Q5D细胞库--中英文对照

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March 1998

CPMP/ICH/294/95

生物制品的质量：用于生物技术产

Quality of Biotechnological Products: Derivation and

品及生物制品生产的细胞基质的

Characterisation of Cell

来源和特性描述

ICH Topic Q 5 D

Substrates Used for Production of Biotechnological/Biological

Products

1. INTRODUCTION

1.1 Objective

The objective of this guideline is to provide broad

guidance on appropriate standards for the derivation of

human and animal cell lines and microbial cells to be

used to prepare biotechnological/biological products

defined in Section 1.3, Scope, and for the preparation

and characterisation of cell banks to be used for

production. The document, therefore, provides

recommendations on the information in these areas that

should be presented in marketing applications for these

products.

1.2 Rationale

Historically, some quality concerns for cell-derived

biological products have originated from the presence of

adventitious contaminants or from the properties of the

cells used to prepare the product. Recombinant DNA

(rDNA) - derived products also carry quality concerns

regarding the expression construct contained in the cell

substrate. Thus, it is well established that the properties

of the cell substrate and events linked to the cell

substrate can affect resultant product quality and safety

and, further, that effective quality control of these

products requires appropriate controls on all aspects of

handling the cell substrate.

This document complements other guidelines to provide

a comprehensive approach to quality issues arising from

biological aspects of processing products from metazoan

and microbial cell culture.

1．前言

1．1 目的

本指导原则的目的是对用于生产生物

技术产品及生物制品的人和动物细胞

系和微生物细胞系以及为用于生产的

细胞库的制备和鉴定建立标准提供指

导。该文件还提供了在这些领域里申请

产品上市应上报哪些资料的建议。

1．2 基本原理

在历史上，由于细胞源性的生物制品存

在外来的污染物或出于对用于制备产

品的细胞性质方面的考虑，引发了一些

对产品质量的关切。重组DNA(rDNA)

制品的质量也与细胞基质中构建的表

达载体有关：由此，人们达成共识，认

为细胞基质的特性以及与之相联系的

事件将影响产品的质量和安全性，为有

效地控制产品质量，需对操作细胞基质

的各方面进行严格控制。

本文件对从后生动物和微生物细胞培

养物制备的制品在生物学方面产生的

有关质量问题作了补充。

1.3 Scope

This guideline covers cell substrates having a cell

banking system. In this document, “cell substrate” refers

to microbial cells or cell lines derived from human or

animal sources that possess the full potential for

generation of the desired biotechnological/biological

products for

human in vivo or ex vivo use. Reagents for in vitro

diagnostic use are outside the scope of this document.

Animal sources of cell lines include all those of

metazoan origin. Both continuous cell lines of indefinite

in vitro lifespan and diploid cells of finite in vitro

lifespan are included.

Microbial sources include bacteria, fungi, yeast, and

other unicellular life forms.

“Biotechnological/biological products” refers to any

products prepared from cells cultivated from cell banks

with the exception of microbial metabolites such as, for

example, antibiotics, amino acids, carbohydrates, and

other low molecular weight substances. Cell banks used

to prepare gene therapy products or vaccines should

follow the recommendations presented in this document.

Some biological products, such as certain viral vaccines,

are prepared in primary cell cultures derived directly

from animal tissues or organs. Primary cells are not

banked and therefore are not addressed by this

document. However, other considerations which may

apply to primary cells are discussed further in Appendix

1 of this document.

2. GUIDELINES

2.1 Source, History, and Generation of the Cell

Substrate

2.1.1 Introduction

It is important to provide supportive documentation

which describes the history of the cell substrate that is

used in the manufacture of a biotechnological/biological

product, as well as any parental cell line from which it

was totally or partially derived. Events during the

research and development phases of the cell substrate

may contribute significantly to assessment of the risks

associated with the use of that particular cell substrate

for production. The information supplied in this regard is

meant to facilitate an overall evaluation which will

ensure the quality and safety of the product.

Careful records of the manipulation of the cell substrate

1．3 范围

本指导原则涵盖了所有具备细胞库系

统的细胞基质。在文件中，“细胞基质”

是指微生物细胞或来自于人或动物的

细胞系，它们具有生产供人类在体内或

体外使用的生物技术产品及生物制品

的全部潜能。体外诊断试剂不在此文件

的范围之内。动物来源的细胞系是指所

有后生动物，包括体外无限生长的传代

细胞系以及体外有限代次的二倍体细

胞。微生物的来源包括细菌、真菌、酵

母和其他单细胞生物。

“生物技术产品及生物制品”是指从细

胞库培养的细胞中制备的任何产品，不

包括细胞代谢物如抗生素、氨基酸、碳

水化合物和其他低分子物质。用于制备

基因治疗产品或疫苗的细胞库也应该

遵循本文件的原则。某些生物制品，如

某些病毒疫苗，是直接从动物组织或器

官的原代细胞培养物中制备的，原代细

胞不建库，因此，在本文中未提及。但

适用于原代细胞的其他要求，在本文的

附录l 中作了进一步的讨论。

2．指导原则

2．1 细胞基质的来源、历史和产

生

2．1．1 前言

为确保制品的质量和安全性，需提供一

份支持性文件，记载生产生物技术产品

及生物制品的细胞基质的历史，还应记

载它们全部或部分来源于母细胞系的

历史，以便将细胞基质在研究和开发阶

段所发生的事件与生产制品所用的特

定细胞基质相联系来全面评估其危险

性。

在开发过程中应详细记录细胞基质的

操作。对细胞历史的记载仅是鉴定细胞

基质的内容之一，一般来说，对细胞历

史资料的缺乏不会影响产品的批准，但

should be maintained throughout its development.

Description of cell history is only one tool of many used

for cell substrate characterisation. In general,

deficiencies in documented history may not, by itself, be

an impediment to product approval, but extensive

deficiencies will result in increased reliance on other

methods to characterise the cell substrate.

2.1.2 Origin, Source, and History of Cells

The source of cells (laboratory or culture collection)

from which the cell substrate was derived should be

stated, and relevant references from the scientific

literature should be cited.

Information obtained directly from the source laboratory

is preferred. When this is not available, literature

references may be utilised.

For human cell lines, it is relevant to describe the

following characteristics of the original donor: Tissue or

organ of origin, ethnic and geographical origin, age, sex

and general physiological condition. If known, the state

of health or medical history of the donor should be

reported along with the results of any tests of the donor

for pathogenic agents. Specifically for human diploid

fibroblasts, the age of the donor may influence the in

vitro lifespan of the cell line and this information should

be provided if available. For animal cell lines, relevant

descriptions of the source include species, strains,

breeding conditions, tissue or organ of origin,

geographical origin, age and sex, the results of tests for

pathogenic agents, and general physiological condition

of the original donor.

For microbes, manufacturers should describe the species,

strain, and known genotypic and phenotypic

characteristics of the organism from which the cell

substrate was derived.

Manufacturers should also describe the pathogenicity,

toxin production, and other biohazard information, if

any.

The cultivation history of the cells should be

documented. The method originally used for the isolation

of the cells should be described as well as the procedures

used in the culturing of the cells in vitro and any

procedures used to establish cell lines (for example, use

of any physical, chemical, or biological procedure, or

added nucleotide sequences). A description of any

genetic manipulation or selection should be provided. All

会使人们更多的依赖其他方法对细胞

基质进行鉴定。

2．1．2 细胞的起源、来源和历史

应说明所用细胞基质的细胞来源(实验

室制备或来源于菌种库)，引证科学文献

上的相关参考资料。直接从实验室获得

的资料是首选的，如果没有这些资料，

则应利用文献。

对于人细胞系，应相应描述原供体的下

述特征：组织或器官的起源、人种和地

理位置、年龄、性别和一般的生理状况。

如有材料，应将供体致病因子的检查结

果与健康状况或病史一并报告。特别是

人二倍体成纤维细胞，由于供体的年龄

可能影响细胞系的体外寿命，应尽可能

提供。至于动物细胞系的来源，应提供

物种、品系、饲养条件、组织或器官的

起源、地理位置、年龄和性别、致病因

子的检查结果以及原供体的一般生理

状况。

对于微生物细胞，应提供产生细胞系的

物种、株和已知的遗传和表型特征。如

可能，还应提供其致病性、毒素产物和

其他生物危害方面的资料。

应记录细胞的培养历史，包括最初分离

细胞的方法、细胞体外培养的方法以及

建立细胞系的方法(例如，任何物理、化

学或生物学的方法，或附加的核苷序

列)。应提供所有对基因操作和筛选的描

述、细胞内源性和外源性因子的鉴别、

特性和检测结果等资料。

对于来源于后生动物的传代细胞系，通

常通过估计其群体的倍增数，或在规定

的稀释倍数下的传代次数或所需天数，

来决定其培养周期。对于体外有限代次

的二倍体细胞系，应在整个研究、开发

和生产阶段，准估计其群体倍增数。对

于微生物细胞，只需记录细胞基质产生

后的传代次数。

关于细胞基质的产生，应详细研讨制备

方法中接触于传染因子的步骤。应提供

available information regarding the identification,

characteristics, and results of testing of these cells for

endogenous and adventitious agents should be provided.

For continuous cell lines of metazoan origin, it is usually

adequate to quantitate culture duration by estimation of

either number of population doublings, or number of

subcultivations at defined dilution ratio, or time in days.

For diploid cell lines possessing finite in vitro lifespan,

accurate estimation of the number of population

doublings during all stages of research, development,

and manufacturing is important. For microbial cells,

documentation of subcultivation frequency after cell

substrate generation is considered adequate.

Regarding the generation of cell substrates, applicants

should provide a thorough discussion of procedures

which would provide exposure to infectious agents.

Constituents of the culture medium should be described,

in particular, information regarding exposure of the cells

to materials of human or animal origin such as serum,

enzymes, hydrolysates, or other living cells. The

description should include the source, method of

preparation and control, test results, and quality

assurance. Relevant literature on these points may be

referenced when available. This information will allow a

detailed analysis of potential entry routes for

adventitious agents from these sources, and will be part

of the risk-benefit analysis of the product.

2.1.3 Generation of the Cell Substrate

A crucial step is the choice of a suitable parental cell

line. For recombinant products, a parental cell line is

typically the untransfected recipient cell line. The use of

characterized parental cell banks is suggested, but is not

considered essential. A characterised parental cell bank

may be of benefit, especially when multiple cell

substrates are generated from the same parental cell type,

by providing a set of information on which the quality

assessment of the Master Cell Bank (MCB) can be

based . For example, the myeloma cell line may be

banked as a parental cell line for hybridomas.

During the generation of the cell substrate, one or more

specific procedures may be utilised in the ultimate

development of the desired characteristics. These may

include, for example, cell fusion, transfection, selection,

colony isolation, cloning, gene amplification, and

adaptation to specific culture conditions or media.

培养基的组分，特别是提供关于细胞接

触人或动物源的物质如血清、酶、水解

产物或其他活细胞方面的资料。该资料

还应包括来源、制备和质控方法、试验

结果和质量保证。还应提供这方面的相

关文献。这些资料将用于详细分析外源

因子来源的可能途径，并成为制品的利

弊分析的一部分。

2．1．3 细胞系的产生

对于重组产品，选择合适的母细胞系是

关键的一步。母细胞系通常是未转染的

受体细胞系。建议采用经鉴定的母细胞

库，但这不是必需的，特别是当多个细

胞系产生于同一种母细胞系时，采用鉴

定的母细胞库能提供一套对主细胞库

(MCB)进行质量评估的信息，如骨髓瘤

细胞系可以作为母细胞系制库来生产

杂交瘤细胞系。

在细胞系的构建期间，可利用一种或多

种特定的方法使细胞系具有所需的特

性。些方法包括细胞融合、转染、筛选、

克隆分离、克隆、基因扩增和对特定培

养条件或培养基的适应。

用于开发细胞系的方法学资料有助于

了解细胞系的详细历史。有些细胞系如

人二倍体成纤维细胞可能不需在制库

前作额外的处理或克隆。

Information regarding the methodologies utilised in

developing the cell substrate can help to provide a clear

understanding of the history of the cell substrate. Some

cell substrates such as human diploid fibroblasts may not

need extensive manipulation or cloning prior to cell

banking.

For recombinant products, the cell substrate is the

transfected cell containing the desired

sequences, which has been cloned from a single cell

progenitor. For further information on generation of

rDNA-modified cell substrates, consult other relevant

(e.g., regional or

international) guidelines. For non-recombinant products

or non-recombinant vaccines, the cell

substrate is the cell from the parental cell line chosen for

preparation of the MCB without further modification.

For products derived from hybridomas, the cell substrate

is the hybridoma cell line derived by fusion of the

parental myeloma cell line with other parental cells, e.g.,

immune spleen cells.

2.2 Cell Banking

One of the most important advantages of using serially

subcultivated cells to produce

biotechnological/biological products is the ability to have

a characterised common starting source for each

production lot, i.e., the preserved bank of cells.

Manufacturers may prepare their own cell banks, or may

obtain them from external sources. Manufacturers are

responsible for ensuring the quality of each cell bank and

of the testing performed on each bank.

2.2.1 Cell Banking System

The concept of a two-tiered cell bank, in which the MCB

which is used to generate Working Cell Banks (WCBs),

is generally accepted as the most practical approach to

providing a supply of cell substrate for continued

manufacture of the product. Manufacturers should

describe their strategy for providing a continued supply

of cells from their cell bank(s), including the anticipated

utilisation rate of the cell bank(s) for production, the

expected intervals between generation of new cell

bank(s), and the criteria for qualification of cell bank(s).

Generally, the MCB is made first, usually directly from

an initial clone or from a preliminary cell bank derived

from an initial clone. It is not considered necessary to

prepare cell banks from clones for certain types of cells

对于重组产品，细胞系是指克隆自一个

祖细胞且含有所需序列的转染细胞。关

于生产重组DNA 修饰细胞系的更多资

料，可参阅其他有关(如地区或国际性)

指导原则。对于非重组产品或非重组疫

苗，细胞系是指来源于那些选择用作制

备主细胞库且未经进一步修饰的母细

胞系的细胞。对于源于杂交瘤的产品，

细胞基质是指骨髓瘤母细胞系和其他

母细胞系融合后产生的杂交瘤细胞系，

如免疫脾细胞。

2．2 细胞库

用连续传代培养的细胞生产生物技术

产品及生物制品的最大优点是使每批

产品都有一个经检定过的共同起源，即

细胞的储备库。生产商可制备自己的细

胞库，也可从外部获得。

生产商有责任对每个细胞库进行检验，

以确保其质量。

2．2．1 细胞库系统

两级细胞库的概念，即用主细胞库生产

工作细胞库(WCB)，通常被认为在连续

生产产品时供应细胞基质的最实际方

法。生产商应说明从自己的细胞库中连

续供应细胞的方案，包括生产用细胞库

的预期利用率、制备新细胞库所预期的

间隔时间以及细胞库的合格标准。

通常，MCB 直接从最初的克隆或源于

最初克隆的初级细胞库中制得。对某些

类型的细胞，不必从克隆制备细胞库(如

二倍体细胞，它们的体外寿命有限或其

他的技术因素使细胞克隆不切合实际；)

或末被克隆的细胞群体，对某种用途来

讲已足够均质。

WCB 来源于一支或多支MCB。WCB

(e.g., diploid cells, where limited in vitro life span or

other technical factors make cell cloning impractical) or

where the uncloned cell population is already adequately

homogeneous for the intended use.

A WCB is derived from one or more containers of the

MCB. It is the WCB which is typically used to directly

provide cells for the manufacturing process. Additional

WCBs are generated from the MCB as needed. A newly

prepared WCB should be appropriately qualified by

characterisation and testing.

It should be noted that the MCB and WCB may differ

from each other in certain respects, e.g., culture

components and culture conditions. Similarly, the culture

conditions used to prepare the MCB and WCB may

differ from those used for the production process. If

changes in cell culture process do not affect product

quality, it is not considered necessary to recline the cells

or to rebank the MCB or WCB. It is important that a

characterised bank provides a consistent product.

A single-tiered banking system consisting only of the

MCB but no WCBs could be used in principle, for

example, if relatively few containers were needed each

year to produce the desired product.

In some microbial expression systems, a new

transformation is performed for each new cell substrate

container lot, based upon using aliquots of thoroughly

tested host cell banks and plasmid banks for each new

transformation and on testing of each transformed cell

substrate bank. This transformed cell substrate bank is

considered the MCB, and it is used as the source of cell

substrate for production. Host cell banks, plasmid banks,

and MCBs are maintained by appropriate preservation

methods. This alternative system is considered adequate

because the

transformation of bacteria and yeast is generally a very

reproducible and easily performed process, unlike the

events needed for transfection of metazoan cells.

Manufacturers should provide information on the host

cells, rDNA molecules (such as plasmids), method of

transformation and of cell banking, and the results of

characterisation studies.

2.2.2 Cell Banking Procedures

It is important to prevent a contaminated cell substrate

(or bank) from being used in

production and to avoid a loss of product availability or

是直接提供生产用细胞。当需要时，可

从MCB中生产更多支的WCB。新制备

的WCB应通过鉴定和试验证明符合要

求。

必须指出，MCB 和WCB 在诸如培养

基组分和培养条件方面也许有差别，制

备MCB 和WCB 的培养条件也许与产

品生产时不同，如细胞培养条件的改变

不影响产品质量，就不必重新克隆细胞

或重新制备MCB 或WCB 库。重要的

是经过鉴定的细胞库可生产稳定的产

品。

如每年生产的产品仅需有限的几支细

胞时，仅建有MCB 而没有WCB 的单

级细胞库，原则上亦是允许的。

在一些微生物表达系统中，对每批新细

胞基质进行新的转化，这种新的转化要

使用经过彻底检验的宿主细胞库和质

粒库，每次转化的细胞基质库都经过检

验。这个转化的细胞基质库可被看做为

MCB，它被用作生产用细胞基质的来

源。宿主细胞库、质粒库和MCB 均需

用合适的方法予以保存。因为细菌和酵

母的转化通常是重复性好、操作容易，

不像转染后生动物细胞那样复杂，因

此，这种经过改变的表达系统是符合要

求的。生产商应提供有关宿主细胞、重

组DNA 分子(如质粒)、转化及细胞建库

的方法以及鉴定结果等方面的资料。

2．2．2 细胞建库的方法

要避免被污染的细胞基质(或细胞库)用

于生产。这是十分重要的，否则，由于

受污染的细胞库不能使用，必须重新生

产细胞库，这将延误产品上市或开发的

development time resulting from the need to recreate a

cell bank found to be unusable due to contamination. It is

recognised that no cell bank testing regimen is able to

detect all potential contaminants; therefore, use of these

preventive principles during cell banking is important to

provide reasonable assurance of the absence of

contamination and to provide a reliable source of the cell

substrate.

Manufacturers should describe the type of banking

system used, the size of the cell bank(s), the container

(vials, ampoules, or other appropriate vessels) and

closure system used, the methods used for preparation of

the cell bank(s) including the cryoprotectants and media

used, and the conditions employed for cryopreservation

and storage.

Manufacturers should describe the procedures used to

avoid microbial contamination and cross-contamination

by other cell types present in the laboratory, and the

procedures that allow the cell bank containers to be

traced. This should include a description of the

documentation system as well as that of a labelling

system which can withstand the process of preservation,

storage, and recovery from storage without loss of

labelling information on the container.

Manufacturers should describe their cell banking

procedures. Cells are generally prepared for banking by

expanding cultures in a progressively greater number or

larger size of vessel until a pool of cells can be obtained

which is sufficient to generate enough containers for the

bank.

To ensure the uniform composition of the contents of

each container, a single pool of cells for banking should

be prepared by combining the cells from all of the

culture vessels, if more than one vessel is used.

Cells suspended in preservation medium are aliquoted

from the single pool into sterilized containers which are

then sealed and stored under appropriate conditions. For

example, animal cells in media containing a

cryoprotectant are frozen in the sealed containers under

defined and controlled conditions, and then transferred to

storage in the vapor or liquid phase of liquid nitrogen or

at equivalent ultra low temperatures. Other methods of

preservation and storage may be adequate depending on

the organism used, but they should be capable of

maintaining a level of cell viability upon reconstitution

时机。应该认识到没有哪一种细胞库检

测方案能检测出所有可能潜在的污染；

因此，只有在细胞建库期间采取预防措

施，才是确保细胞不被污染及提供可靠

的细胞基质的合理保障。

生产商应说明所用制库系统的类型、细

胞库的大小、所用的容器(瓶子、安额或

其他合适的器皿)和密闭系统，用于制备

细胞库的方法包括所用的防冻剂和培

养基以及细胞保存和贮藏的条件。

生产商应阐明避免微生物污染和避免

被实验室中其他类型细胞交叉污染的

方法，还应介绍可追踪细胞库容器的方

法，包括记录系统，以及能经受保存、

贮藏和从贮藏中复苏时容器的标签不

会丢失。

生产商应说明细胞建库的方法。细胞建

库通常是逐步增加容器数量或扩大容

器培养以获得足够量的细胞，并将其分

装到足够量的容器中的。为了确保每个

容器中的内容物完全一致，如培养细胞

采用几个器皿的，应把所有培养器皿中

的细胞混合成单批。

将悬浮在培养基中的单批细胞，定量分

装到无菌容器中并在适宜条件下密封

和贮藏。例如，含有细胞防冻剂的培养

基中的动物细胞，在规定和控制的条件

下，分装到密封容器中冷冻，然后转移

到液氮的气体或液态中或在相当的超

低温条件下保存。其他的保存和贮藏方

法也是可用的，但要根据细胞阶性质而

定。总之应使细胞保持一定的生存能

力，以便在复苏时适用于生产及产品一

致性的要求。

为确保持续正常生产，生产商应采取措

施，防止如火灾、断电和人为错误等事

故的发生，使细胞库不能用于生产。生

产商应提供预防计划；例如，在多个冷

藏罐中分别保存细胞库，使用备用电

源，使用自动液氮填充系统，将部分

MCB 和WCB 保存在较远的场所，及

MCB的再生等。

在生产中体外细胞的寿命应从解冻一

个或多个容器的MCB 开始算起。对于

二倍体细胞系，其体外寿命应以细胞的

which is both consistent and adequate for production use.

To ensure continuous, uninterrupted production of

pharmaceuticals, manufacturers should carefully

consider the steps that can be taken to provide for

protection from catastrophic events that could render the

cell bank unusable. Examples of these events include

fires, power outages and human error. Manufacturers

should describe their plans for such precautions; for

example, these may include redundancy in the storage of

bank containers in multiple freezers, use of back-up

power, use of automatic liquid nitrogen fill systems for

storage units, storage of a portion of the MCB and WCB

at remote sites, or regeneration of the MCB.

The starting point of reference for estimates of in vitro

cell age during manufacturing should be the thawing of

one or more containers of the MCB. For diploid cell

lines, in vitro lifespan should be estimated in terms of

population doubling levels. The population doubling

level at which senescence occurs should be determined

for diploid cells.

2.3 General Principles of Characterisation and

Testing of Cell Banks

The characterisation and testing of banked cell substrates

is a critical component of the

control of biotechnological and biological products.

Characterisation of the MCB allows the manufacturer to

assess this source with regard to presence of cells from

other lines, adventitious agents, endogenous agents and

molecular contaminants (e.g., toxins or antibiotics from

the host organism). The objective of this testing is to

confirm the identity, purity, and suitability of the cell

substrate for manufacturing use. In some cases,

additional testing such as tumorigenicity or karyology

may be useful. The testing program chosen for a given

cell substrate will vary according to the biological

properties of the cells (for example, growth

requirements), its cultivation history (including use of

human-derived and animal-derived biological reagents)

and available testing procedures. The extent of

characterisation of a cell substrate may influence the type

or level of routine testing needed at later stages of

manufacturing. Manufacturers should perform tests for

identity and purity once for each

MCB, and tests of stability during cell cultivation once

for each product to be registered. In addition, tests of

倍增水平估算，也应测定二倍体细胞发

生衰老时的细胞倍增水平。

2．3 细胞库鉴定和检测的一般原

则

对建库细胞基质的鉴定和检测是生物

技术产品及生物制品质量控制的重要

组成部分。通过对MCB 的鉴定，生产

商可对是否存在来自其他细胞系的细

胞、外来和内在因子以及其他污染物(如

来自于宿主的毒素或抗生素)进行评估。

检测的目的是为了证实用于生产的细

胞基质的特性、纯度和可用性。在有些

情况下，其他方面如致癌性或胞核学的

检测也是有用的。

对某一细胞基质选择何种检测方案要

根据细胞的生物学特性(如生长的需

求)、它的培养历史(如人源和动物源性

的生物试剂的使用)和可应用的试验方

法而定。细胞基质的鉴定程度将影响以

后的生产阶段所需的常规试验的类型

或水平。生产商应对每个MCB 作一次

纯度试验和鉴别实验，对将要注册的每

一产品在细胞培养期间作一次稳定性

试验。此外，还要对每一个WCB 作纯

度和有限的鉴别试验。申报者亦应参考

ICH 指导原则中有关病毒安全性方面

的内容，进行以下所列的有关试验，并

在申请制品上市时将试验内容和试验

结果一并上报。

purity and limited tests of identity should be performed

once on each WCB.

Also, applicants should consult the ICH guideline on

viral safety. Relevant tests among those described below

should be performed and described in the market

application, along with the results of the testing.

For cell lines containing exogenously assembled

expression constructs, the relevant ICH guideline on

rDNA expression constructs should be consulted for

guidance on the characterisation of nucleotide and amino

acid sequences. It may also be useful to examine, by

similar methods, the coding sequences in some

non-recombinant DNA-derived cell lines

where the gene sequences have been characterised and

are well understood. However, it is not considered

necessary to carry out investigations of the sequences

encoding complex natural products, for example,

families of related gene products, microbial vaccine

antigens, or monoclonal antibodies from hybridomas.

Manufacturers are also encouraged to employ

“state-of-the-art” methods and technological

improvements in cell substrate characterisation and

testing as they become available, as long as the

specificity, sensitivity, and precision of the newer

methods are at least equivalent to those of existing

methods.

The manufacturer may choose to characterise the WCB

instead of the MCB, if justified.

2.3.1 Tests of Identity

Appropriate tests should be performed to determine that

the banked cell is what it is represented to be. Either

phenotypic or genotypic characteristics may be used in

identity testing. It is not considered necessary to do all

the possible tests. Tests of identity are generally

performed on the MCB. In addition, limited identity

testing is generally performed on each WCB.

2.3.1.1 Metazoan Cells

For human or animal cells which grow attached to a

substratum, morphological analysis may be a useful tool

in conjunction with other tests. In most cases, isoenzyme

analysis is sufficient to confirm the species of origin for

cell lines derived from human or animal sources; other

tests may be appropriate depending on the history of the

cell line. Other technologies may be substituted to

对于含有外源构建的表达载体的细胞

系，应参照ICH 指导原则中有关心

rDNA 表达载体方面的内容，作为核首

酸和氨基酸序列鉴定方面的指南。也可

用类似的方法对基因序列明确并已鉴

定的某些非重组DNA 来源的细胞系的

编码序列进行分析。但是，没有必要对

那些编码复杂的天然产品的序列进行

分析，如相关的基因家庭的产品、微生

物疫苗抗原或来自于杂交瘤的单克隆

抗体。

如条件具备，应鼓励生产商在细胞基质

鉴定和测试中使用先进方法和技术更

新，但新方法的特异性、灵敏度和精密

度至少应与现有方法相当。

如说明理由，生产商可选择鉴定WCB，

而不鉴定MCB。

2．3．1 鉴别试验

应采用合适的试验证明建库的细胞正

是它本身。鉴别试验可利用细胞的表型

或遗传型的特征，但不必作所有的试

验。通常对MCB 作鉴别试验，而对每

个WCB 仅作有限的鉴别试验。

2．3．1．1 后生动物细胞

对于贴壁生长的人或动物细胞，可采用

形态学分析与其他试验相结合的方法。

在大多数情况下，同工酶分析足以确证

人或动物细胞库的种属采源；依据细胞

系的历史情况，可采用其他合适的试

验。对于种族来源亦可用其他替代技术

予以确认，如用染色体条带分析或种属

特异性抗血清的方法。替代方法可显示

其独特的标志物。例如，通过使用染色

体细胞遗传学检测独特的标记染色体，

confirm species of origin, including, for example,

banding cytogenetics or use of species-specific antisera.

An alternative strategy would be to demonstrate the

presence of unique markers, for example, by using

banding cytogenetics to detect a unique marker

chromosome, or DNA analysis to detect a genomic

polymorphism pattern (for example, restriction fragment

length polymorphism, variable number of tandem

repeats, or genomic dinucleotide repeats). Either

confirmation of species of origin or presence of known

unique cell line markers is considered an adequate test of

identity. Expression of the desired product may represent

a complementary approach to confirmation of identity.

2.3.1.2 Microbial Cells

For most microbial cells, analysis of growth on selective

media is usually adequate to confirm host cell identity at

the species level for the host cell bank and the

transformed cell bank. For E. coli, where a variety of

strains may be used, biological characterisation methods

such as phage typing should be considered as

supplementary tests of identity. For plasmid banks,

identity assessment can be accomplished as described by

the ICH document on analysis of the expression

construct. Expression of the desired product is also

considered adequate to confirm the identity of the

microbial expression system.

2.3.2 Tests of Purity

A critical aspect of cell development and banking is the

assessment that the MCB and WCB are biologically

pure, i.e., are free from adventitious microbial agents and

adventitious cellular contaminants. The impact of

selective agents and antibiotics on the detection of

adventious microbial contaminants should be considered

when planning and performing these tests.

2.3.2.1 Metazoan Cells

Tests for the presence of bioburden (bacteria and fungi)

should be performed on individual containers (1% of the

total number but not less than two containers) of the

MCB and WCB. In all other aspects, the current

methodologies described in either the European

Pharmacopoeia (Ph. Eur.), the Japanese Pharmacopoeia

(JP) or the U.S. Pharmacopoeia (U.S.P.) for testing

microbial limits or microbial sterility may be considered

adequate.

Tests for the presence of mycoplasma should be

或用DNA 分析来检测基因组多态性

(例如，限制性片断长度多态性，串联重

复数目的变化，或基因组双核苷酸重复

数目)。只要能确证其起源物种或某种已

知细胞系独特性标记物的存在，就可认

为是适宜的鉴别试验方法。目标产品的

表达可作为鉴别试验的一种补充手段。

2．3．1．2 微生物细胞

对于大多数微生物细胞，分析细胞在选

择性培养基上生长情况就可用以确证

宿主细胞库的宿主细胞种族特性。当使

用大肠杆菌时，由于有许多品系，可考

虑采用生物学鉴别方法如噬菌体分类，

作为鉴别试验的补充。对于质粒库，可

按ICH 文件中有关表达载体分析的内

容进行特性评估。目标产品的表达也可

用以确证微生物表达系统的特性。

2．3．2 纯度试验

评估MCB 和WCB 的生物学纯度，即

无外源性微生物因子和外源性细胞污

染是细胞研制和建库的关键部分。在设

计和进行这些试验时，应考虑选择性试

剂和抗生素对检测外源微生物污染的

影响。

2．3．2．1 后生动物细胞

应用MCB 和WCB 单个容器(容器总数

的1％，但不少于2 个)检测生物负荷(细

菌和真菌)的存在。在其他方面，目前在

欧洲药典、日本药局方或美国药典所记

载的用于测试微生物限度或无菌试验

的方法亦是适用的。

应检测MCB 和WCB 是否存在文原

体。目前所用的包括琼脂和肉汤培养基

的方法以及指示剂细胞培养法是适用

的。用于支原体测试的方法在欧洲药

典、日本药局方和FDA“用于生产生物

制品的细胞系鉴定的考虑要点”一文

(FDA，CBER。1993)中部有记载。仅用

1支MCB 或W 哪来源的细胞进行测试

performed on the MCB and WCB. Current procedures

considered adequate include both the agar and broth

media procedures as well as the indicator cell culture

procedure. Current methods for mycoplasma testing are

described in Ph. Eur., JP, and “Points to Consider in the

Characterization of Cell Lines Used to Produce

Biologicals” (FDA, CBER, 1993). Testing cells derived

from a single container is generally considered adequate.

For non-mammalian animal cell lines, alternative

controls and/or assay conditions may be appropriate;

manufacturers should consult with the national/regional

regulatory authority for appropriate methodology.

If future efforts to harmonise bioburden and mycoplasma

assays are fruitful, then the scientifically appropriate

harmonised assay should be used.

Virus testing of cell substrates should be designed to

detect a wide spectrum of viruses by using appropriate

screening tests and relevant specific tests, based on the

cultivation history of the cell line, to detect possible

contaminating viruses. Applicants should consult the

ICH guideline on viral safety. For product classes not

covered by the viral safety guideline, the current World

Health Organisation (WHO) documents for use of

animal cells may be consulted.

The purity of cell substrates can be compromised

through contamination by cell lines of the same or

different species of origin. The choice of tests to be

performed depends upon whether opportunities have

existed for cross-contamination by other cell lines. In

some cases, it may be necessary to maintain growing

cultures of different cell lines in the same laboratory.

During procedures in cell banking where open

manipulations are performed, care should be taken to

ensure that simultaneous open manipulations of other

cell lines are avoided to prevent cross-contamination.

Whenever another cell line was present in the cell

banking room at the same time that open cell banking

procedures were being performed (such as cell

expansion, pooling, or aliquoting of the chosen cell line),

the cell banks should be tested for the presence of cells

from (or products derived from) the second cell line. In

general, the methods described in Section 2.3.1.0 to

assess cell identity are also considered adequate tests to

detect cross-contamination by other cell lines. Additional

assurance of lack of crosscontamination can be provided

就可以了。对非哺乳动物细胞系，供选

择的测试方法确/或条件应适当，采用哪

种方法，生产商应咨询当地国家/地区管

理机构。

如果生物负荷和支原体测定方法通过

三方进一步协调取得一致意见，则应采

用科学协商后的测定方法。

细胞基质中可能污染病毒的检测，应根

据细胞系的培养史，通过筛选和相关的

特异性试验，选用可检测广谱病毒的方

法。申请者应就病毒安全性问题参考

ICH 指南，对于那些尚未在该指南中涉

及的产品类别，可参考WHO 们关于使

用动物细胞的有关资料。

细胞基质的纯度会由于污染相同或不

同品系的细胞而降低。选择什么样的纯

度试验方法取决于该细胞是否存在被

其他细胞系交叉污染的可能性。有时，

在同一实验室会同时培养几种不同的

细胞系，在细胞建库过程中，当进行开

放性操作时，应注意避免与其他细胞系

同时开放操作导致交叉污染。在建库的

开放性操作过程中(如细脑扩增、细胞混

合或细胞分装等)，只要在此操作间有另

一种细胞系存在，就应检测该细胞库细

胞中是否污染该细胞系的细胞。

在2．3．1 节中介绍的细胞特性的检测

方法一般亦适用于检测是否与其他细

胞系存在交叉污染。另外，从细胞基质

中成功地制得所预期的产品是说明无

交叉污．染的另一种旁证。

by successful preparation of the intended product from

the cell substrate.

2.3.2.2 Microbial Cells

The design and performance of specific tests for

adventitious microbial agents and adventitious cellular

contaminants in microbial cell banks should take into

account the properties of the banked cell, the likely

contaminants based upon scientific literature, source,

methods and materials used for cultivation, and other

organisms present in the banking laboratory. For

example, visual examination of the characteristics of

well-isolated colonies is suggested, using several

microbiological media, of which some do and some do

not support growth of the cell substrate. However, it is

not intended that manufacturers necessarily characterise

resistant mutants of the cell substrate arising from such

studies, or other artifacts of such assays. Rather, the

purpose of such assays is to detect existing contaminants.

2.3.3 Cell Substrate Stability

Another dimension to cell characterisation is

appropriateness for intended use in production.

There are two concerns for cell substrate stability:

Consistent production of the intended product and

retention of production capacity during storage under

defined conditions.

For the evaluation of stability during cultivation for

production, at least two time points should be examined,

one using cells which have received a minimal number

of subcultivations, and another using cells at or beyond

the limit of in vitro cell age for production use described

in the marketing application. The limit of in vitro cell

age for production use should be based on data derived

from production cells expanded under pilot plant scale or

commercial scale conditions to the proposed limit of in

vitro cell age for production use or beyond. Generally,

the production cells are obtained by expansion of cells

from the WCB; cells from the MCB could be used with

appropriate justification. This demonstration of cell

substrate stability is commonly performed once for each

product marketing application.

Evaluation of the cell substrate with respect to the

consistent production of the intended product of interest

should be the primary subject of concern. The type of

testing and test article(s) used for such assessments will

depend on the nature of the cell substrate, the cultivation

2．3．2．2 微生物细胞

在设计检测微生物细胞库中外源性微

生物因子和外源性细胞污染物的特异

性试验方法时，应考虑以下问题：建库

细胞的性质、科技文献中报道的可能存

在的污染物、用于细胞培养的细胞来

源、方法和材料以及存在于建库实验室

中的其他生物等等。例如，可考虑采用

几种微生物培养基对该细胞进行培养，

其中有些培养基可使细胞生长，有些则

不能，培养后采用目视法对分离的菌落

进行鉴定。由于试验的目的是检测是否

存在污染物，因此不要求生产商鉴定研

究过程中出现的抗性突变株，或弄清楚

方法中出现的某些假象。

2．3．3 细胞基质的稳定性

细胞鉴定的另一个角度是细胞在生产

中的适用性。对细胞基质稳定性主要应

从两个方面进行考虑，即生产产品的一

致性和贮存在规定条件下细胞能否维

持其生产能力。

评估培养期间的稳定性，至少应考核两

个时间点，一是能使用的最少传代数的

细胞，另一个是上市申请生产时使用达

到细胞体外传代限度或超过该限度的

细胞。用于生产的细胞的体外传代限

度，应根据从试生产规模或生产规模的

细胞扩增代数到建议的生产代次限度

或以上所得到的资料来定。生产用细胞

一般是通过WCB 细胞扩增获得；有适

当理由时亦可直接用MCB 细胞扩增。

细胞基质稳定性的验证通常在每种产

品上市申请时进行一次。

生产预期产品的一致性是对细胞基质

进行评估的主要项目。用于此类评估的

试验类型和试验项目由细胞基质的性

质、培养方法和所生产的产品而定。对

于含有重组DNA 表达构建体的细胞

methods, and the product. For cell lines containing

recombinant DNA expression constructs, consistency of

the coding sequence of the expression construct should

be verified in cells cultivated to the limit of in vitro cell

age for production use or beyond by either nucleic acid

testing or product analysis, as described in the relevant

ICH guideline. For nonrecombinant cell lines in which

the coding sequence for the desired product has already

been analysed at the MCB or WCB level, invariability of

the protein coding sequence during production should be

verified in the production cells cultivated to the proposed

limit of in vitro cell age for production use or beyond by

either nucleic acid testing or analysis of the purified

protein product.

Where the product cannot be analysed as described

above, other specific traits which may include, for

example, morphological characteristics, growth

characteristics, biochemical markers, immunological

markers, productivity of the desired product, or other

relevant genotypic or phenotypic markers may be useful

for the assessment of cell substrate stability.

In some cases, where direct comparison of the

characteristics of the MCB with those of the production

cells at or beyond the limit of in vitro cell age is difficult

or impossible, one may compare the characteristics of

cells at the initial stages of cultivation or production to

those of cells at or beyond the limit of in vitro cell age

for production use in order to assess cell stability during

production. Indices such as, for example, oxygen or

glucose consumption rates, ammonia or lactate

production rates may be useful for such testing. Increases

in the defined limit of in vitro cell age for production use

should be supported by data from cells which have been

expanded to the proposed new limit of in vitro cell age.

For diploid cell lines, data should be presented that

establish the finite in vitro lifespan of the cells from the

WCB under conditions representative of those employed

for manufacturing use.

Evidence for banked cell stability under defined storage

conditions will usually be generated during production of

clinical trial material from the banked cells. Data from

the determination of cell viability when the preserved

cells are reconstituted for production of clinical trial

supplies will verify that the revived cells have survived

the preservation process. Data from the preparation of

系，在其培养达到或超过体外代次限度

时，应按ICH 有关指南中的方法，通过

核酸检测或产品分析，对表达构建上编

码序列的一致性进行验证。对于非重组

细胞系，其预期产品的编码序列已在建

立MCB 或WCB 时分析过，在生产中

细胞培养达到或超过体外代次限度时，

应通过核酸检测或纯化蛋白质制品的

分析验证其蛋白质编码序列未有改变。

如果产品不能按上述方法进行分析，则

其他特征诸如形态学特性、生长特性、

生化标志、免疫标志、对预期产品的生

产能力或其他相关的表型或遗传型标

志等都可用于评估细胞基质的稳定性。

在某些情况下，将MCB细胞的特性直接

与达到或超过细胞体外代次限度的生

产用细胞比较有一定困难或不可能时，

评估生产过程中细胞的稳定性，可将培

养或生产的最初阶段的细胞与达到或

超过细胞体外代次限度的生产用细胞

进行特性比较。某些指数，如氧或葡萄

糖的消耗率、氨或乳酸的产生率可用于

这类试验。如要延长用于生产的细胞的

体外代次限度，应有体外代次已达到所

建议的新限度的生产资料支持。对于二

倍体细胞，应提供拟用于生产条件下建

立WCB 细胞的体外有限代次的资料。

在规定贮藏条件下建库细胞的稳定性

一般需通过生产临床试验用制品来验

证。当储存的细胞复苏生产供临床试验

样品时，对细胞存活力的测定数据可证

明这些复苏细胞在储存过程中仍然保

存活力。通过对临床试验用制品的生产

可证实复苏细胞能用于生产所需的产

clinical materials will demonstrate that the revived cells

can be used to prepare the desired product. Available

data should be clearly documented in the application

dossiers, plus a proposal for monitoring of banked cell

stability should be provided. The proposed monitoring

can be performed at the time that one or more containers

of the cryopreserved bank is thawed for production use,

when the product or production consistency is monitored

in a relevant way, or when one or more containers of the

cryopreserved MCB is thawed for preparation of a new

WCB (and the new WCB is properly qualified), as

appropriate. In the case when production does not take

place for a long period of time, viability testing on the

cell bank used as a source of the production substrate

should be performed at an interval described in the

marketing application. If the viability of the cell

substrate is not significantly decreased, generally no

further testing of the MCB or WCB is considered

necessary.

2.3.4 Tests for Karyology and Tumorigenicity

Utilisation of karyology and tumorigenicity testing for

evaluating the safety of a diploid cell line or

characterising a new cell line may be useful depending

on the cells, the nature of the product and the

manufacturing process. Extensive analysis to determine

the relative abundance of aneuploid cells has not been

found to be useful. Karyology need not be determined

for rodent cell lines or new cell lines known to be

non-diploid. However, cytogenetic analysis may be an

adequate method to assess cell substrate identity or

purity as described in Sections 2.3.1 and 2.3.2.

Repetition of tumorigenicity testing for cells with

already documented evidence of tumorigenicity is not

considered necessary.

For products that are highly purified and that contain no

cells, karyology and tumorigenicity testing are generally

not considered necessary, provided that appropriate

limits for residual host cell DNA are shown to be

consistently met by either process validation studies or

by lot release testing.

In general, products for which the presence of live cells

cannot be excluded or which have little downstream

purification (for example, some conventional live virus

vaccines) will need such characterisation of the cell

substrate. The utility of tumorigenicity testing and

品。以上这些资料需收载到申报卷宗中

去，并附上对建库细胞稳定性监测的方

案。这种监测应在一支或多支冷冻保藏

的细胞库细胞被解冻以制备生产用细

胞时进行，因为这时产品或生产过程的

监测方式是相近的。或当一支或多支冷

冻保藏的MCB 被解冻并用于制备新

WCB 时进行。

当在长时间末进行生产的情况下，应按

上市申请时所述的间隔时间对生产用

细胞库进行活力测试。如果细胞基质的

活力没有明显的减退，一般不需对MCB

或WCB 作进一步测试。

2．3．4 胞核学和致瘤性试验

根据细胞类型、产品的性质以及生产工

艺，决定是否必要用胞核学和致瘤性试

验评价二倍体细胞系的安全性或鉴定

一个新的细胞系。采用广泛的分析方法

测定非整倍染色体细胞的相对丰度未

必有用。啮齿类动物细胞系或已知为非

二倍体的新细胞系不需要作胞核学测

定，因细胞遗传学分析足以评估细胞基

质的特性或纯度。对文献已记载具有致

瘤性的细胞不需重复进行致瘤性试验。

chromosomal analysis for new cell substrates for

unpurified products should be evaluated on a

case-by-case basis. Use of cell lines known to be

tumorigenic or to possess abnormal karyology should be

evaluated in terms of risk-benefit for each product

application when the product contains cells or when not

highly purified.

Products that are manufactured in genetically unmodified

MRC-5 or WI-38 cells do not need characterisation of

these cell substrates by karyology or tumorigenicity

since extensive characterisation has already been

performed and published for these cell lines. However,

for each MRC-5 and WI-38 WCB generated,

manufacturers should confirm, once, that the cells grown

in the manner to be used in production are diploid and

have the expected lifespan.

For new or previously uncharacterised diploid cell

substrates, confirmation of diploid karyology should be

presented and tumorigenic potential should be

established, using cells from the MCB.

APPENDIX 1

PRIMARY CELL SUBSTRATES

I. Introduction

The principles contained in this document apply in

general to biotechnological/biological products prepared

from characterised banked cells. However, a number of

biological products, in particular certain viral vaccines,

are prepared using primary cells.

Because primary cell cultures are used within the first

passage after establishment from the tissue of origin, it is

not possible to carry out extensive characterisation of the

cells prior to their use as is done for banked cell

substrates. In addition, biological products produced

using primary cell substrates often do not undergo

extensive processing (e.g., purification). Despite these

differences, the approach taken to assure the suitability

and safety of primary cell substrates for production of

biologicals is analogous, in many respects, to that

outlined in this document and in other guidelines.

This Annex outlines cell substrate-related information

that should be included in marketing applications for

biological products prepared using primary cells. This

information falls into three general categories: (1)

Information concerning the source tissue (or organ) and

对于高度纯化和不含细胞的制品，只要

通过生产工艺验证或通过批签发试验，

表明残留的宿主细胞DNA 符合限度要

求，通常不必进行胞核学和致瘤性试

验。

一般而言，对不能去除活细胞或几乎无

下游纯化工艺(如一些传统的活病毒疫

苗)的制品需要做细胞基质的胞核学和

致瘤性试验。用于非纯化制品的新细胞

基质，是否需进行致瘤做试验和染色体

分析，应针对具体案例逐一评估。用已

知是致癌的或具有异常细胞核的细胞

系生产含有细胞或未高度纯化的制品

时，应在每个产品申报时作利弊关系的

评估。

用遗传上未修饰的MRC-5 或WI-38 细

胞生产时，不必对这些细胞进行胞核学

或致瘤性的鉴定，因为对这些细胞系已

作了大量的鉴定工作，并有资料发表。

但是，对于产生的每个MRC-5 或WI-38

工作细胞库，生产商应再证实生产所用

的细胞其生长方式是二倍体，并有所期

望的代次。

对于新建的或先前未经鉴定的二倍体

细胞，应用MCB 的细胞证实其具有二

倍体细胞核，并确定其潜在致瘤性。

胞核学和致瘤性分析的方法可参见

WHO 文献“WHO：动物细胞做为体外

细胞基质用于生物制品生产的有关要

求”，见日内瓦世界卫生组织“WHO 生

物制品标难化专家委员会第47 次报

告”(WHO 技术报告丛书)。

附件：原代细胞基质

1．前言

收载于该文件中的原则通常适用于采

用鉴定过的建库细胞制备的生物技术

产品及生物制品。然而，许多生物制品，

尤其是一些病毒疫苗是用原代细胞制

备的。

原代细胞从原始组织确立后，第一代就

other animal-derived raw materials used for the

establishment of primary cell substrates, (2) information

concerning the preparation of primary cell substrates,

and (3) testing performed on primary cell substrates to

ensure the safety of the product.

II. Source Tissue and Other Raw Materials

Information should be provided about the animals used

as a source of tissue for the preparation of primary cell

substrates. Tissue should be derived from healthy

animals subjected to veterinary and laboratory

monitoring to certify the absence of pathogenic agents.

Whenever possible, donor animals should be obtained

from closed, specific pathogen-free (when available)

colonies or flocks. Animals used as tissue donors should

not have been used previously for experimental studies.

Animals should be adequately quarantined for an

appropriate period of time prior to use for the preparation

of cells. In some countries, animals may need to be

quarantined in the country where the primary cells are

prepared.

Manufacturers should consult with national/regional

authorities for specific requirements.

Information on materials and components used for the

preparation of primary cell substrates should be

provided, including the identity and source of all

reagents of human or animal origin. A description of

testing performed on components of animal origin to

certify the absence of detectable contaminants and

adventitious agents should be included.

III. Preparation of Primary Cell Substrates

Methods used for isolation of cells from tissue,

establishment of primary cell cultures and maintenance

of cultures should be described.

IV. Testing of Primary Cell Substrates

Tests performed on primary cell substrates to qualify

them for use in production should be described. As

noted, the nature of primary cell substrates precludes

extensive testing and characterisation prior to use.

Testing to demonstrate the absence of adventitious

agents in these substrates is therefore conducted

concurrently and may include: Observation of production

or uninfected control cultures before, during, and beyond

the period of production; inoculation of culture fluids

from production and uninfected control cultures into

various susceptible indicator cell cultures capable of

开始使用，不必像鉴定建库细胞那样在

生产之前对其进行全面鉴定。此外，用

原代细胞生产的生物制品通常不进行

深加工处理(如纯化)。尽管存在着这些

不同，但对用于生物制品生产的原代细

胞基质进行适用性和安全性的检测方

法在很多方面与本文件和其他指南中

所介绍方法类同。

本附件列出了用原代细胞生产的生物

制品在申请上市时所需提供的与细胞

基质有关的资料。这些资料分为三大

类：(1)有关原代细胞的组织来源及用于

制备原代细胞基质的动物源性原材料

的资料；(2)有关原代细胞制备的资料；

(3)为证制品的安全性对原代细胞进行

检测的资料。

2．原代细胞的组织来源和其

他原材料

应提供制备原代细胞所用组织来源的

动物资料。这些组织通常取自健康、并

经兽医和实验室检测证明无致病因子

的动物。如有可能，供体动物应从隔离

的无特殊致病原的群体中获得。已用于

实验研究的动物不能作为供体。在用于

制备细胞之前的适当时期，对动物进行

检疫。在有些国家，动物应在制备原代

细胞的国家检疫。对一些特殊要求，生

产商应咨询当地有关国家地区的管理

机构。

应提供用于制备原代细胞基质的物料

及成分的资料，包括人和动物源性的所

有试剂的特性和来源以及对动物源性

的成分证明检测不出污染物和外源因

子的检测方法。

3．原代细胞基质的制备

detecting a wide range of relevant viruses, followed by

examination for cytopathic changes and testing for the

presence of hemadsorbing viruses; and other tests for

specific agents (such as relevant retroviruses) as

necessary.

Additional information concerning specific viral tests

may be found in the relevant

national/regional/international guidelines.

Appropriate testing regimens and test methods for cells

used in the production of specific products will vary

depending on the donor species used as a source of

tissue, adventitious agents potentially present, the nature

of the product, its intended clinical use, aspects of the

manufacturing process, and the extent of testing

performed on the final product. Applicants should

explain and justify the approach taken with respect to

their specific product.

3. GLOSSARY

Cell bank: A cell bank is a collection of appropriate

containers, whose contents are of uniform composition,

stored under defined conditions. Each container

represents an aliquot of a single pool of cells.

Cell line: Type of cell population which originates by

serial subculture of a primary cell population, which can

be banked.

Continuous cell line: A cell line having an infinite

capacity for growth. Often referred to as “immortal” and

previously referred to as “established”.

Diploid cell line: A cell line having a finite in vitro

lifespan in which the chromosomes are paired (euploid)

and are structurally identical to those of the species from

which they were derived.

Host cells: See Parental cells.

In vitro cell age: Measure of time between thaw of the

MCB vial(s) to harvest of the

production vessel measured by elapsed chronological

time, by population doubling level of the cells, or by

passage level of the cells when subcultivated by a

defined procedure for dilution of the culture.

Metazoan: Organism of multicellular animal nature

应提供从组织分离细胞、建立原代细胞

并维持其培养的方法。

4．原代细胞基质的检测

应提供对原代细胞基质检测的资料以

证明其用于生产是合格的。前已提及，

原代细胞在应用之前不必对其性质作

全面的检测和鉴定。细胞基质的外源因

子检测可与生产过程同时进行，包括在

生产前、中、后期对生产的培养物或非

感染的对照细胞培养物进行观察；在生

产的培养液或非感染的对照培养液中

分别加入各种敏感的指示细胞培养液，

该培养液能检测一

定范围的相关病毒，然后检查其细胞病

变和检测其血吸附病毒；必要时，还有

用其他的方法检查特殊因子（如相关的

反转录病毒）。其他有关特异性病毒检

测的资料，可从有关国家/地／国际指南

中获得。

要根据制备细胞的供体品系、可能存在

的外源因子、产品的性质、临床用途、

生产工艺以及对终产品检测的程度等

情况，对于生产特殊制品的细胞制定相

应的检测方案及方法。申请者应对特殊

制品所采用的检测方法进行解释并阐

明其理由。

3．术语

细胞库 是内容物组成一致、贮存在规

定条件下的等量分装的容器集合。每个

容器代表一个单批细胞的一等份。

细胞系 能建库的原代细胞群进行一系

列传代得到的细胞群体类型。

传代细胞系 有无限生长能力的细胞

系。常被称作“永生化”细胞系，以前

MCB (Master Cell Bank): An aliquot of a single pool

of cells which generally has been prepared from the

selected cell clone under defined conditions, dispensed

into multiple containers and stored under defined

conditions. The MCB is used to derive all working cell

banks. The testing performed on a new MCB (from a

previous initial cell clone, MCB or WCB) should be the

same as for the MCB unless justified.

Parental cells: Cell to be manipulated to give rise to a

cell substrate or an intermediate cell line. For microbial

expression systems, it is typical to also describe the

parental cells as the host cell. For hybridomas, it is

typical to also describe the parental cells as the cells to

be fused.

WCB (Working Cell Bank): The Working Cell Bank is

prepared from aliquots of a homogeneous suspension of

cells obtained from culturing the MCB under defined

culture conditions.

被称为建株细胞。

二倍体细胞系 染色体成双(整倍体)、体

外寿命有限的细胞系，其结构与它们所

起源的细胞是一致的。

宿主细胞 见母细胞。

体外细胞传代期 指从MCB 小瓶细胞

融化至生产容器收获的这段时间，它可

用培养所耗的时间来量度，或用细胞数

的倍增水平来量度，当细胞以规定的步

骤稀释培养物进行传代培养时，也可用

细胞的传代次数来表示。

后生动物 具多细胞动物特征的生物

体。

MCB(主细胞库) 在指定条件下从选定

的细胞克隆制备而得的单批细胞，它被

分装到多个容器中，并在规定的条件下

贮藏。 MCB 通常用于制备所有的工作

细胞库。对一个新的MCB(来自先前最

初细胞克隆、MCB 或WCB)进行的试验

应与原有MCB 相同。

母细胞 通过制备过程可用于产生细胞

基质或中间细胞系的细胞。对于微生物

表达系统，通常把母细胞称作宿主细

胞。对于杂交瘤，通常把母细胞称作融

合细胞。

WCB(工作细胞库) 在规定的培养条件

下，从培养MCB 中的细胞获得相同来

源的匀质细胞悬液等量分装的容器集

合。