Castilho Leda R., Moraes Angela Maria (Ed.) Animal Cell Technology: From Biopharmaceuticals to Gene Therapy

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The ability to obtain cell clones that respond to cytokines and serve as

source material for bioassays has aided the development of bioassays. The

cell lines, obtained mainly from human or murine cancers, often depend

on a cytokine for their growth and tend to be immortal, thus providing a

unique homogeneous cell source, which may be distributed among labora-

tories (Mire-Sluis and Thorpe, 1998). The advent of recombinant DNA

technology has enabled the cloning of speciﬁc cytokine receptors and their

expression in any cell line (Canosi et al., 1996). In this way, a cell line can

be created that responds speciﬁcally to almost any cytokine, and avoids

the necessity to isolate a cell line with an endogenous receptor.

13.5.3 Experimental design

An in vitro bioassay can be designed in several ways, but requires

statistical validity. A one point assay is not valid. The bioassay should be

designed to consider factors that introduce variability, and the analysis

should test such variability. A measurement series of a test sample should

be compared to an equivalent series of the reference material, carefully

considering the comparisons between the linear portions of the dose-

response curves (Mire-Sluis et al., 1996). To test validity of a bioassay

inter- and intra-assay variability should be considered in both preparation,

and in the case of multiwell plates, the variability between each plate. To

reduce the positional effect in plate tests, it is advisable to distribute the

points on the curves randomly and also to include a reference standard in

each plate (Gaines-Das and Meager, 1995). One of the most widely used

techniques to validate a bioassay’s performance is to include internal

duplicates. The data arising from the comparison can be important in

assessing the test’s variability.

Products manufactured by different companies, even with the same

source materials, can present very different speciﬁc activities. Conse-

quently, the concept of a universal standard of potency has been developed

by the World Health Organization (WHO) and this has proved extremely

valuable to reduce the variability of results between laboratories, as well as

to improve the comparability of clinical studies and research (Mire-Sluis

et al., 1996).

The international units and standards are vital when establishing the

biological potency of a preparation. The mass cannot be used, since it is

practically impossible to weigh a drug, especially if it contains excipients,

as occurs with most biotherapeutic products. To establish international

standards, a strict process is followed according to the regulations of

WHO. The importance of this is reﬂected in the establishment of new

standards, the Reference Reactives (RRs) of the WHO, for cytokines and

growth factors. These are prepared according to the WHO protocol. Each

standard is monitored so that it does not lose activity during lyophiliza-

tion, and its activity remains stable. However, the potency of a therapeutic

product measured by an in vitro test is not used to establish clinical doses,

because this may not reﬂect in vivo activity, which can only be established

by clinical trials.

344 Animal Cell Technology

13.5.4 Statistical analysis

The most widely used type of simultaneous assay is the one in which the

response has a homoscedastic linear regression on a logarithmic scale.

Homoscedasticity means that the variance of all experimental groups is the

same. For such an assay, the condition of similarity requires that the

straight lines of the standard and the samples should be parallel. Other-

wise, the condition of similarity between the sample and standard is not

established, that is, it would not be valid to assume that dilutions of one

behave the same as dilutions of the other, which is the assay’s underlying

principle.

This type of test is called parallel-line assay and is based on the compari-

son of a sample response with that of a reference standard (Finney, 1978).

In general, it determines the response – at least by duplicates – of a series

of dilutions of each preparation (sample and standard) while plotting the

means of their corresponding doses on a logarithmic scale. As this test

requires analysis of a linear portion of the curves, at least three points of

each curve belonging to such a portion should be selected. The more

selected points, the better the comparison.

If successive doses for each preparation show the same logarithmic

spacing this enables a mathematical transformation that facilitates the

calculation without modifying the ﬁnal result. With these new values

assigned to the doses on the abscissa axis and the mean response values on

the ordinate axis, the straight lines of dose-response are replotted, and

must be parallel to allow comparison. This graph allows a quick potency

estimate, and also protects against serious mistakes or misinterpretations

of statistical analysis. The horizontal distance between both straight lines

provides an estimate of the difference in the transformed abscissa values,

between doses with the same response. Finally, by expressing this differ-

ence in terms of doses, the sample’s potency value is obtained. For this

estimate to be valid, the variance analysis – more commonly known as

ANOVA – must be performed. The existence of homoscedasticity is

determined by Bartlett’s test (Finney, 1978).

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Quality control of biotechnological products 347

Regulatory aspects

Maria Teresa Alves Rodrigu es and Ana Maria Moro

14.1 Introduction

The scientiﬁc and technological achievements of the last decades have

allowed the discovery and production of new biological medicines, or

substitution of those previously extracted from animal sources, like insulin

and growth hormone. Most of those medicines are proteins obtained by

animal cell cultivation, which differs from bacteria and yeast in that animal

cells carry out post-translational modiﬁcations needed for biological

activity and similar to the natural protein. Biopharmaceutical production

in mammalian cells, including hormones, monoclonal antibodies (mAbs),

vaccines, and other molecules with medical interest, involves high cost

processes due to factors such as:

• low cell yield

• complex culture media

• shear sensitivity

• low resistance to toxic metabolites

• complex puriﬁcation

• rigorous control of all phases

• sophisticated methodologies of process control and ﬁnal product

control.

Concepts like quality, safety, and efﬁcacy should be taken into con-

sideration during process development, since it is not enough to inspect

and test only the ﬁnal product. Good Manufacturing Practice (GMP), as

well as quality assurance systems are essential to guarantee safety, quality,

and economic feasibility during the whole process and therefore to obtain

a marketing license.

Biopharmaceuticals, like synthetic pharmaceuticals, can cause adverse

effects. Protein molecules can elicit immunological reactions in patients,

which can be enhanced by small modiﬁcations of the molecule. Thus, it is

very important to proceed with in-process controls, ﬁnal product testing,

and process validation. By the use of reﬁned analytical methodologies it is

possible to change dogmas such as ‘‘the process deﬁnes the product,’’

which is imperative for old biological products composed of complex and

less puriﬁed mixtures. The more recent biopharmaceuticals might be con-

sidered as ‘‘well characterized.’’ During the last few years considerable

discussions have occurred between regulatory agencies and manufacturers

concerning the requirements needed for product safety, so regulations are

constantly evolving due to advances in research. In this chapter we present

the latest positions concerning animal cell-derived biological products

adopted by the regulatory agencies of the major pharmaceutical markets in

the world – the FDA (Food and Drug Administration) in the United

States and EMEA (European Medicines Evaluation Agency) in the Eur-

opean Union – as well as by the WHO (World Health Organization) and

ICH (International Conference on Harmonization of Technical Require-

ments for Registration of Pharmaceuticals for Human Use).

14.2 Good Manufacturing Practices and quality assurance

Quality, efﬁcacy, and safety of a medicine are attributes to be planned and

established throughout the production process. This concept is especially

valid for the production of biologics to assure the fundamental character-

istics of efﬁcacy and safety to the patient in each vial of every lot. Every

step of pharmaceutical manufacturing should follow high standards in-

tended for production consistency and product quality. The principles

deﬁning these standards are described in the ‘‘Good Manufacturing

Practices’’ (GMP) publications.

GMP principles were originated in the US, by the FDA, in 1978. The

principles are generally accepted throughout the world. Regulatory autho-

rities verify the adherence and compliance to these principles, regularly

inspecting the production plants, and determine whether a manufacturing

license is maintained or revoked. GMP principles are generic and applic-

able to all pharmaceutical processes, but may be complemented by speciﬁc

conditions depending on products or systems. As an example, the Eur-

opean Union publication (EC, 1998a) includes the following chapters:

‘‘Quality management’’, ‘‘Personnel,’’ ‘‘Premises and equipment,’’ ‘‘Doc-

umentation,’’ ‘‘Production,’’ ‘‘Quality control,’’ ‘‘Manufacture and analy-

sis contract,’’ ‘‘Complaints and product recall,’’ and ‘‘Self-inspection.’’ In

addition to the main chapters, there is a series of annexes: ‘‘Production of

sterile products,’’ ‘‘Production of biological products for human use,’’

‘‘Production of medicines in development intended for clinical trials,’’

‘‘Production of human blood and plasma derived products,’’ ‘‘Computer-

ized systems,’’ ‘‘Parametric release,’’ among others. Similarly, the FDA

publishes the ‘‘Points to Consider’’ (PTC) and ‘‘Letters to Industry’’, and

WHO publishes speciﬁc annexes.

The GMP principles are part of a wider concept that encompasses many

aspects that can inﬂuence product quality; this concept constitutes the

‘‘quality assurance’’ system (QA). QA is the sum of systematic actions,

which are intended to guarantee quality standards expected for a given

product. The QA system concerns all steps that can inﬂuence biopharma-

ceutical quality, such as: research and development; project evaluation

under GMP rules; designation of responsibilities with deﬁnition of posi-

tions and functions; selection of suppliers; raw material storage; process

development; validation; pilot scale production; clear description of pro-

duction and control operations following GMP requirements; testing and

control of raw materials, intermediate product, bulk product, and ﬁnished

product; records; labeling; distribution; and complaints. Self-inspection is

also part of the program aiming to evaluate the effectiveness and application

of QA system. The QA system should include formal training of produc-

350 Animal Cell Technology

tion and QA personnel, making the whole staff conscious of these practices

and their importance for quality attainment. The concepts of QA, GMP,

and quality control (QC) are inter-related aspects of quality management.

QC is a segment of the QA system and encompasses sampling, inspec-

tion, test and release of raw materials, packaging materials, and intermedi-

ate, bulk, and ﬁnished products. The ﬁnal product release depends on

aspects like critical examination of production conditions, results of in-

process assays, manufacturing documentation, adherence to the speciﬁca-

tions, and examination of ﬁnal packaging. The QC department should be

independent of other departments and be granted access to the production

areas whenever considered necessary for sampling or investigation, and

should be involved in all decisions related to product quality (EC, 1998b;

Christiansen, 1999; ANVISA, 2003).

The manufacture of biological products, unlike that of pharmaceuticals,

uses materials that present variability. The active substance is generally

produced in small quantities and needs to be separated from complex

mixtures containing several types of contaminants. Lots are generally small

and quality control tests are usually based on biological techniques that

present higher variability than physicochemical assays. The processes are

also susceptible to microbial contamination. The in-process controls are of

fundamental importance to detect quality deviations that cannot be

assessed through assays performed only on the ﬁnal products.

The development of biological products obtained through animal cell

culture, such as recombinant proteins and mAbs, has resulted in well-

characterized, high purity therapeutic products. The puriﬁcation pro-

cesses generally include various chromatography steps, and the products

are analyzed by sophisticated analytical techniques and validated by

biological activity assays. In 1995, the concept of a ‘‘well-characterized

product’’ (WCP) was introduced by the FDA, being deﬁned as an entity

whose purity, potency, and quantity can be determined and controlled.

For a biological compound to be considered a WCP, certain tests and

data are not needed, making it simpler to introduce changes to the

manufacturing process. To be granted a WCP proﬁle, a product must

meet some criteria deﬁned by the industry and the FDA. The manufac-

turing process should be robust, reproducible, and validated. Validation

is used to demonstrate product safety when produced with consistent

standards (for example, demonstration of the removal of impurities and

adventitious agents through validated analytical tests). Also, the product

should be safe from a toxicological perspective and should pass character-

ization tests to conﬁrm identity, purity, and potency (Foulkes and

Traynor, 1999; Schiff, 2004). For recombinant DNA-derived proteins,

the identity can be assessed by primary and secondary structure determi-

nation, including amino acid sequence analysis, presence of disulﬁde

bonds, and post-translational modiﬁcations such as glycosylation. mAbs

can be identiﬁed by physicochemical and immunochemical assays. Purity

and impurities should be quantiﬁable and the impurities should be

identiﬁed whenever possible; the quantity and biological activity should

be measurable. When a product satisﬁes these criteria, it is considered

‘‘well characterized’’ and can be regulated with less interference from the

FDA (Henry, 1996).

Regulatory aspects 351

14.3 Regulatory agencies

The US regulatory authority for medicines is the FDA. Inside its organiza-

tional structure, biological products such as vaccines, blood-derived pro-

ducts, antisera, toxins, and antitoxins intended for therapeutic use are

under control of the Center for Biologics Evaluation and Research

(CBER). mAbs for in vivo utilization, immune modulators, and therapeu-

tic proteins like cytokines, growth factors, and enzymes are generally

under the surveillance of the Center for Drug Evaluation and Research

(CDER). The FDA regulates clinical trials, evaluates investigational new

drug (IND) applications, analyzes clinical data, checks commercial li-

censes, inspects and approves industrial installations, and veriﬁes GMP

adherence.

In the European Union, the regulatory authority is the European

Commission (EC), composed of representatives of each participating

country. The role of the EC is to propose legislation for the European

Union. For pharmaceuticals it has a ten-volume series, named The Rules

Governing Medicinal Products in the European Union (EC, 2007). These

volumes comprise all aspects of pharmaceutical activity. In 1995 the

creation of the EMEA (European Medicines Evaluation Agency, later

named European Medicines Agency), based in London, simpliﬁed the

requirements needed for new drug registration. EMEA, like the FDA,

receives applications for the marketing of pharmaceutical products.

Although they have similar functions, the FDA has the authority to grant

a license, while EMEA does not. Through the CPMP (Committee for

Proprietary Medicinal Products), the EMEA advises on product licensing,

but only the EC has authority to grant a marketing license that covers the

European Union, which constitutes the largest pharmaceutical market in

the world, followed by the USA and Japan. The PAB (Pharmaceutical

Affairs Bureau) regulates the pharmaceutical industry in Japan.

In Brazil, the biological products are regulated by ANVISA (National

Agency for Sanitary Vigilance), which has the authority for regulation,

control, and inspection of products and services that can be hazardous to

public health. It is linked to the Health Ministry, and was created in

January 1999. The resolution RDC 315 (ANVISA, 2005) regulates bio-

logical products including vaccines, hyperimmune sera, blood-derived

products, biomedicines, and allergens. The biomedicines include products

obtained from biological ﬂuids or tissues of animal origin, as well as mAbs

and others obtained through biotechnological processes. Biological pro-

ducts from other countries can be registered in Brazil if they are registered

and licensed in their country of origin. New medicine registration and

approval is based on the Medicines Technical Chamber report.

14.4 Harmonization

The goal of the pharmaceutical industry to compete on the international

market is hampered by differences among the regulatory requirements. To

bring uniformity, the ICH has been promoting a process of harmonization

of the technical requirements for pharmaceuticals registration among

pharmaceutical industries and regulatory authorities from Europe, the

352 Animal Cell Technology

USA, and Japan since 1990. This effort is supported by the pharmaceutical

companies because it will make for more efﬁcient regulation in the most

attractive markets and will also be beneﬁcial to patients with faster

availability of new drugs. The ICH aims to eliminate duplication of assays

during research and development of new drugs, as well as provide guide-

lines intended to unify the requirements for approval (Walsh, 2004a;

Foulkes and Traynor, 1999).

14.5 Premises

14.5.1 Clean rooms

Due to the inherent variability of animal cell-based systems, in-process

control and strict adherence to GMP are of critical importance for

obtaining quality products. Carefully planned facilities, together with

bioreactor design and in-process control systems, determine product

quality at feasible manufacturing costs. The regulatory agencies (EC,

1998a; CFR, 2003) clearly state that the premises and equipment must

follow GMP standards.

As most biopharmaceuticals are delivered by the parenteral route,

production plants must follow rules for ‘‘injection’’ quality. Critical

operations are done in clean areas. Other facilities, such as storage,

packaging, and quality control, should be adequately built and maintained

within appropriate conditions of cleaning and hygiene (Walsh, 2004b).

Clean areas are deﬁned by the number of particles per unit volume of air.

Thus, the main element of a clean area is ﬁltered air. Clean rooms are

designed to protect the product from contamination by microorganisms

or particles, which may be derived from various sources, air-borne,

personnel, or equipment. HEPA (high efﬁciency particulate air) ﬁlters are

used to ﬁlter the air and remove particulate matter. Sterile products are

made in rooms with positive pressure intended to avoid the entry of

contaminants from outer rooms. For manipulating pathogens, toxic sub-

stances or radioisotopes, the recommendations are to conduct operations

in a negative pressure area, with air ﬂow exhaustion through sterilizing

ﬁlters that should be treated after use (usually incinerated).

Each operation of a production process takes place in a different room

within the clean area and might require a speciﬁc and deﬁned environment

and cleanliness. Rooms should be designed to maintain a degree of

cleanliness higher than required when not in use, so they are within limits

during operational use. Clean rooms intended for production of sterile

products are classiﬁed according to the total and live particles in the

environment. In Europe (EC, 2003) clean rooms used to be classiﬁed as A,

B, C, and D; A being for the most critical operations like ﬁlling and aseptic

connections, while C and D areas were intended for most of the opera-

tions, upstream, downstream, and solutions preparation. The correspond-

ing US classiﬁcation (IES, 1992) was to designate a clean room as class 100

(equivalent to A/B above), class 10.000 (level C) or class 100.000 (level D).

Brazilian rules (ANVISA, 2003) used to follow the European and WHO

norms (WHO, 2002).

Regulatory aspects 353