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ELSEVI ER Pharmaceutics Acta Helvetiae 71 (1996) 421-438
Quality assurance for biopharmaceuticals: An overview of regulations,
methods and problems
Kristian M. Miiller a,1, Mathias R. Gempeler b, Max-Werner Scheiwe , B. Tatjana Zeugin d3*
Biochemisches I nstitut der Uniuersitiit Ziirich, Winterthurerstr. 190, CH-8057 Zurich, Switzerland
b Pentapharm AG, Engelgasse 109, CH-4002 Basel, Switzerland
Mepha AC, Dornacherstrasse 114, Post$ach, CH-4147 Aesch, Switzerland
Regionale Fachstelle fir Heilmittelkontrolle, Missionsstrasse SO, Postfach, CH-4012 Basel, Switzerland
Received 8 May 1996; revised 18 June 1996; accepted 21 August 1996
Abstract
This paper provides an overview of regulations with special regard to Good Manufacturing Practice (GMP) of biopharmaceuticals in
different countries (Switzerland, European Union, USA). Problems during the set-up and maintenance of a cell bank and expression
system, fermentation, protein purification, and product testing are outlined as well as methods and techniques for quality assurance during
these processes. Freeze-dlying and aseptic filling of the finished dosage form are discussed.
Keywords: Biopharmaceuticals; Guidelines; Quality assurance; GMP; Fermentation: Purification; Lyophilization: Formulation
1. Introduction
Biopharmaceuticals, here defined as products manufac-
tured by biotechnological means and genetical engineering,
have become an important tool in medical therapy. Al-
though they are relatively young products and knowledge
on the handling of them is still growing they are consid-
ered as safe and efficient as conventional drugs by physi-
cians, patients and a wide part of the population (e.g.
antibiotics, insulin, interferons).
However, compared to conventional drugs most bio-
pharmaceuticals must be administered by parenteral route.
They mostly require careful handling and manufacturing
methods like lyophilization and aseptic filling and must be
stored at low temperatures to preserve stability. Biophar-
maceuticals are still developing products that demand
progress in analytical and manufacturing methods and
techniques, and they also demand new considerations of
how quality, safety and efJicacv can be achieved.
2. Regulations, authorities, quality assurance and GMP
Abbreviations: CPMP: Committee for Proprietary Medicinal Prod-
ucts; EC: European Commission; FDA: Food and Drug Administration;
IOCM/IKS: Intercantonal Office for the Control of Medicines/Interkan-
tonale Kontrollstelle ftir Heilmittel; ISO: International Standard Organiza-
tion; NIH: National Institutes of Health; OECD: Organization for Eco-
nomic Cooperation and Development; PIC: Pharmaceutical Inspection
Convention; WHO: World Health Organization
* Corresponding author. Tel.: +41-61.2672390; Fax: +41-61-
2672385.
E-mail: kristian@bioc.unizh.ch.
Biopharmaceuticals open the way to new forms of
therapy. They require a specific collaboration among uni-
versity, industry, and authorities. Regulations in this field
are manifold and it is not always easy to have an overview
on them, since their status in the legal hierarchy may
spread from law to strong recommendation. Due to this
fact the term legal requirements must be understood in a
widespread sense in this paper.
0031-6865/96/$15.00 Copyright 0 1996 Elsevier Science B.V. All rights reserved.
PII SOO31-6865(96)00050-7
422 K.M. Miiller et al. / Pharmaceutics Acta Heluetiae 71 (1996) 421-438
2.1. The role of regulatory bodies
Extensive documentation on chemical, pharmaceutical,
toxicological and clinical characteristics of the product are
required and examined by the authorities before a product
gets approval. This to make sure that the patient will get a
safe and efficient product of the quality needed. Since
biotechnological products are fast and steadily developing
drugs, every new product enhances the know-how in this
field. Thus regulatory bodies profit out of this fact, compil-
ing problems and solutions, and getting a good overview
on the latest findings and pivotal problems in the whole
field.
The mandate of regulatory authorities is to approve
products of state of the art quality, safety and efficacy. The
ongoing of worldwide collaboration in this field becomes
evident by the fact that in December 1995 the third
International Conference on Harmonization (ICH) took
place in Yokohama, Japan. Scientists and experts from
regulatory bodies and industry met there to discuss harmo-
nization issues concerning the approval requirements in
Europe, USA and Japan, however, implementation of har-
monized guidelines must still be adopted by the competent
authorities in the relevant countries.
Table 1 shows legal requirements concerning conuen-
tional and biotechnological products. Basically one can
say that biotechnologically and genetically engineered
products must fulfill the same criteria of quality, safety and
efficacy like conventional drugs. The structure of legal
requirements is world-wide approximately the same,
namely Good Clinical Practice (GCP), Good Laboratory
Practice (GLP), Good Manufacturing Practice (GMP),
biosafety requirements and application requirements, but
they have not always the same wording. Differences are
quite common and may jeopardize registration of a product
depending on the approving country. International meet-
ings like the ICH-Conference try to harmonize all the
various registration requirements. Table 1 also shows that
application requirements are a compilation of data from
particular requirements as GCP, GLP, GMP and biosafety
evaluation including environmental impact evaluation and
protection of the population and employees as well.
Two guidelines concerning the quality of biotechnologi-
cal products were adopted at the ICH meeting at Yoko-
hama, namely:
- Analysis of the expression construct in cells used for
production of r-DNA derived protein products
(CPMP/ICH/139/95, 1995).
* Stability testing of biotechnological/biological prod-
ucts (CPMP/ICH/138/95, 1995).
Further, one guideline concerning quality was given free as
a draft document for discussion, namely:
* Viral safety evaluation of biotechnology products de-
rived from cell lines of human and animal origin
(CPMP/ICH/295/95 Draft, 1995).
These three CPMP/ICH guidelines refer to the quality of
biotechnological products. Quality is one of the pivotal
issues when regulatory authorities examine the documents
of application. But examination is not enough. They will
also inspect the companies to have an insight on good
manufacturing practices (GMP) at the production site may
it be for the bulk substance or the bulk and finished
product.
Other ICH-guidelines that concern conventional and
Table 1
Legal requirements a concerning efficacy, safety and quality of pharmaceuticals
Legal requirements Purpose/function
Good Clinical Practice (GCP)
_
evaluation of the eflcacy and safety of the product
- safety for the test person (ethic requirements)
_
protection of the patients
Good Laboratory Practice (GLP) (OECD) - evaluation of the toxicological safety
_
protection of test persons/patients
Good Manufacturing Practice (GMP) - evaluation of the manufactured (built in) qualify of a product
_
protection of the product (safety, efficacy)
_
protection of the patient (safety, efficacy)
Biosafety requirements
- Environmental requirements
_
evaluation of the environment protection
_
protection of the environment and population
- Preventive health requirements - protection of the employees
Application requirements (registration)
_
requirements concerning the documentation to be submitted for examination to the authority
- documentation contains: compilation of sajery, efficacy and quality data
a See Section 2.
K.M. Miiller et al. /Pharmaceutics Acta Hehetiae 71 (1996) 421-438 423
biotechnological products were adopted in December 95 as
well. They regulate toxicological (GLP) and clinical topics
(GCP). So the titles were on
* Clinical study reports format and content
(CPMP/ICH/137/95, 1995).
- Guideline on the need for carcinogenicity studies of
pharmaceuticals (CPMP/ICH/140/95, 1995).
- Reproductive toxicology: toxicity to male fertility,
(CPMP/ICH/136/95, 1995).
* Stability testing requirements for new dosage forms
(CPMP/ICH/280/95, 1995).
- Validation of analytical procedure methodology
(CPMP/ICH/281/95, 1995).
.
Impurities of new drug products
(CPMP/ICH/282/95, 1995).
- Clinical safety data management: periodic safety up-
date reports for marketed drugs (CPMP/ICH/288/95,
1995).
Before quality assurance and GMP requirements will be
discussed in detail some short comments on GCP and GLP
requirements are given.
GCP requirements deal with the clinical evaluation of
the biopharmaceutical or drug and answer questions on
eficacy including potency and adverse reactions (safety).
GCP regulations have been adopted in Switzerland on 18
November 1993 by the IKS (IOCM/IKS, 1993). This
regulation adopted by IKS strongly involves the 26 can-
tons of Switzerland in applying the statements of this
regulation. This regulation is above all based on the Guide-
lines to Good Clinical Practice for Clinical Trials of
Medicines of July 1991 of the European Communities
(EC, 1991).
GLP requirements mustnt be mixed up with analytical
laboratory requirements concerning the control of bulk
substance, bulk product and finished product. Those qual-
ity control requirements are a part of GMP.
The connotation GLP is still dedicated to the first good
practice regulations that dealt with the conducting of non-
clinical laboratory studies (OECD, 1981; CFR/FDA,
1995a). Therefore GLP includes in vitro and in vivo
laboratory studies that determine the safe9 of a product
whereas GMP related analyses mainly determine quality.
2.2. Quality assurance and GMP requirements
Quality assurance (QA) is a system involving all re-
sources (manpower, machines, know-how, knowledge, etc.)
of a company to produce a product of the quality required
by the customer/authority and considered safe and effi-
cient by the authorities to the benefit of the employees, the
company and the patient.
QA involves all divisions of a firm and to be successful
QA must be aware of the key problems in the manufactur-
ing of drugs/biopharmaceuticals. QA must know what
level of quality has to be assured with the existing and
steadily progressing techniques/methods. Further, due to
the steadily progressing biotechnological and recombinant
techniques and even more recent ways of therapeutic
progress the firm and QA must steadily acquire new
knowledge and keep up with science and legal require-
ments. Science, development, research and legal/regu-
latory requirements are linked to each other, since develop-
ment will change the current state of the art, and regula-
tions will and must alter accordingly.
This definition shows that quality assurance is a quality
system applicable to conventional drugs as well as to
biopharmaceuticals. Development of quality concerning
aspects began with the quality control of a product, where
quality was analyzed into the product. Modem concepts
include quality assurance (QA) and quality management
(e.g. total quality management (TQM) and IS0 9000).
Basic requirements of GMP are applied to conventional
drugs as well as to biotechnological products, since the
aim to produce quality and the way to get there are
basically not different. So the following points are of great
importance:
The quality assurance (QAI department is responsible
to build up a quality system with the support of the
companys upper management. QA has a coordinating
function between the intercepts of a company and must
involve following points into a quality system:
- a documentation system,
- appropriate equipment and facilities, including engi-
neering and technical supply,
- well-defined and verified production processes and
verification that they lead to a suitable quality (valida-
tion, qualification),
* verification of quality control laboratories and verifica-
tion that their analyses/methods are reliable (valida-
tion, qualification),
- verification of the quality of design, dellelopment and
research (aims),
- need for skilled. well-trained personnel with adequate
and steadily growing knowledge and know-how,
- others: like hygiene-requirements, warehousing, elec-
tronic data processing (EDP), registration relevant re-
quirements, purchase and sales, legal (patents) and
medical department requirements.
2.3. GMP in Switzerland and Europe
All legal requirements undergo a certain hierarchy and
so do regulations on GMP in Switzerland as well as in
Europe and the USA (Table 2).
424 KM Miller et al. /Pharmaceutics Acta Heluetiae 71 (1996) 421-438
The manufacture of immunobiological (e.g. vaccines)
and blood and most blood-derived products in Switzerland
is regulated by the Federal Office of Health (BAG),
whereas all other medicinal products are regulated by the
Cantons. Switzerland is divided in 26 Cantons (comparable
to Bundeslander in Germany or States in the USA).
These 26 Cantons have founded a concordat called In-
terkantonale Vereinbanmg wherefrom the Intercantonal
Office for the Control of Medicines/Interkantonale Kon-
trollstelle fur Heilmittel (IOCM/IKS) emerged.
IOCM/IKS, on behalf of the cantons, is responsible for
the approval of medicinal products. GMP-matters, how-
ever, are still cantonal or regionally structured, e.g. there
already exist regional concordats of several cantons. In the
field of GMP the IOCM/IKS has issued Manufacturing
Guidelines (latest revision 18 May 1995) (IOCM/IKS,
1995) which adopt the Guide to Good Manufacturing
Practice of May 1992 issued by PIC and also consider
recommendations of the World Health Organization
(WHO). PIC is the Pharmaceutical Inspection Convention
that was founded in 1970 by EFTA-countries (Switzerland
was one of the founding countries). In brief one can say
that the GMP-Guidelines on Good Manufacturing Practice
for Medicinal Products are mandatory in the countries of
the European Union as well as in Switzerland.
Table 2
General regulations on GMP
IOCM/IKS Switzerland European Union
European directives and guidelines but also interna-
tional regulations (e.g. WHO, European Pharmacopoeia)
are considered in Swiss legislation and they are often
accordingly adapted into Swiss law.
2.3.1. Guidelines on GMP
Basic GMP-guidelines are the same for conventional
products as well as for biopharmaceuticals. With the bio-
pharmaceuticals some supplementary requirements have to
be considered. So the PIC- and EU-Guide (Guide to Good
Manufacturing Practice for Pharmaceutical Products, Doc-
ument PH 5/89 and amendment 5/92; The Rules govern-
ing Medicinal Products in the European Community, Vol.
IV, Guide to Good Manufacturing Practice for Medicinal
Products (EC Document 111/3093/92-EN, 1992)) are ap-
plicable to biopharmaceuticals and have virtually the same
wording with some minor exceptions. These two guide-
lines consist of the following chapters which point out the
pivotal items to consider in manufacturing, namely:
(1) Quality management and quality assurance
The requirements to get a product on the market are
complex and therefore quality management and quality
assurance are essential for a company. In fact without a
QA-system that systematizes and coordinates the manifold
issues, it is not possible nowadays to produce a GMP-con-
FDA; USA
Interkantonale Vereinbarung liber die Kontrolle
der Heilmittel (3rd June 19711 (IOCM/IKS,
1971) (Concordat of 26 Swiss Cantons = IKS)
Regulation on the Control of medicinal products
of 25th May 1972; amended 23rd November
1995 (I~CM/IKS, 1972/1995)
Richtlinien der IKS betr. die Herstellung von
Arzneimitteln (Manufacturing Guidelines of 18th
May 1995) (IOCM/IKS, 1995)
Convention for the Mutual Recognition of In-
spection in Respect of the Manufacture of Phar
maceutical Products: PIC-Guide to Good Manu-
facturing Practice for Pharmaceutical Products
PH 5/89 May 1989 (PIG, 1989); amended/sup-
plemented 1992 (PIG, 1992b) and adapted to
respective EC-regulations
Community procedures for the authorization and
supervision of medicinal products for human and
veterinary use and establishing a European
Agency for the Evaluation of Medicinal Products
(EMEA) + Council Regulations
Commission of the European Communities: Reg-
ulations or administrative action relating to pro-
prietary medicinal products: EC Council Direc-
tive 65/65/EEC (19651, amendments: EC
Council Directive 83/570/EEC; 87/21/EEC;
87/22/EEC; 89/341/EEC; 75/318/EEC:
75/319/EEC
Commission Directive 91/356/EEC: Guidelines
of good manufacturing practice for medicinal
products for human use (EC Directive
91/356/EEC, 19911
Committee for Proprietary Medicinal Products
(CPMP): The Rules Governing Medicinal Prod-
ucts in the European Community Volume IV:
Good Manufacturing Practice for Medicinal
Products (1989); amended/supplemented EC
Document 111/3093/92-EN, 1992
Federal Food, Drug and Cosmetic Act of 1983
(Congress)
Code of Federal Regulations 21 CFR: 21 CFR
200-299 (CFR/FDA, 1995b): 21 CFR 600-699
(CFR/FDA, 1995~1; Biologics
guidelines, guides
Several more specific guidelines, notes for guidance, points to consider (PTC).
K.M. Miiller et al./Phannaceutica Acta Heloetiae 71 (1996) 421-438 425
form product. QA and GMP begin even before product
development begins namely with project management and
planning.
(2) Personnel
The know-how of the personnel is most important, but
the company must also ensure that the know-how can be
kept on a high level. Therefore training is essential as well.
Further, there is not only the know-how on methods,
techniques, etc., the hygiene aspect must flow into every-
days behavior in the firm.
(3) Premises and equipment
Premises and equipment must be maintained and cali-
brated. But before maintenance becomes an issue equip-
ment must be qualified, carrying out installation and opera-
tion qualification where the proof must be given that they
fulfill the purpose they are supposed to do. This is true for
heating, ventilation, air supply, water supply and so on.
(4) Documentation
Everything that one carries out must be written down to
give proof that the work was really done. This means that
documentation has sometimes even an implicit role in QA.
Less stringent forms may be seen in other branches, but
not in the pharmaceutical field.
This means that already planning must be reported,
carrying out of the planned must be reported and finally
the conclusion drawn must be reported in a concise and
informative way. Therefore manufacturing protocols must
be rather detailed. It does not suffice if one writes down
that e.g. sugar was added, it must be said when it was
added and under which circumstances (e.g. temperature,
stirring speed and so on). Relevant documents are SOPS
(Standard Operating Procedures), master formulas, batch
records, specifications, certificates of analysis, analytical
methods, qualification and validation reports, development
reports and others.
(5) Production
This chapter describes items like prevention of cross-
contamination, which is linked to cleaning validation, vali-
dation, choosing of starting materials, processing opera-
tions of intermediate and bulk products, packaging materi-
als and operations, the finished product and rejected, re-
covered and returned material. Validation of production
processes has become a very important item and recently a
new PIC guideline (PH I /96 (PIC, 1996)) has been issued
on Principles of Qualification and Validation in Pharma-
ceutical Manufacture. This guideline comprises recom-
mendation on four topics:
- validation master plan,
- installation and operational qualification,
- non-sterile process validation,
- cleaning validation.
In the above mentioned guidelines process validation is
defined as: Documented verification that the integrated
system functions as intended, in its normal operating envi-
ronment. Validation is a project requiring collaboration
of experts of various disciplines and it is important and
more important than ever that specialists work together on
the verification of a process, piece of equipment and so on.
This might also be the challenge in todays world where
everybody gets trained as a specialist within its field and
must cooperate with others in their field. Therefore valida-
tion is a most demanding challenge.
(6) Quality control
Here we finally get to the laboratory and the control of
products and release of them. Quality control has changed
its status in the release of products since its unique impor-
tance to determine quality has been replaced by the con-
cept to directly produce quality and to already detect
quality problems during manufacturing applying in process
controls (IPC) and qualification and validation. However,
quality control confirms quality and it is therefore still the
last check before the products come to the market. Confir-
mation of quality or certifying quality is still an important
task and may be crucial.
(7) Contract manufacture and analysis
The outcontracting of process steps must thoroughly
define the contract givers and the contract acceptors
responsibilities, since misunderstandings could lead to a
product of insufficient quality.
(8) Complaints and product recall
Complaints may give hints with regard to problems
occurring during the manufacturing process. Therefore
there should be a system to review these complaints
according to written procedures. Further, a preventive sys-
tem should be established in case that a deficiency leads to
a recall from the market.
(9) Self inspection
Whether a system built up by a company does work,
should be checked internally carrying out self inspections.
The supplementary guideline that may be applied to
parenterally applied biopharmaceuticals and conventional
drugs is integrated in the above mentioned GMP-guides
and deals with the manufacturing of sterile pharmaceutical
products (PIG, 1992a).
Other guidelines that are relevant for biopharmaceuti-
cals are the supplementary EC/PIG-guideline to the
EU/PIC-GMP-Guide 012 manufacture of biological phar-
maceutical products for human use and others like
- EC Council Directive 87/22/EEC (1987) on the ap-
proximation of national measures relating to the placing
on the market of high-technology medicinal products,
particularly those derived from biotechnology.
- Ad hoc working party on biotechnology/pharmacy
111/3477/92 Note for Guidance concerning Production
426 K.M. Miiller et al. / Pharmaceutics Acta Heluetiae 71 (1996) 421-438
and Quality Control of Medicinal Products derived by
recombinant DNA technology.
2.4. GMP in the USA
The regulations and points of view of the US regulatory
authority Food and Drug Administration (FDA) are cer-
tainly important for every manufacturer all over the world.
Therefore we will first give a short overview how quality
relevant FDA regulations concerning the production of
drugs/biopharmaceuticals are organized (Fig. 1).
The Congress enacts the Food, Drug and Cosmetic Act
(FD and CA) and FDAs existence is due to the mandate
to approve drugs based on the demonstration of safety,
efficacy and quality. FDA is divided in five divisions,
however, we are only interested in two of them, namely
CBER (Center for Biologics Evaluation and Research) and
CDER (Center for Drug Evaluation and Research). Nor-
mally CBER evaluates biotechnological products, but not
all biotechnologically manufactured products have been
Congress
c
Food, Drug & Cosmetic Act (FD&C Act)
I
f
FDA
(Food and Drug Administration)
// \\\
Food CBER CDER CVM Med. Devices
CBER: Center for Biologics Evakption and Research
CDER: Center for Drtk# Ev$uation aryJ Research
CVM: Center of etennary Medme
FDA
(FD&C Act)
c
Laws / Regulations
e.g. US Code of Federal Regulations
PlCFR; Parts 200 - 299: Drugs
Parts 600 - 699: Biologics
Guidelines / Guides / Points to Consider
W-1
(e.g. Biotechnology inspection Guide Nov. 91)
Fig. 1.
examined by CBER. For example, erythropoetin was regu-
lated by CDER.
In general, FDA like the European and Swiss authori-
ties has stated that drugs produced by the new biotech-
nology will be regulated in the same way as conventional
drugs.
As already mentioned in Table 2 laws and regulations
undergo a certain hierarchy, therefore the Code of Federal
Regulations (CFR) is on a higher legal level compared to
the guidelines and guides which have the status of recom-
mendations, however, most stringent recommendations.
Points to consider are even on a lower level, since they are
the reaction of the authority to keep up with the latest
trends in the pharmaceutical and/or biotechnological field.
If you can prove that you get a safe and efficient drug of
good quality with other adequate means than recom-
mended, then you neednt follow the recommendations,
but often the recommendations show you the latest level of
the state of the art and therefore they will become a must.
2.4.1. Specific guidelines on GMP / biotechnological
guidelines
In general GMP-guidelines must be applied with
biotechnological products as well. This is evident when we
take out the citations of other regulations from the Biotech-
nology Inspection Guide (Nov. 1991). The following
guidelines are cited in it:
Guideline on General Principles of Process Validation,
May 1987 (FDA, 1987a).
Guide to Inspections of High Purity Water Systems,
July 1993 (FDA, 1993a).
Guide to Inspections of Validation on Cleaning Pro-
cesses, July 1993 (FDA, 1993~).
Guideline on Sterile Drug Products Produced by Asep-
tic Processing, June 1987 (FDA, 1987b).
Guide to Inspections of Lyophilization of Parenterals,
1993 (FDA, 1993b).
U.S.P. Antimicrobial Preservative Effectiveness Test.
NIH (National Institute of Health&Guidelines for re-
combinant DNA research (1987, 1988).
Specific guidelines for biopharmaceuticals will be dis-
cussed in the subsequent part.
3. Problems and methods in the fermentation and pu-
rification of a recombinant protein
3.1. Quality assurance and control of the host and expres-
sion system
The variety of hosts used in research and industrial
biotechnology is enormous and includes species from all
K.M. Miiller et al. / Pharmaceutics Acta Helretiae 71 lI 996) 421-438 421
kingdoms. This enormous number of hosts can be nar-
rowed down to a handful of organisms and cell types,
mainly used as model systems in research for some time,
when biopharmaceuticals are considered. Typical hosts are
E. coli K12 derivatives, Saccharomyces cereuisiae, and
cell lines like CHO or C127. The difference originates
from the stringent requirements employed in the testing
and evaluation of each compound of the production pro-
cess, including the host and expression system.
The choice of the host is often determined by the
product as not all organisms can produce every protein.
This selection also greatly influences the whole down-
stream processing procedure. The same holds true for the
expression system, which is specific for each host. It is
obvious that an expression system that allows the purifica-
tion of the desired protein from a defined supematant or
that results in a high ratio of product to cell debris will
ease purification. Furthermore, and in the case of every
technology prerequisites for using an organism and its
expression system are knowledge, familiarity of staff with
the required procedures and availability of the necessary
equipment.
3.1.1. Problems that may encounter
The precise identification of the host can be a difficult
task when it has to be separated from closely related
organisms or cell types or in the case of an occurrence of a
genetic drift. Problems in detecting contaminating organ-
isms such as phages, related cells, bacteria including my-
coplasma, (retro-) viruses, and fungi increase with the
homology to the host, the decreasing size of these organ-
isms and with their ability to hide within the host. The
stability of the expression system is also a major concern,
especially if the product is toxic and leads to selective
stress for the host. Several types of stabilities, such as
segregational stability for plasmids, insertional stability
(transposon), structural stability (deletion, rearrangement),
sequence stability (base pair substitution, frameshift) have
also to be considered. In this respect, DNA whether de-
rived from the expression system itself or from an exoge-
nous source, can also be seen as a contamination. Unstable
or undefined hosts or expression systems may result in
production levels varying beyond specifications or even
lead to alterations that are dangerous and difficult to detect
in the product.
3.1.2. Approaches of the QA-system
Several guidelines and points to consider notes of the
authorities on how to maintain and identify a defined cell
stock have been published (PTC/FDA, 1987; PTC/FDA,
1994; EC Document 111/3477/92, 1994; FDA, 1991). A
seed lot system with standardized protocols for selection,
preparation, preservation, storage, and adventitious agent
testing of stock cultures should ensure the identity and
safety of the starting raw material.
According to FDA (PTC/FDA, 1987; FDA, 1991) a
cell seed or master cell bank (MCB) is defined as a
quantity of cells derived from a single colony (bacteria.
yeast) or a single eucaryotic cell. The MCB is stored
cryogenically (- 70C or below) in sufficient aliquots, one
or more of which would be used for the production of the
manufacturers working cell bank (WCB). The cell seed
for a diploid cell line should be prepared from cells at a
low doubling level or passage number. The working cell
bank is defined as a quantity of cells derived from one or
more ampoules of the master cell bank, stored cryogeni-
cally and used to initiate the production batch.
In addition a description of the cell strain including its
genealogy and identification characteristics (genotype,
phenotype) must be available and is part of a product
application (PTC/FDA, 1987; FDA, 1991). The origin,
form, storage, use, and expected duration at the anticipated
rate of use must be described in full for all cell banks (EC
Document 111/3477/92, 1994). A record of cell history
should be kept and the population doubling should not
exceed an upper limit specified on the basis of perfor-
mance of the cell culture (FDA, 1991; PTC/FDA, 1987).
Potential viral contamination, cross-contamination with
other cell lines and genetic instabilities should be antici-
pated.
The genetic part of a recombinant product requires the
same attention. A detailed description of the cloned gene
including its sequence and details of origin, identification
and isolation should be available. The origin and structure
of the expression vector should be known and a detailed
map with a complete annotated sequence of functionally
relevant regions is essential. Regions created by ligation
during vector construction and that affect the expression of
the inserted gene should be confirmed by sequencing. The
sequenced parts and those deduced from literature have to
be indicated. The development of modem sequencing tech-
nology should allow to sequence whole standard cloning
vectors.
The status of recombinant DNA within the host cell
should be described. For extrachromosomal expression
systems the copy number and proportion of host cells
retaining the expression construct should be determined.
For integrated copies of a gene the product should be
tested at the mRNA level (EC Document 111/3477/92,
1994). Immortalisation procedures should be described
(FDA, 1991).
EC regulations require that during the establishment of
the banks no other cell lines should be handled simultane-
ously in the same laboratory suite or by the same person
428 K.M. Miiller et al. /Pharmaceutics Acta Heluetiae 71 (1996) 421-438
(EC Document 111/3477/92, 1994). Presently FDA gener- continuous (multiple harvest fermentation). Each fermenta-
ally requires an approved product license application or tion starts with a small starting culture used to inoculate
amendment before a new master cell bank can be gener- the main culture with a volume varying from a few liters
ated (FDA, 1991). to several thousand liters.
3.1.3. Methods concerning host and expression systems
Checks concerning the identity of the host and of the
expression system should address both the genetic and the
protein levels. Table 3 shows tests that may be applied in
various combinations.
3.2.1. Problems that may be encountered
Newly developed expression constructs or cell lines
may help to minimize the problems of instability and false
product formation. For example, for E. coli the stability of
plasmids could be enhanced by the use of segregation loci
on plasmids, like the pat-B locus from the plasmid pSClO1
(Skogman et al., 1983) or postsegregational killing systems
like the hok/sok system (Gerdes, 1988). Recombinant
genes with host codon usage may reduce the incorporation
of incorrect amino acids, while optimized cell strains with
low by-product formation and defined deficiencies, for
example in proteases, may allow higher reproducibility in
expression.
Various factors may influence the reproducibility of a
fermentation run, e.g. varying composition of the media,
non-host contamination, genetic alteration of host or ex-
pression system, loss of expression system, and bad con-
trol of run and set-up parameters. In the case of the raw
materials, especially (bovine-) sera for mammalian cell
culture are of particular concern as they may harbor organ-
isms such as mycoplasm, the hoof and mouth disease or
the bovine spongioform encephalopathy (BSE) (FDA,
1991). Biosafety aspects and environmental concerns in-
crease with the potential toxicity of product or host and the
fermentation volume. The upscale of fermentation can
easily lead to alterations of the product, especially using
mammalian cells.
3.2. Quality assurance and control of the fermentation
process
There are several types of fermentation, which gener-
ally can be described as batch, fed batch (single harvest) or
3.2.2. Approaches of the QA-system
To achieve a high reproducibility of the fermentation
and the final cell suspension, all factors which may inter-
fere with growth and protein production have to be elimi-
nated.
A first step is the stringent control of the raw materials
Table 3
Methods concerning host and expression systems
Points to consider Test/method
Identity of the host and expression system
(especially cloned fragment) on the genetic level
Genomic integration of the vector
Transcription
Translation
Identity of the host and expression system
on the phenotype level
Microbial, viral, mycoplasma contamination
Stability of the expression system
- restriction enzyme mapping
- DNA fingerprinting
_
sequence analysis
- Southemblot analysis
- mRNA or cDNA sequencing
_
protein characterization according to Table 6 in product section
- karyological monitoring
_
characterization of nutrient requirements
_
isoenzyme analysis (e.g. by electrophoreses)
_
growth (doubling time)
_
morphological characterization
_
monitoring reproducibility of product production
_
contamination assays according to Table 6 in product section
_
continuous preparation and quantification of plasmids
_
restriction digest
- sequencing
_
nutritional markers
_
resistance markers (antibiotics)
_
plasmid contents by monitor enzymes (e.g. beta-lactamase),
reproducibility of protein production, and analysis of protein variants
K.M. Miiller et al./ Phamaceutica Acta Heluetiae 71 (1996) 421-438 429
calculation of
specific growth rate
@ = f(F, V, Cco,, Co,)
_<, ,^ **,,
process control
data monitoring
*:,:, .,_
I.
standard controls
glucore ammonia
fed batch mode:
&point for substrate pumps
_, .
offgas-analyzer
gasflow ratio controller
dosage controller
calculation of feeding rate
of growth limiting substrate
Fig. 2. Scheme of a fermentor for E. coli fed batch culture with equipment that allows continuous monitoring for reproducible results (courtesy of D.
Riesenberg and colleagues, HKI Jena).
including water. Sterilization of media prior to use and
aseptic inoculation, transfer and harvesting should prevent
contamination with other organisms. To avoid chemical
contamination the fermentation equipment should prefer-
ably be sterilized with steam. If the facility is used for
different fermentations additional testing for cross contam-
ination is required. Extensive in-process monitoring of the
fermentation parameters facilitates a uniform process and
where necessary an earlier rejection. An example for E.
coli is given in Fig. 2 (Horn et al., in press).
For single harvest production the generation number or
population doubling level should be specified on the basis
of the stability of the host-vector system. For a multiple
harvest production the period of fermentation should be
specified.
In most countries the biosafety requirements are regu-
lated by law and technical notes (NIH, 1986; NIH, 1987;
NIH, 1988). Exempt host-vector systems with inherent
biological safety such as non-pathogenicity, auxotrophy
and non-transmissible vector are generally regarded as safe
(GRAS) (Hasskarl, 1990; BG Chemie, 1991; BG Chemie,
1992). In general the Good Large-Scale Practice (GLSP)
applies (FDA, 1991).
3.2.3. Methods concerning the fermentation process
Table 4 describes methods used in fermentation.
Defined media based upon inorganic and synthetic
chemicals which have been developed in the last number
of years for bacterial (Horn et al., in press), yeast, and
mammalian cell culture (Jumarie and Malo, 199 1) simplify
the control and sterilization of the raw material and also
the task of downstream processing.
3.3. Quality assurance and control of the purification
process
The protein purification starts either with the culture
supematant as for example in the case of monoclonal
antibodies from hybridoma culture or the harvested cells of
the fermentation, e.g. E. coli or Saccharomyces cerevisiae,
in cases where no secretion systems are available. The aim
of purification is to remove all impurities such as product-
related proteins, other proteins, lipids, carbohydrates, nu-
cleic acids and all organisms, especially viruses.
Cells are generally disrupted by physical force, as in the
case of high pressure release in a French- or Gaulin press,
but enzymatic or chemical methods may also be applied.
Filtration or ultrafiltration are usually the first purification
step to concentrate the supematant or to separate the
desired product from cell debris. Further, purification steps
usually take advantage of well established chromatography
430 K.M. Miiller et al./Pharmaceutica Acta Helvetiae 71 (1996) 421-438
such as ion exchange and gel filtration. The most desired
purification is selective for the active protein like affinity
chromatography for antibodies (Scopes, 1994; Deutscher,
1990). Proteins from mammalian cell culture are normally
treated with an additional virus inactivation step, as viruses
are seen as one of the most dangerous contaminations and
therefore requiring special attention.
3.3.1. Problems that may be encountered
During the set-up phase of a purification procedure the
most crucial point is to select the right purification steps
with a capacity allowing to produce pure and active pro-
tein. Once a purification scheme has been established and
upscaled, the main challenge for quality assurance is the
performance of purification steps which are non-robust,
and the ageing of the filter and chromatographic material.
Chromatography media and biological ligands (antibodies)
in particular may leach and exhibit poorer performances.
Depending on the reproducibility of the previous fermenta-
tion and initial purification steps the amount of impurities
may vary, thus demanding constant control. Repeatedly
used columns are a potential source of microbial contami-
nation and bacterial endotoxins.
3.3.2. Approaches of the QA-system
One of the important points for the administration of the
purification process is that the correct validation of all
procedures is achieved. As the procedure behavior may
change with scale, tests should be performed with the final
set-up.
In most countries, chromatographic media in general
must be approved for pharmaceutical production. Media
with biological contents should be produced according to
pharmaceutical standards (e.g. immunoaffinity columns
must be virus free). The sanitation procedure of all mate-
rial involved in purification should be monitored with
challenge tests during the set-up stage and with continuous
controls in the production process. Data collected during
set-up and validation should be used to set the lifetime of
the material.
3.3.3. Methods concerning the purification process
Already well-known methods are generally easier to
validate than new and specific procedures (such as e.g.
affinity chromatography) that may, however, give better
results. A short list of the principle methods is presented in
Table 5.
The physico-chemical properties of the support mate-
rial have to be considered with regard to sanitation, ther-
mostability, etc.
Expression in inclusion bodies yielding highly enriched
protein in aggregates and refolding are techniques em-
ployed in recombinant expression in E. coli, which allow
the expression of disulfide containing proteins and the
circumvention of protease problems and toxic stress on the
host (Rudolph, 1990). In recent years the use of engineered
peptide or protein fusions have become a popular research
tool for protein purification due to their general usefulness
in purification (Nygren et al., 1994). Among these the
His-tag, consisting of five to six histidines fused to either
end of the protein, has been used in several promising
approaches to yield pharmaceutical grade protein (Casey et
al., 1995; Kaslow and Shiloach, 1994).
3.4. Quality assurance and control of the product and in
process controls tI PCJ
This section presents an overview of analytical methods
confirming protein product quality. Tests are most rigor-
Table 4
Methods concerning the fermentation process
Points to consider Test/method
Sterility of facility, media, and gas (aeration) - in place steam sterilizing is the method of choice for the facility
- media should be autoclaved, if impossible, filtration might be used
_
sterile filtration of gas
Filter validation
_
challenge test with Pseudomonas diminutu (FDA, 1987a; FDA, 1987b; FDA, 1987~)
In process control of fermentation - monitoring of growth, product formation and contamination: optical density, pH, temperature,
oxygen pressure, outlet gas, foam, nutrients, waste, by-product level, viscosity, addition of chemicals,
density, flow injection analysis is a good way to monitor metabolites
Lot test with respect to genetic drift
Lot test with respect to contamination
- see host and expression system section in Table 3
- fatty acid profile
- see also product section Table 6
K.M. Miiller et al. / Phannaceutica Acta Helvetiae 71 (1996) 421-438 431
ously applied during the set-up phase and mainly used for
product testing, but are also suitable as in-line process
controls. The more physical and biological properties of
the protein can be exploited and the more specific tests for
impurities are available the better. Although physico-
chemical methods might fully characterize a protein, con-
firmation of quality by biological testing in vitro or in vivo
is often desirable, because they often better reflect real
application. However, the advantage of the latter is some-
times overcome by consistency problems.
3.4.1. Problems challenging the QA-system
Testing protein drugs with their biological impurity
profile and special basic characteristics differs from con-
ventional drug testing. Proteins are of high molecular
weight ranging from lo4 to lo6 g/mol for proteins such
as IgM. The primary peptide sequence folds to secondary
and tertiary structures that might complex to a quartemary
structure. Intra and inter chain disulfide bridges, as well as
Table 5
Methods concerning the purification process
posttranslational modifications such as N- or O-glycosyla-
tion or the hydroxylation of proline are important for
function and immunogenicity. These highly sophisticated
properties give rise to many problems during synthesis,
purification, and product-filling. Proteins may form aggre-
gates or be partially degraded by proteases or thermally
denatured. The glycosylation pattern can vary with fermen-
tation conditions quite easily and wrong disulfide bridges
may be formed. Some amino acids are likely targets for
chemical modification: methionine is easily oxidized and
asparagine can be deamidated to aspartyl- or isoaspartyl-
rests. Trace amounts of cleaning agents or plasticizers may
also react with the product.
Apart from these product-related impurities, contamina-
tions from the host or expression system that are not
removed by the purification and adventitious organisms
require the same attention. As previously stated a virus
disease is most worrisome as it may not be limited to the
treated patient. At present blood derived products contami-
Points to consider Test/method
Purification
Chromatographic purification
Validation of purification
Cleaning and sanitation of
chromatographic media
Validation of sanitation
Virus removal/inactivation
Validation of virus removal
- precipitation of product or contaminants (e.g. ammoniumsulfate)
- filtration
_
ultrafiltration
_
centrifugation
- ion exchange chromatography (weak to strong anion or cation exchanging functional groups)
_
size exclusion chromatography (SEC) (also named gel filtration or gel permeation chromatography (GPC))
_
affinity chromatography (pseudo-) substrate affinity, immunoaffinity, immobilized metal affinity,
protein affinity (e.g. protein A))
- hydrophobic interaction chromatography (various short aliphatic or aromatic groups)
- adsorption chromatography (e.g. thiophilic adsorption, hydroxyapatite)
- Overloading/spiking of process steps with specific contaminants such as DNA or host cells might be a
way to demonstrate their ability
_
sodium hydroxide may be used to remove proteins, nucleic acids, endotoxins, and viruses
_
sodium chloride for the removal of proteins and nucleic acids
- detergents are used to remove hydrophobic proteins and lipids
_
challenge tests with various organisms e.g. Staphylococcus aweus (Gram-positive bacteria),
Escherichia coli (Gram-negative bacteria), Candida albicans (yeast) and Aspergillus niger
(mould) (Adner and Sofer, 1994)
- pH 3-4.5 is robust and yields a reported log virus removal of 3-3
- heat is robust and yields a reported log virus removal of 4
_
solvent/detergent is robust and yields a reported log virus removal of 5
_
filtration (0.02-0.04 pm) is robust and yields a reported log virus removal of 4-8
- detergent is not robust and yields a reported log virus removal of 4
_
affinity or ion exchange chromatography is not robust and yields a reported log virus removal
0f l-5 WC/FDA, 1994)
- for a virus challenge test, a group of viruses exhibiting a range of purification-relevant
physico-chemical properties should be selected and mixed with the crude preparation:
special guidelines cover the removal of viruses
432 K.M. Miiller et al. / Pharmaceutics Acta Heluetiae 71 (1996) 421-438
Table 6
Methods concerning quality control and IPC of the product
Points to consider Test/method
Physical and biological identity - chemical or enzymatic peptide mapping
_
sodiumdodecylsulfate polyacrylamide gel electrophoresis (SDS PAGE)
_
isoelectric focusing (IEF)
_
2-dimensional electrophoresis (IEF combined with SDS-PAGE)
_
high performance liquid chromatography (HPLC); especially reversed phase
_
enzyme linked immunosorbent assay (ELISA), radioactive immunoassay (RIA)
_
immuno blot (Western blot)
_
terminal protein sequencing
_
total amino acid analysis
_
mass spectroscopy (MS)
- capillary electrophoresis (CE)
- modem analysis combines several methods like peptide mapping with MS or CE
_
circular dichroism (CD) spectroscopy
_
nuclear magnetic resonance (NMR) spectroscopy
- X-ray crystallography
Quantity
Carbohydrates
Potency/activity
Protein contamination
Pyrogen contamination
Viral contamination
_
classical protein assays according to Lowry, Biuret, Bradford and their derivatives
_
ultra violet (UV) or fluorescence spectrophotometry
- HPLC
- SDS-PAGE
- glycosidases analysis in combination with SDS gel shift or mass spectrometry
_
immuno detection of specific glycosilation moieties
_
whole animal bioassay
_
cell culture bioassay
_
receptor based immunoassay
_
identification of agents that may adversely affect potency
_
evaluation of functional activity and antigen/antibody specificity (various immunodiffusion methods,
immunoblotting/radio- or enzyme linked immunoassay)
- HPLC validated to correlate certain peaks to biological activity
- SDS-PAGE with Coomassie staining or, for improved sensitivity, silver or fluorescent staining
- HPLC
- contaminant specific ELISA, RIA
- Western blot
_
USP United States Pharmacopoeia 23 (1995) rabbit pyrogen test
_
Limulus amebocyte lysate assay (LAL) (FDA, 1987~)
- endogenous pyrogen assay
_
cytopathic effect in several cell types
- hemabsorption
_
embryonated egg testing
_
polymerase chain reaction (PCR)
_
viral antigen and antibody immunoassay
_
mouse antibody production (MAP)
- reverse transcriptase assay
Microbial contamination
_
total plate count
(bacteria including mycoplasma, fungi, yeast)
- heterotrophic plate count and total yeast and molds
- Mycoplasma test (purification processes should show the clearance of at least six logs of
mycoplasma (Huxsoll, 199411
Nucleic acid detection
Chemical contamination
- hybridisation
_
whole DNA assay
_
same tests as with conventional drugs
K.M. Miiller et al./Phannaceutica Acta Heluetiae 71 (1996) 421-438 433
nated with HI-virus still leave their footsteps in the death
statistics of patients. Other impurities may lead to im-
munogenic reactions or unforeseen side effects as exempli-
fied in the tryptophan case (Mayeno and Gleich, 1994).
Other sources of contamination must be considered, e.g.
allergens, petroleum oils residual solvents, cleaning mate-
rials, and metal ions.
3.4.2. Approaches of the QA-system
Several points to consider are helpful for the develop-
ment of test procedures and in setting specification limits
(PTC/FDA, 1985; PTC/FDA, 1987; PTC/FDA, 1992).
Analysis data should be collected during the set-up and
from about five initial bulk lots of the product character-
ized to fullest available extent. These data give the basis
for the specification of the product. For routine analysis a
test set ensuring identity, purity, potency and stability of
the product should be selected (EC Document
111/3477/92, 1994). The extent of the contamination re-
moval should be confirmed by both validation studies and
end product testing. Assays used to qualify and quantify
the biological and physical properties of proteins should be
used to evaluate lot-to-lot consistency of the drug product
and to monitor the stability of the product with time. The
basic criterion for a standardized and reliable product is
the demonstration of lot-to-lot consistency with respect to
certain predetermined release specifications.
Tests for potency should consist of in vitro or in vivo
tests, which have been specifically designed for each prod-
uct so as to its potency. A reference preparation for
biological activity should be established and used to deter-
mine the bioactivity of the final product. In house biologi-
cal potency standards should be cross-referenced against
international standards (e.g. WHO, Ph. Eur. and USP
standards).
Although DNA content in biological drug products is
evaluated on case by case basis, FDA (CBER) guidelines
require a DNA assay to have a sensitivity in the order of
10 pg DNA per dose, and the WHO guidelines require less
than 100 pg per dose (CPMP, 1987).
Table 1
Compatibility of excipients
Topic Examples
3.4.3. Methods concerning quality control and IPC of the
product
Tests that are needed will depend on the process and the
intended use of the product. Aspects that should be consid-
ered and tested are shown in Table 6.
Viral assays that first employ amplification of the virus
and then detection in a cell culture system are more
sensitive than direct cell culture assays and thereby pro-
vide greater assurance of the absence of viruses.
From this wealth of tests only a few are used for routine
testing. E.g. Epogen from Amgen is tested in bulk by
SDS/Western blot, isoelectric focusing, size exclusion
chromatography, tryptic mapping, N-terminal sequencing,
radio immunoassay for activity, in vivo assay for activity,
HPLC for purity, DNA assay, immunoassay for contami-
nating proteins, mycoplasma assay, sterility, protein, and
endotoxin assay. The final product is checked for appear-
ance, activity in RIA, protein content, sterility, safety,
pyrogenicity/endotoxin content, identity, and amount of
excipients.
4. Quality aspects on development and formulation of
the final product (lyophilizate)
4.1. Pre-validation during development
The quality of the final product depends on the quality
and safety of each step of action during development and
manufacture of the product (Wichert, 1993). Validation
should begin as soon as possible (Laicher, 19891, e.g.
much of the validation should already be commenced
during the development. This may also be helpful when
more data are demanded during the approval process or in
any questions concerning product quality and safety later.
Thus the pharmaceutical, technical and developmental
steps dealing with compatibility studies, formula develop-
ment, and with the manufacturing are essential and are
looked at in more detail.
Solution medium, solubility, cryoprotectants,
other protectants, pH, buffers, temperature, light
Solubilizers
Impurities in medium
Packaging materials
water (WPI), polyethyleneglycol, NaCl, dimethylsulfoxide, hydroxyethylstarch, glycerol,
hypromellose, sorbitol, mannitol, xylitol, glucose, gelatine, glycine, alanin, valin
polysorbate ethoxylated ricinoleate, pluronics, cyclodextrins
metal ions
siliconized glass, plastics, colors in plastics, residual monomers
434 KM. Miller et al. /Pharmaceutics Acta Helvetiae 71 (1996) 421-438
4.2. Compatibility Table 8
In this stage the basic parameters necessary for formula-
tion have to be clarified. These parameters should include
activities necessary and related to any type of lyophilizate
for injection, and should be supplemented with specific
requirements for the products discussed. Table 7 summa-
rizes necessary activities on compatibility.
Formulation: How difficulties may be solved
Effect May be overcome by:
Degradation, chemical
Hydrolysis, deamidation, oxidation, - CaCl,
disulfide bonds exchange - low 02-level
- buffer-ions
The function of proteins is related to the secondary as
well as to the tertiary and quarternary structure; the spatial
structure influences surface effects and may influence pH
and temperature.
Stabilization in aqueous solutions is e.g. possible by
human serum albumin (HSA), sugar alcohols and sugars
(mannitol, xylitol, sorbitol, glucose, maltose), by peptides,
aminoacids (glycine, valine, alanine), polysorbate, and
buffers.
Degradation, physical
Adsorption, aggregation, precipitation, - ionic strength of buffers
loss of activity - cryoprotectants
Sticking to glass surface - polysorbate
Unstable at room temperature -pHnear7
_
sugar, sugaralcohols,
peptides, aminoacids
Sticking to glass surfaces may be overcome by polysor-
bate and decomposition by metal ions when using CaCl,
(woog, 1993).
4.3. Formulation
Due to the compatibility study, the choice of inactive
ingredients and conditions for establishing a galenical for-
mula may be defined. In this stage, the appropriate combi-
nation of excipients, pH, isotonicity, concentration, wall
material and rubber closure has to be found resulting in
adequate solubility, feasibility of lyophilization, stability
and sufficient dissolution properties (Eckardt, 1993; Woog,
1993) (Table 8).
It should be pointed out, that during freezing the pH
and the concentration of salts may change very much
(Kijrber and Scheiwe, 1983; Woog, 1993), but this can be
modified by cryoprotectants (Kiirber and Scheiwe, 1980)
or the selection of salts in buffers (Eckardt, 1993). These
effects are well known from the freezing of living eukary-
otes and prokaryotes. Since a living cell is more complex
than a single protein, the mechanisms and means for
cryopreservation established may possibly give hints how
to optimize the freeze-drying process for pharmaceutically
used proteins.
4.4. Lyophilization process
tively complex freeze-drying process largely affects and
determines product properties and should be validated
thoroughly. The temperature of glass formation and eutec-
tic crystallization should be established for the solution
used, and the process parameters must be adjusted (Franks,
1990). Adaption of the freeze-drying cycle to these de-
mands avoids collapsing of the lyophilization cake (result-
ing in irreversible insolubility and stickiness) and can be
achieved by avoiding any holding time near the glass-tran-
sition point. Respective data are discussed in the literature
(e.g. Korber and Scheiwe, 1980). Residual channels in the
lyophilization cake are necessary for the transport of vapor
and should be optimized in size and stability and to assure
redissolving. For example Rupprecht showed that cooling
with a rate of 3C/min gives 4 pm of diameter whereas
25C/min results in 1 pm pores in a dextran-solution
(Rupprecht, 1993). Further, the residual humidity is essen-
tial for proteins. An optimized water content for human
growth hormone has been found to be in the range of 0.6%
to 2.8% (Eckardt, 1993). Experts dont always consider
lyophilization as an advantageous possibility for stabilizing
a certain drug. It is pointed out that the lyophilization cake
has a very large surface and is thus susceptible to interac-
tions from the surrounding (e.g. germs). Also the residual
moisture may cause difficulties during stability testing. An
alternative could be embedding of proteins/peptides in
quickly dissolving polymer matrices. Up to now, however,
such formulations to our knowledge are not on the market.
The lyophilization process includes the following steps:
dissolution, sterilization of the bulk, filling and partial
stoppering, transport to the lyophilization chamber, freez-
ing, vacuum application and relief and complete stoppering
of the vials.
The set of physico-chemical parameters of the rela-
Using aseptic conditions, bioburden should be kept as
low as possible and environmental surveillance as well as
media fills are necessary tools to manage the system. The
equipment used offers several possible contamination
sources like leakage of the chamber during vacuum appli-
cation or contamination of the media employed for cooling
and heating. Finally the rather complex equipment may fail
KM. Miiller et al. / Pharrnaceutica Acta Helvetiae 71 (1996) 421-438 435
Table 9
Controls of lyophilizate and reconstituted product
Topic Necessary activity
Dry substance
Optical homogeneity
Weight
Moisture of cake
Isotropy of cake
Incomplete sublimation
Inner surface
Reconstituted solution
Optical appearance
Solubility in reconstitution medium
Physical parameters
Filtration
Chemical
Microbiological
_
visual control
_
net-weight
_
sorption-isotherms, residual equilibrium moisture, actual moisture
_
crystalline/amorphous state (differential scanning calorimetry, DSC)
_
tendency of meltback
- helium-pycnometer
_
turbidity, opalescence, lightscattering
_
clarity, turbidity, opalescence time for complete solution
- pH, viscosity, density, optical density, clearness, oxygen content
_
active loss on passing injection minifilter
_
content, activity, potency (bioassay)
_
sterility testing, pyrogen testing
or malfunction during the long lasting process. On the
other hand sterile filtration of the final product is often not
possible and mostly a preservative is not present in the
dissolving solution. From this situation the need arises for
thorough planning and validation of the aseptic steps of the
process (FDA, 1993b; FDA, 1987b).
Table 10
Formulation examples in the market
Recombinant alpha-2 b Recombinant alpha-2 a Interferon beta from fibroblasts
Intron-A Schering-Plough Roferon-A Roche Naferon Sclavo
Ingredients
Sodium chloride
Sodium phosphate anhydride
Sodium phosphate monobasic
Glycol
Human albumine
Mannitol
9.0 mg
2.27 mg
0.55 mg
20 mg
1 mg 5 mg 292
40 mg
Solwnt
Benzylic alcohol
Water for injection ad
9mg
1 ml 1 ml 1 ml
Urokinase
Kisolv Mundipharm
human Urokinase
Alfakinasi Alfa-Wasserm. a Urokinase Choay Purochin Sclavo
human Urokinase
Ingredients
Dextrane 100 mg
Sodium chloride 17.5 mg
Sodium-phosphate monosodium 2H,O 34 mg
Mannitol 20 mg 10-13.33 mg
Na-Edetat
2mg
Sodium phosphate 2.4 mg 9.5 mg
human albumin 25 mg
Solvent
Sodium chloride 18
mg
45
mg
9mg
Sodium phosphate monobasic 0.507 mg
Sodium-phosphate dibasic 7.79 mg
Water for injection ad 2 ml 2 ml 5 ml 2 ml
a Same formulation: Persolv Richter (Lepetit); Ukidan (Serono); Urokinasi (Iketon).
436 K.M. Miiller et al. / Phannaceutica Acta Helvetiae 71 (1996) 421-438
In summary, the following points must be considered
concerning the manufacture of products used for clinical
trials as well as products for the market:
Need for aseptic processing; environmental monitoring;
media fills with regard to filling, transportation into the
chamber and handling of the final stopper&g, etc. The
number of units filled during media fill should at least
be adequate to the normal load.
Sterilization and maintaining sterility in the lyophilizer.
Identification of procedures in case of malfunction of
the system, like failure of vacuum-pump, leakage, or
too much ice on the condenser.
Discussion of the rationale of a chosen cycle.
Lyophilizator design, including assurance of integrity of
air or gas filters, sterilization of the rods for stoppering
in the chamber, contamination of the condenser, and
opening in a clean area only.
Meltback and poor solubility of the final product result-
ing in losses of active ingredient on injection through
the normally used microfilters, and crystallinity status.
The FDA-Guide (FDA, 1993b) gives insights in the
problems that may occur during lyophilization and should
already be applied in the developmental stage (Table 9).
4.5. Formulation examples
From (1Informatore Farmaceutico, 1994) some actual
formulations are known, e.g. interferon and urokinase, they
are listed in Table 10.
The formulations show among normally used excipients
of parenteral lyophilizates also amino acids (glycine), hu-
man serum albumin, and NaEDTA. Some cryoprotectants
are used as well (e.g. glycol, mannitol, dextrane).
5. Conclusion
This paper may have revealed that the know-how on
biopharmaceuticals is steadily growing and that keeping up
with state of the art technologies and methods is not only
compelling for the manufacturers but also for the authori-
ties. Therefore, regulatory means like requirements on a
basically low enforcement level as e.g. points to consider
help to transfer the latest information in the relevant field.
Further, a certain degree of collaboration and harmoniza-
tion all over the world is urgently needed and the ICH is a
helpful tool for regulating authorities as well as the indus-
try. Harmonization, collaboration and coordination within
the company is of great importance to allow an adequate
transfer of know-how. QA- and GMP-issues may have
shown the complexity of requirements on the manufacture
of a product to achieve the quality required.
In the main part problems and methods in fermentation
and purification of a recombinant protein have been dis-
cussed trying to make QA- and GMP-related problems
evident, since QA can only be efficient and stake out its
claims if it knows the crucial points in good manufacturing
practice. Tables should enable the reader to get a fast
overview on methods used with the host and expression
system, with fermentation, purification and quality control
as well as IPC.
And finally some hints concerning the lyophilization of
the final product may have shown that from the active bulk
substance to the final product there is still a tremendous
developmental work left. Not to forget that from the final
formulation to the finished dosage form that comes to the
market there are still some further steps to consider.
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