微镜下观察培养瓶长成致密单层，细胞呈梭形，生长液为橙色 Under microscope observation the cells have grown into a compact single layer, the cells are shuttle-shaped and the growth liquor is orange in color. 显微镜下观察培养瓶长成致密单层，细胞呈梭形，生长液为橙色 The cell grow is good. 细胞生长状态良好 The cell grow is good. 细胞生长状态良好 The cell grow is good. 细胞生长状态良好 The cell grow is good. 细胞生长状态良好 Under microscope observation the cells have grown into a compact single layer, the cells are shuttle-shaped and the growth liquor is orange in color. 显微镜下观察培养瓶长成致密单层，细胞呈梭形，生长液为橙色 Under microscope observation the cells have grown into a compact single layer, the cells are shuttle-shaped and the growth liquor is orange in Observation at 16 bottles site in 3L bottles 16瓶 3L瓶中现场观察 Day 5 第5天 Microscopic inspection 显微观察 Observation at 16 bottles site in 3L bottles 16瓶 3L瓶中现场观察 Day 6 (if necessary) 第6天（如需）第6天（如需） Microscopic inspection 显微观察 Observation at 16 bottles site in 3L bottles 16瓶 3L瓶中现场观察 Day 7 (if necessary) 第7天（如需） Microscopic inspection 显微观察 Observation at 16 bottles site in 3L bottles 16瓶16瓶 3L瓶中现场观察 Day 1 第1天 Day 2 第2天 Day 3 第3天 Day 4 第4天 C30-3L转瓶第三次培养 Day 5 第5天 Microscopic inspection 显微观察 Microscopic inspection 显微观察 Microscopic inspection 显微观察 Microscopic inspection 显微观察 Observation at site in 3L bottles 3L瓶中现场观察 Observation at site in 3L bottles 3L瓶中现场观察 Observation at site in 3L bottles 3L瓶中现场观察 Observation at site in 3L bottles 3L瓶中现场观察 20 bottles 20瓶 20 bottles 20瓶 20 bottles 20瓶 20 bottles 20瓶 Microscopic inspection 显微观察 Observation at 20 bottles site in 3L bottles 20瓶 3L瓶中现场观察 Day 6 (if necessary) 第6天（如需） Microscopic inspection 显微观察 Observation at 20 bottles site in 3L bottles 20瓶 3L瓶中现场观察名称 Title Process Validation (PV) Protocol for XXX XXX工艺验证方案 Page 页码 Doc. No. 文件编号 color. 显微镜下观察培养瓶长成致密单层，细胞呈梭形，生长液为橙色 Under microscope observation the cells have grown into a compact single layer, the cells are Microscopic shuttle-shaped and the inspection growth liquor is orange in 显微观察 color. 显微镜下观察培养瓶长成致密单层，细胞呈梭形，生长液为橙色 Microscopic inspection 显微观察 The cell grow is good. 细胞生长状态良好 Day 7 (if necessary) 第7天（如需） Observation at 20 bottles site in 3L bottles 20瓶 3L瓶中现场观察 Day 1 第1天 Day 2 第2天 Microscopic inspection 显微观察 The cell grow is good. 细胞生长状态良好 Day 3 第3天 Microscopic inspection 显微观察 The cell grow is good. 细胞生长状态良好 Day 4 第4天 Microscopic inspection 显微观察 The cell grow is good. 细胞生长状态良好 Under microscope observation the cells have grown into a compact single layer, the cells are shuttle-shaped and the growth liquor is orange in color. 显微镜下观察培养瓶长成致密单层，细胞呈梭形，生长液为橙色 Under microscope observation the cells have grown into a compact single layer, the cells are shuttle-shaped and the growth liquor is orange in color. 显微镜下观察培养瓶长成致密单层，细胞呈梭形，生长液为橙色 Under microscope observation the cells have grown into a compact single layer, the cells are shuttle-shaped and the growth liquor is orange in Observation at site in 15L bottles 15L瓶中现场观察 Observation at site in 15L bottles 15L瓶中现场观察 Observation at site in 15L bottles 15L瓶中现场观察 Observation at site in 15L bottles 15L瓶中现场观察 20 bottles 20瓶 20 bottles 20瓶 20 bottles 20瓶 20 bottles 20瓶 C31-15L瓶第一次培养 Day 5 第5天 Microscopic inspection 显微观察 Observation at site in 15L bottles 15L瓶中现场观察 20 bottles 20瓶 Day 6 (if necessary) 第6天（如需） Microscopic inspection 显微观察 Observation at site in 15L bottles 15L瓶中现场观察 20 bottles 20瓶 Day 7 (if necessary) 第7天（如需） Microscopic inspection 显微观察 Observation at site in 15L bottles 15L瓶中现场观察 20 bottles 20瓶名称 Title Process Validation (PV) Protocol for XXX XXX工艺验证方案 Page 页码 Doc. No. 文件编号 color. 显微镜下观察培养瓶长成致密单层，细胞呈梭形，生长液为橙色 Day 1 第1天 Microscopic inspection 显微观察 The cell grow is good. 细胞生长状态良好 Observation at site in 15L bottles 15L瓶中现场观察 Observation at site in 15L bottles 15L瓶中现场观察 Observation at site in 15L bottles 15L瓶中现场观察 Observation at site in 15L bottles 15L瓶中现场观察 20 bottles 20瓶 Day 2 第2天 Microscopic inspection 显微观察 The cell grow is good. 细胞生长状态良好 20 bottles 20瓶 Day 3 第3天 Microscopic inspection 显微观察 The cell grow is good. 细胞生长状态良好 20 bottles 20瓶 Day 4 第4天 Microscopic inspection 显微观察 The cell grow is good. 细胞生长状态良好 Under microscope observation the cells have grown into a compact single layer, the cells are shuttle-shaped and the growth liquor is orange in color. 显微镜下观察培养瓶长成致密单层，细胞呈梭形，生长液为橙色 Under microscope observation the cells have grown into a compact single layer, the cells are shuttle-shaped and the growth liquor is orange in color. 显微镜下观察培养瓶长成致密单层，细胞呈梭形，生长液为橙色 Under microscope observation the cells have grown into a compact single layer, the cells are shuttle-shaped and the growth liquor is orange in color. 显微镜下观察培养瓶长成致密单层，细胞呈梭形，生长液为橙色 20 bottles 20瓶 C32-15L瓶第二次培养 Day 5 第5天 Microscopic inspection 显微观察 Observation at site in 15L bottles 15L瓶中现场观察 20 bottles 20瓶 Day 6 (if necessary) 第6天（如需） Microscopic inspection 显微观察 Observation at site in 15L bottles 15L瓶中现场观察 20 bottles 20瓶 Day 7 (if necessary) 第7天（如需） Microscopic inspection 显微观察 Observation at site in 15L bottles 15L瓶中现场观察 20 bottles 20瓶  Sample No. 样品编码 Table 29 Sample Nos. for Resurrection and Passage

表格 30复苏和传代样品编码

名称 Title Process Validation (PV) Protocol for XXX XXX工艺验证方案 Page 页码 Doc. No. 文件编号 Process Step 工艺过程 Resurrection 复苏 C25 - First square bottle incubation C25-方瓶第一次培养 C26 - second square bottle incubation C26-方瓶第二次培养 C27 - Kolle flask incubation C27-克氏瓶培养 C28 - First 3L rotary bottle incubation C28-3L转瓶第一次培养 C29 - second 3L rotary bottle incubation C29-3L转瓶第二次培养 C30 - third 3L rotary bottle incubation C30-3L转瓶第三次培养 C31 - first 15L bottle incubation C31-15L瓶第一次培养 C32 - second 15L bottle incubation C32-15L瓶第二次培养 Sample No. 样品编码 CR-IN-01 C25-ME-01 C26-ME-01~04 C27-ME-01~02 C28-ME-01~02 C29-ME-01~16 C30-ME-01~20 C31-ME-01~20 C32-ME-01~20  Sampling and testing method取样及检测方法 The sampling operations at this step are alls performed in cell containing bottles or test tubes. The entire testing process is performed at site without causing any damages. 本过程取样均在细胞盛装的瓶中或试管中，检测过程均为无损现场检测。  Test records检测记录

The test results are to be summarized in the Attachment 10 – Cell Growth Inspection Test Record. The original test records shall be attached to this protocol.

检测结果统计在附表10-细胞生长情况检查记录表，原始检测记录作为附件附在本方案后。 11.3 Harvest liquid test 收获液检测  Purpose 目的

Perform the harvest liquid obtained through 20 successive harvests. The test items include HBsAg expression and sterility. The tests are used to verify the quality of the harvest liquid. 对连续20次收获的收获液进行检查，检测项目为HBsAg表达量和无菌检查，通过检测，确定收获液的质量。

 Acceptance Criteria可接受标准

Table 31 Specifications of Harvest Liquid

名称 Title Process Validation (PV) Protocol for XXX XXX工艺验证方案 Page 页码 Doc. No. 文件编号表格 32收获液标准

Test Items 检测项目 Sterility test 无菌检查 HBsAg expression level HBsAg表达量 Acceptance criteria 合格标准 Conforming to the specifications 应符合规定 ELISA test, no less than 1.0mg/L ELISA法检测，应不低于1.0mg/L Reference specifications: Specifications for Intermediates of Recombinant Hepatitis B Vaccine (CHO cell) (Document No.: 1106.031-14-01)

参考标准：重组乙型肝炎疫苗（CHO细胞）中间品质量标准（文件编号：1106.031-14-01）  Sampling and testing method取样及检测方法

When taking the above samples, all samples for sterility test shall be taken and tested following the SOP for Sterility Test (No.: 1302.001-16-02).

样品取样时，无菌检测的，按照无菌检查标准操作规程（编号：1302.001-16-02）的要求进行取样和检验。

The HBsAg content shall be tested as specified in SOP for the Assay of Recombinant Hepatitis B Vaccine(CHO cell) HBsAg (ELISA Test) (Document No.: 1302.013-16-02). HBsAg含量测定按照《重组乙型肝炎疫苗（CHO细胞）HBsAg含量测定(酶联免疫法)标准操作规程》（文件编号：1302.013-16-02）要求执行。  Test records检测记录

A Sterility Test Record (Membrane Filtration Method) (Document No.: 2102.001-16-02) or a Sterility Test Record (Direct Inoculation Method) (Document No.: 2102.002-16-02) shall be filled for each of the sample for sterility test.

无菌检测的，记录填写在《无菌检查（薄膜过滤法）记录》（文件编号：2102.001-16-02）或无菌检查(直接接种法)记录（文件编号：2102.002-16-02）。

The HBsAg content test results shall be recorded in a Record for the Assay of Recombinant Hepatitis B Vaccine (CHO cell) HBsAg (ELISA Test) (Document No.: 2102.014-16-02). HBsAg含量测定记录填写在《重组乙型肝炎疫苗（CHO细胞）HBsAg含量测定（酶联免疫法）记录》（文件编号：2102.014-16-02）

The test results are to be summarized in Attachment 11 Harvest Liquid Test Result Summarization Table. The original test records shall be attached to this protocol. 检测结果统计在附表11-收获液检测记录统计表。原始检测记录作为附件附在本方案后。

名称 Title Process Validation (PV) Protocol for XXX XXX工艺验证方案 Page 页码 Doc. No. 文件编号 11.4 Clarification Filtration Efficacy Test 澄清过滤效果检测  Purpose 目的

Check the cell residue filtration efficacy for the 20 consecutive runs to verify that there are no cell residues or cells in the filtrate and prove that the filtration efficacy conforms to the requirements.

对连续20次去细胞残渣过滤效果进行检查，确定过滤液中没有细胞残渣或细胞，证明过滤效果符合要求。

 Acceptance Criteria可接受标准

Table 33 Specifications of Clarification Filtration

表格 34澄清过滤标准

Test Items 检测项目 Visual inspection 目测 Microscopic inspection 镜检 Acceptance Criteria 合格标准 It shall be a kind of clear and transparent liquid. 应为澄清透明液体 No dead/live cells 无死/活细胞  Sampling and testing method取样及检测方法 Take 100ml from each of the filtrate after clarification filtration. Perform a visual inspection first and then inspect the sample under a microscope.

在澄清过滤后的液体中每次取100ml，先进行目检，在放于显微镜下观察。  Test records 检测记录

The test results are to be summarized in Attachment 12 Clarification Filtration Efficacy Test Record.

检测结果记录在附表12-澄清过滤效果检测记录表。 11.5 UF Concentration 1 Efficacy Test超滤浓缩1效果检测  Purpose 目的

Perform protein content test on the liquid above the membrane and that below the membrane of the UF concentration of the 20 consecutive clarification filtration runs (UF concentration 1) to verify that the UF membrane can effectively retain proteins with a molecular weight above 100Kd.

对连续20次对澄清过滤后的超滤浓缩（简称为超滤浓缩1）后的膜上液和膜下液进行蛋白含量检测，证明超滤膜能够有效的截留100Kd以上蛋白。  Acceptance Criteria可接受标准

Table 35 Specifications of UF Concentration 1

名称 Title Process Validation (PV) Protocol for XXX XXX工艺验证方案 Page 页码 Doc. No. 文件编号表格 36超滤浓缩1标准

Test Items 检测项目 Liquid above Membrane 膜上液 Membrane filtrate 膜下液 Acceptance Criteria 合格标准 The calculated protein content is the same as the total protein content in the harvest liquid 经过与收获液含量计算，蛋白总量一致 The protein content test result is negative. 蛋白含量检测为阴性 Calculation formula for liquid above membrane: harvest volume (L) X harvest concentration (mg/L) = volume of liquid above membrane (L) X protein concentration of liquid above membrane (mg/L)

膜上液这算公式：收获液体积（L）×收获液浓度（mg/L）=膜上液体积（L）×膜上液蛋白浓度（mg/L）

 Sampling and testing method取样及检测方法

Take 20ml of sample from the liquid above membrane and the membrane filtrate respectively after UF concentration 1 each time and perform the test as specified in the SOP for Protein Content Test (Lowry Test) for Recombinant Hepatitis B Vaccine (CHO cell).

膜上液和膜下液每次在超滤浓缩1完成后，取样20ml，按照重组乙型肝炎疫苗(CHO细胞) 蛋白质含量测定（Lowry法）标准操作规程（文件编号：1302.004-16-02）进行检测  Test records检测记录

The protein content test results shall be recorded in a Record for Protein Content Test (Lowry Test) for Recombinant Hepatitis B Vaccine (CHO cell) (Document No.: 2102.005-16-02).

蛋白含量记录填写在重组乙型肝炎疫苗(CHO细胞)蛋白质含量测定（Lowry法）记录（文件编号：2102.005-16-02）。

The test results are to be summarized in Attachment 13 UF Concentration 1 Efficacy Test Result Summarization Table. The original test records shall be attached to this protocol. 检测结果统计在附表13-超滤浓缩1效果检测记录统计表。原始检测记录作为附件附在本方案后。

11.6 Ammonium sulfate precipitation efficacy test 硫酸铵沉淀效果检测  Purpose 目的

Perform the test on the supernatant obtained from 20 consecutive ammonium sulfate

名称 Title Process Validation (PV) Protocol for XXX XXX工艺验证方案 Page 页码 Doc. No. 文件编号 precipitation runs to verify that the target proteins can be precipitated completely during the ammonium sulfate precipitation process or the predetermined efficacy can be achieved. 对连续20次对硫酸铵沉底的上清液进行检测，确定硫酸铵沉淀时可以将目标蛋白完全沉淀，或达到预期的效果。

 Acceptance Criteria可接受标准

Table 37 Specifications of Ammonium Sulfate Precipitation

表格 38硫酸铵沉淀标准

Test Items 检测项目 Protein content in supernatant 上清液蛋白含量 Acceptance Criteria 合格标准 Only the actual values are recorded. 本次测试仅记录实际值  Sampling and testing method取样及检测方法 Take 20ml of sample from the supernatant after ammonium sulfate precipitation and perform the test as specified in the SOP for Protein Content Test (Lowry Method) for Recombinant Hepatitis B Vaccine (CHO cell) (Document No.: 1302.004-16-02).

硫酸铵沉淀结束后，取上清液20ml，按照重组乙型肝炎疫苗(CHO细胞) 蛋白质含量测定（Lowry法）标准操作规程（文件编号：1302.004-16-02）进行检测  Test records检测记录

The protein content test results shall be recorded in a Record for Protein Content Test (Lowry Test) for Recombinant Hepatitis B Vaccine (CHO cell) (Document No.: 2102.005-16-02).

蛋白含量记录填写在重组乙型肝炎疫苗(CHO细胞)蛋白质含量测定（Lowry法）记录（文件编号：2102.005-16-02）。

The test results are to be summarized in Attachment 14 Ammonium Sulfate Precipitation Efficacy Test Result Summarization Table. The original test records shall be attached to this protocol.

检测结果统计在附表14硫酸铵沉淀效果检测记录统计表。原始检测记录作为附件附在本方案后。

11.7 Potassium bromide extraction efficacy test 溴化钾提取效果检测  Purpose 目的

Perform potassium bromide extraction for 8 times and then perform the low speed

名称 Title Process Validation (PV) Protocol for XXX XXX工艺验证方案 Page 页码 Doc. No. 文件编号 centrifugation. Test the supernatant to verify the output of the potassium bromide extraction step.

对连续20次溴化钾提取8次后并低速离心后的上清液进行检测，确定溴化钾提取的产量。  Acceptance Criteria可接受标准

Table 39 Specifications of Potassium Bromide Extraction

表格 40溴化钾提取标准

Test Items 检测项目 Protein content in supernatant 上清液蛋白含量 Acceptance Criteria 合格标准 Only the actual values are recorded. 本次测试仅记录实际值  Sampling and testing method取样及检测方法 After 8 potassium bromide extraction runs, combine the supernatant and perform the low speed centrifugation on the supernatant. After completing centrifugation, take 20ml of sample from the supernatant and perform the test as specified in the SOP for Protein Content Test (Lowry Method) for Recombinant Hepatitis B Vaccine (CHO cell) (Document No.: 1302.004-16-02).

溴化钾完成8次提取后，合并上清液，对上清液进行低速离心，离心完成后，取上清液20ml，按照重组乙型肝炎疫苗(CHO细胞) 蛋白质含量测定（Lowry法）标准操作规程（文件编号：1302.004-16-02）进行检测  Test records检测记录

The protein content test results shall be recorded in a Record for Protein Content Test (Lowry Test) for Recombinant Hepatitis B Vaccine (CHO cell) (Document No.: 2102.005-16-02).

蛋白含量记录填写在重组乙型肝炎疫苗(CHO细胞)蛋白质含量测定（Lowry法）记录（文件编号：2102.005-16-02）。

The test results are to be summarized in Attachment 15 Potassium Bromide Extraction Efficacy Test Result Summarization Table. The original test records shall be attached to this protocol.

检测结果统计在附表15-溴化钾效果检测记录统计表。原始检测记录作为附件附在本方案后。 11.8 First density gradient centrifugation efficacy test 一次密度梯度离心效果检测  Purpose 目的

名称 Title Process Validation (PV) Protocol for XXX XXX工艺验证方案 Page 页码 Doc. No. 文件编号 Check the efficacy of the first density gradient centrifugation to verify the output of the first density gradient centrifugation.

对连续20次一次密度梯度离心的效果进行检测，确定一次密度梯度离心的的产量。  Acceptance Criteria可接受标准

Table 41 Specifications for First Density Gradient Centrifugation

表格 42一次密度梯度离心标准

Test Items 检测项目 Protein content in the antigen solution 抗原溶液蛋白含量 Acceptance Criteria 合格标准 Only the actual values are recorded. 本次测试仅记录实际值  Sampling and testing method取样及检测方法 After completing the first density gradient centrifugation, combine the antigen solution and take 20ml of sample from the antigen solution. Perform the test as specified in the SOP for Protein Content Test (Lowry Method) for Recombinant Hepatitis B Vaccine (CHO cell) (Document No.: 1302.004-16-02).

一次密度梯度离心完成后，合并抗原溶液，取抗原溶液20ml，按照重组乙型肝炎疫苗(CHO细胞) 蛋白质含量测定（Lowry法）标准操作规程（文件编号：1302.004-16-02）进行检测  Test records 检测记录

The protein content test results shall be recorded in a Record for Protein Content Test (Lowry Test) for Recombinant Hepatitis B Vaccine (CHO cell) (Document No.: 2102.005-16-02).

蛋白含量记录填写在重组乙型肝炎疫苗(CHO细胞)蛋白质含量测定（Lowry法）记录（文件编号：2102.005-16-02）。

The test results are to be summarized in Attachment 16 First Density Gradient Centrifugation Efficacy Test Result Summarization Table. The original test records shall be attached to this protocol.

检测结果统计在附表16-一次密度梯度离心效果检测记录统计表。原始检测记录作为附件附在本方案后。

11.9 Second density gradient centrifugation efficacy test 二次密度梯度离心效果检测  Purpose 目的

名称 Title Process Validation (PV) Protocol for XXX XXX工艺验证方案 Page 页码 Doc. No. 文件编号 Check the efficacy of the second density gradient centrifugation to verify the output of the second density gradient centrifugation.

对连续20次二次密度梯度离心的效果进行检测，确定二次密度梯度离心的的产量。  Acceptance Criteria可接受标准

Table 43 Specifications for Second Density Gradient Centrifugation

表格 44二次密度梯度离心标准

Test Items 检测项目 Protein content in the antigen solution 抗原溶液蛋白含量 Acceptance Criteria 合格标准 Only the actual values are recorded. 本次测试仅记录实际值  Sampling and testing method取样及检测方法 After completing the second density gradient centrifugation, combine the antigen solution and take 20ml of sample from the antigen solution. Perform the test as specified in the SOP for Protein Content Test (Lowry Method) for Recombinant Hepatitis B Vaccine (CHO cell) (Document No.: 1302.004-16-02).

二次密度梯度离心完成后，合并抗原溶液，取抗原溶液20ml，按照重组乙型肝炎疫苗(CHO细胞) 蛋白质含量测定（Lowry法）标准操作规程（文件编号：1302.004-16-02）进行检测  Test records 检测记录

The protein content test results shall be recorded in a Record for Protein Content Test (Lowry Test) for Recombinant Hepatitis B Vaccine (CHO cell) (Document No.: 2102.005-16-02).

蛋白含量记录填写在重组乙型肝炎疫苗(CHO细胞)蛋白质含量测定（Lowry法）记录（文件编号：2102.005-16-02）。

The test results are to be summarized in Attachment 17 Second Density Gradient Centrifugation Efficacy Test Result Summarization Table. The original test records shall be attached to this protocol.

检测结果统计在附表17-二次密度梯度离心效果检测记录统计表。原始检测记录作为附件附在本方案后。 11.10

UF Concentration 2 Efficacy Test 超滤浓缩2效果检测

 Purpose 目的

名称 Title Process Validation (PV) Protocol for XXX XXX工艺验证方案 Page 页码 Doc. No. 文件编号 Perform protein content test on the liquid above the membrane and the membrane filtrate after the UF concentration after completing the second dense gradient centrifugation (UF concentration 2) to verify that the UF membrane can effectively retain proteins with a molecular weight above 100Kd.

对连续20次对二次密度梯度离心后的超滤浓缩（简称为超滤浓缩2）后的膜上液和膜下液进行蛋白含量检测，证明超滤膜能够有效的截留100Kd以上蛋白。  Acceptance Criteria可接受标准

Table 45 Specifications of UF Concentration 2

表格 46超滤浓缩2标准

膜上液这算公式：收获液体积（L）×收获液浓度（mg/L）=膜上液体积（L）×膜上液蛋白浓度（mg/L）

 Sampling and testing method取样及检测方法

Take 20ml of sample from the liquid above membrane and the membrane filtrate respectively after UF concentration 1 each time and perform the test as specified in the SOP for Protein Content Test (Lowry Method) for Recombinant Hepatitis B Vaccine (CHO cell).

The protein content test results shall be recorded in a Record for Protein Content Test (Lowry Test) for Recombinant Hepatitis B Vaccine (CHO cell) (Document No.: 2102.005-16-02).

蛋白含量记录填写在重组乙型肝炎疫苗(CHO细胞)蛋白质含量测定（Lowry法）记录（文件编

名称 Title Process Validation (PV) Protocol for XXX XXX工艺验证方案 Page 页码 Doc. No. 文件编号号：2102.005-16-02）。

The test results are to be summarized in Attachment 18 UF Concentration 2 Efficacy Test Result Summarization Table. The original test records shall be attached to this protocol. 检测结果统计在附表18-超滤浓缩2效果检测记录统计表。原始检测记录作为附件附在本方案后。 11.11

Chromatography purification product test 层析纯化产物检测

 Purpose 目的

Test the chromatographic purification products to verify the quality and the output of the chromatographic purification step.

对连续20次层析纯化的产物进行检测，确定层析纯化的质量而后产量。  Acceptance Criteria可接受标准

Table 47 Specifications of Chromatographic Purification

表格 48纯化产物标准

Test Items 检测项目 Acceptance Criteria 合格标准 Operation Procedures 操作规程 SOP for Protein Content Test (Lowry Test) for Recombinant Hepatitis B Vaccine (CHO cell) (Document No.: 1302.004-16-02). 重组乙型肝炎疫苗(CHO细胞) 蛋白质含量测定（Lowry法）标准操作规程（文件编号：1302.004-16-02） Test records 检测记录记录 Record for Protein Content Test (Lowry Test) for Recombinant Hepatitis B Vaccine (CHO cell) (Document No.: 2102.005-16-02). 重组乙型肝炎疫苗(CHO细胞)蛋白质含量测定（Lowry法）记录（文件编号：2102.005-16-02） Record for Specific Protein Belt Test (SDS-polyacrylamide Gel Electrophoresis) for Recombinant Hepatitis B Vaccine (CHO cell) (Document No.: 2102.006-16-02). 重组乙型肝炎疫苗(CHO细胞)特异蛋白带检测（SDS-聚丙烯酰胺凝胶电泳法）记录（文件编号：2102.006-16-02） Record for HBsAg Purity Test (HPLC) for Recombinant Hepatitis B Vaccine (CHO cell) (Document No.: 2102.007-16-02). 重组乙型肝炎疫苗(CHO细Protein content 蛋白质含量 It shall be 100 to 200μg/m (Method II of Appendix VI B) 应为100～200μg/ml（附录VI B 第二法） Specific protein belt 特异蛋白带 Use the reducing SDS polyacrylamide gel electrophoresis (Appendix IV C). There shall be belts of proteins with molecular weights of 23kD and 27kD. There can be belts of proteins with a molecular weight of 30kD and HBsAg polymer proteins. 采用还原型SDS聚丙烯酰胺凝胶电泳法（附录IV C），在分离胶中应有分子量23kD、27kD蛋白带，可有30kD蛋白带及HBsAg多聚体蛋白带 Use the SEC-HPLC method and the result shall be no less than 95.5%. SEC-HPLC法，应不低于95.5% SOP for Specific Protein Belt Test (SDS-polyacrylamide Gel Electrophoresis) for Recombinant Hepatitis B Vaccine (CHO cell) (Document No.: 2102.006-16-02). 重组乙型肝炎疫苗(CHO细胞)特异蛋白带检测（SDS-聚丙烯酰胺凝胶电泳法）（文件编号：2102.006-16-02） Purity 纯度 SOP for Purity Test (HPLC) for Recombinant Hepatitis B Vaccine (CHO cell) (Document No.: 1302.006-16-02). 重组乙型肝炎疫苗(CHO细名称 Title Process Validation (PV) Protocol for XXX XXX工艺验证方案 Page 页码 Doc. No. 文件编号胞) 纯度检测（高效液相色谱法）标准操作规程（文件编号：1302.006-16-02） SOP for Bacterial Endotoxin Test (Gel Limit Test) for Recombinant Hepatitis B It shall be less than 10EU per Vaccine (CHO cell) 10μg protein (Appendix XII (Document No.: E gel limit test) 1302.010-16-02). 每10μg蛋白质应小于10EU重组乙型肝炎疫苗（CHO细（附录Ⅻ E凝胶限度试验）胞）细菌内毒素检查（凝胶限度试验）标准操作规程（文件编号：1302.010-16-02）胞)HBsAg纯度检测（高效液相色谱法）记录（文件编号：2102.007-16-02） Record for Bacterial Endotoxin Test (Gel Limit Test) for Recombinant Hepatitis B Vaccine (CHO cell) (Document No.: 2102.011-16-02). 重组乙型肝炎疫苗（CHO细胞）细菌内毒素检测（凝胶限度试验）记录（文件编号：2102.011-16-02） Bacterial endotoxin test 细菌内毒素检查  Reference specifications: Specifications for Intermediates of Recombinant Hepatitis B Vaccine (CHO cell) (Document No.: 1106.031-14-01)

 参考标准：重组乙型肝炎疫苗（CHO细胞）中间品质量标准（文件编码：1106.031-14-01）  Test records 检测记录

Record the test results in the records listed in the above table. 检测结果记录在上表所列记录中。

The test results are to be summarized in Attachment 19 Chromatographic Purification Product Test Result Summarization Table. The original test records shall be attached to this protocol.

检测结果统计在附表19-层析纯化产物检测记录统计表。原始检测记录作为附件附在本方案后。

名称 Title Process Validation (PV) Protocol for XXX XXX工艺验证方案 Page 页码 Doc. No. 文件编号 12. Bulk Determination原液检验

When the whole process has been completed the determination for finished product should be performed as described in the document, record statistic of the test result into following table. 整个生产工艺结束应按文件规定进行原液检验，检验结果应符合成品质量标准，将统计结果记入测试20-原液检验表中。

The test criteria and the relevant procedures are listed in the table below. 检验标准和相关规程如下表：

Table 49 Specifications of Bulk

表格 50原液标准

Test Items 检测项目 Acceptance Criteria 合格标准 Operation Procedures 操作规程 Standard Operation Procedures for Sterility Test (Document No.: 1302.001-16-02) 无菌检查标准操作规程（编号：1302.001-16-02） Test records 检测记录记录 Record for Sterility Test (Membrane Filtration Method) (Document No.: 2102.001-16-02) 无菌检查（薄膜过滤法）记录（文件编号：2102.001-16-02） Sterility test 无菌检查 Conforming to the specifications 应符合规定 Mycoplasma test 支原体检查 Protein content 蛋白质含量 Specific protein belt 特异蛋白带 Standard Operation Record for Mycoplasma Test Procedures for Mycoplasma (Cultivation Method) Test (Cultivation Method) Conforming to the (Document No.: (Document No.: specifications 2102.003-16-02) 1302.002-16-02) 应符合规定支原体检查(培养法)记录（文支原体检查(培养法)标准操作件编号：2102.003-16-02）规程（编号：1302.002-16-02） SOP for Protein Content Test Record for Protein Content (Lowry Test) for Test (Lowry Test) for Recombinant Hepatitis B Recombinant Hepatitis B Vaccine (CHO cell) Vaccine (CHO cell) It shall be 100 to 200μg/m (Document No.: (Document No.: (Method II of Appendix VI B) 1302.004-16-02). 2102.005-16-02). 应为100～200μg/ml（附录重组乙型肝炎疫苗(CHO细重组乙型肝炎疫苗(CHO细VI B 第二法）胞) 蛋白质含量测定（Lowry胞)蛋白质含量测定（Lowry法）标准操作规程（文件编号：法）记录（文件编号：1302.004-16-02） 2102.005-16-02） Use the reducing SDS polyacrylamide gel Record for Specific Protein electrophoresis (Appendix IV SOP for Specific Protein Belt Belt Test C). There shall be belts of (SDS-polyacrylamide Gel Test (SDS-polyacrylamide proteins with molecular Electrophoresis) for Gel Electrophoresis) for weights of 23kD and 27kD. Recombinant Hepatitis B Recombinant Hepatitis B There can be belts of Vaccine (CHO cell) Vaccine (CHO cell) proteins with a molecular (Document No.: (Document No.: weight of 30kD and HBsAg 2102.006-16-02). 2102.006-16-02). polymer proteins. 重组乙型肝炎疫苗(CHO细重组乙型肝炎疫苗(CHO细采用还原型SDS聚丙烯酰胺胞)特异蛋白带检测（SDS-聚胞)特异蛋白带检测（SDS-聚凝胶电泳法（附录IV C），在丙烯酰胺凝胶电泳法）记录丙烯酰胺凝胶电泳法）（文件分离胶中应有分子量23kD、（文件编号：编号：2102.006-16-02） 27kD蛋白带，可有30kD蛋白2102.006-16-02）带及HBsAg多聚体蛋白带名称 Title Process Validation (PV) Protocol for XXX XXX工艺验证方案 Page 页码 Doc. No. 文件编号 Bovine serum albumin residue 牛血清白蛋白残留量 It shall be no more than 45ng/dose 应不高于45ng/剂（附录ⅧⅠ） SOP for Bovine Albumin Quantitative Analysis (Document No.: 1302.005-14-01) 牛血清白蛋白定量分析标准操作规程（文件编号：1302.005-14-01） SOP for Purity Test (HPLC) for Recombinant Hepatitis B Vaccine (CHO cell) (Document No.: 1302.006-16-02). 重组乙型肝炎疫苗(CHO细胞) 纯度检测（高效液相色谱法）标准操作规程（文件编号：1302.006-16-02） SOP for CHO Cell DNA Residue Test (DNA Probe Marking) for Recombinant Hepatitis B Vaccine (CHO cell) (Document No.: 1302.007-16-02). 重组乙型肝炎疫苗(CHO细胞)CHO细胞DNA残留量测定（DNA探针标记法）标准操作规程（文件编号：1302.007-16-02） SOP for CHO Cell Protein Residue Test for Recombinant Hepatitis B Vaccine (CHO cell) (Document No.: 1302.009-16-02). 重组乙型肝炎疫苗（CHO细胞）CHO细胞蛋白质残留量检查标准操作规程（文件编号：1302.009-16-02） SOP for Bacterial Endotoxin Test (Gel Limit Test) for Recombinant Hepatitis B Vaccine (CHO cell) (Document No.: 1302.010-16-02). 重组乙型肝炎疫苗（CHO细胞）细菌内毒素检查（凝胶限度试验）标准操作规程（文件编号：1302.010-16-02） Record for Bovine Albumin Residual Test (Document No.: 2102.015-14-02) 牛血清白蛋白残留量检测记录（文件编号：2102.015-14-02） Record for HBsAg Purity Test (HPLC) for Recombinant Hepatitis B Vaccine (CHO cell) (Document No.: 2102.007-16-02). 重组乙型肝炎疫苗(CHO细胞)HBsAg纯度检测（高效液相色谱法）记录（文件编号：2102.007-16-02） Record for DNA Residue Test (DNA Probe Marking) for Recombinant Hepatitis B Vaccine (CHO cell) (Document No.: 2102.008-16-02). 重组乙型肝炎疫苗(CHO细胞) DNA残留量测定（DNA探针标记法）记录（文件编号：2102.008-16-02） Record for CHO Cell Protein Residue Test for Recombinant Hepatitis B Vaccine (CHO cell) (Document No.: 2102.010-16-02). 重组乙型肝炎疫苗（CHO细胞）CHO细胞蛋白残留量测定（酶联免疫法）记录（文件编号：2102.010-16-02） Record for Bacterial Endotoxin Test (Gel Limit Test) for Recombinant Hepatitis B Vaccine (CHO cell) (Document No.: 2102.011-16-02). 重组乙型肝炎疫苗（CHO细胞）细菌内毒素检测（凝胶限度试验）记录（文件编号：2102.011-16-02） Purity 纯度 Use the SEC-HPLC method and the result shall be no less than 95.5%. SEC-HPLC法，应不低于95.5% CHO cell DNA residue CHO细胞DNA残留量 Perform the test as specified in Method I in Appendix IX B of CP The results shall be no more than 10pg/dose. 按现行《中国药典》三部“附录IX B第一法”进行。检测结果应不高于10pg/剂 The CHO cell protein residue CHO cell shall be no more than 0.05% protein residue of the total protein content. CHO细胞蛋白CHO细胞蛋白质残留量应不质残留量高于总蛋白质含量的0.05% It shall be less than 10EU per Bacterial 10μg protein (Appendix XII endotoxin test E gel limit test) 细菌内毒素检每10μg蛋白质应小于10EU查（附录Ⅻ E凝胶限度试验） Reference specifications: Specifications for Intermediates of Recombinant Hepatitis B Vaccine (CHO cell) (Document No.: 1106.031-14-01) 参考标准：重组乙型肝炎疫苗（CHO细胞）中间品质量标准（文件编码：1106.031-14-01）

13. Process Validation Summarization工艺验证总结

The process validation results are verified based validation test results and the verification results are filled into the table below.

根据验证检验结果，对本批的工艺验证结果的各步骤进行总结，将确认内容记入测试22-重组乙型

名称 Title Process Validation (PV) Protocol for XXX XXX工艺验证方案 Page 页码 Doc. No. 文件编号肝炎疫苗（CHO细胞）原液工艺验证验证结果总结评价表中。

14. PV Report工艺验证报告

14.1 Process Validation Report 工艺验证报告

A final report will be generated once this protocol has been executed and corrective actions completed. A summary of the results obtained will be compiled and a conclusion drawn based on these results, the report also contains the suggestions of the production. And the formal decision on the acceptance/rejection of the validation results shall be made by the responsible reviewer and the approver.

执行本方案并完成了纠正措施之后，将生成一份最终报告。将编写对所获得结果的总结，并根据这些结果得出结论，另外还将包括对生产的建议。并由负责验证的审核人及批准人做出正式的接受/拒绝验证结果的决定。

The PV report will include the approval page, the test summary, the analysis, and the evaluation, the copy of the test form, the deviation report and the copy of the batch production records.

PV报告将包括签批页、测试总结、分析评价、测试表副本，测试过程中涉及的偏差记录及偏差汇总以及验证批的批生产记录复印件。

14.2 Process Validation Analysis and Evaluation 工艺验证分析与评价

Analyze and evaluate the process validation results through scientific methods. Perform evaluations through sufficient data and perform analyses through vivid graphics to establish the trending scope of the variations in product quality and provide Process ability control index for crucial post. Record the analysis and evaluation results in the report.

用科学的方法对工艺验证结果进行分析和评价，通过充足的数据评估，形象化的图标分析，建立产品质量变化的趋势范围，给出关键工序的工序能力控制指数，并将分析与评价结果记录到报告中。

Summarize the production and final test reports in detail, including the test data concerning test failures. When original data cannot be included, the origin of the original data and the method to access the original materials shall be noted.

对生产和最终检测结果详细总结，包括失败的试验数据，当不能包括原始数据时，应说明原始数据的来历和如何找到原始资料。

Compare and review the results against the preset targets. Any deviations from the validation protocol and their relevant explanations shall be recorded together in the

名称 Title Process Validation (PV) Protocol for XXX XXX工艺验证方案 Page 页码 Doc. No. 文件编号 validation protocol and the validation reports.

将结果和预设目标进行对照和审核，任何与验证方案的偏移都应该和解释一起记录在验证方案及报告中。

15. Requirements on Execution Records 执行记录要求

Record Management Procedural, Document No. 1200.002-14-02 shall be followed for the filling of the records during the execution of the PV.

工艺验证执行过程中的记录填写要求，需按照《记录管理程序》，文件编号1200.002-14-02进行填写。

16. Deviation Handling 偏差处理

During execution all deviations shall be handled following Deviation Management Procedural, document No.: 1202.018-14-01.

如执行过程中出现任何偏差之处，需按照《偏差管理程序》，文件编号1202.018-14-01处理。

17. Change Handling 变更处理

If any changes are required after the PV, Change Control Procedural, document No. 1202.001-14-01, shall be followed.

如工艺验证中存在变更，需按照《变更管理程序》，文件编号1202.001-14-01处理。

名称 Title Process Validation (PV) Protocol for XXX XXX工艺验证方案 Page 页码 Doc. No. 文件编号 18. Test Form测试表

Table 51 List of Test Forms

表格 52测试表列表

Attachment type 附件类别 No. 序号 Test 1 测试1 Test 2 测试2 Test 3 测试3 Test 4 测试4 Test 5 测试5 Test 6 测试6 Test 7 测试7 Test 8 测试8 Test 9 测试9 Test10 测试10 Test executed 测试执行 Test 11 测试11 Test 12 测试12 Test 13 测试13 Test 14 测试14 Test 15 测试15 Doc. No. 文件编号 1305-5.016.00-16-01 1305-5.016.00-16-02 1305-5.016.00-16-03 Doc. Name 文件名称 Personnel Identification 人员确认 SOP Qualification SOP确认表 Training Verification 培训确认 System and Equipment and Instrument Calibration Verification 系统和设备仪表校验确认 Production Related Status Verification 生产相关状态确认 Test Instruments Verification 测试用仪器仪表确认 Analytical Method Verification 检验方法确认 Raw Material and Excipient Verification Sheet 原辅料确认表 Liquid Preparation Test Result Summarization Table 液体配制检测记录统计表 Cell Resurrection and Passage Test Record 细胞复苏及传代检查记录 Harvest Test Record Summarization Table 收获液检测记录统计表 Clarification Filtration Efficacy Test Record 澄清过滤效果检测记录表 UF Concentration 1 Efficacy Test Record 超滤浓缩1效果检测记录表 Ammonium Sulfate Precipitation Efficacy Test Record 硫酸铵沉淀效果检测记录表 Potassium Bromide Extraction Efficacy Test Record 溴化钾提取效果检测记录表 Pages 页数 Prerequisite先决条件 1305-5.016.00-16-04 1305-5.016.00-16-05 1305-5.016.00-16-06 1305-5.016.00-16-07 1305-5.016.00-16-08 1305-5.016.00-16-09 1305-5.016.00-16-10 1305-5.016.00-16-11 1305-5.016.00-16-12 1305-5.016.00-16-13 1305-5.016.00-16-14 1305-5.016.00-16-15 名称 Title Process Validation (PV) Protocol for XXX XXX工艺验证方案 Page 页码 Doc. No. 文件编号 Test 16 测试16 First Density Gradient Centrifugation Efficacy Test Record 一次密度梯度离心效果检测记录表 Second Density Gradient Centrifugation Efficacy Test Record 二次密度梯度离心效果检测记录表 UF Concentration 2 Efficacy Test Record 超滤浓缩2效果检测记录表 Chromatographic Purification Product Test Record 层析纯化产物检测记录表 Bulk Determination 原液检验表 Process Validation Summarization 验证结果总结评价表 Process Risk Analysis List 工艺风险分析表 1305-5.016.00-16-16 Test 17 测试17 1305-5.016.00-16-17 Test 18 测试18 Test 19 测试19 Test 20 测试20 Test 21 测试21 attachment 附表 Attachment1 附表1 1305-5.016.00-16-18 1305-5.016.00-16-19 1305-5.016.00-16-20 1305-5.016.00-16-21 1305-5.016.00-16-21

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