Validation of Bioanalytical Methods

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Patric U. B. Vogel

Validation of bioanalytical
methods
Patric U. B. Vogel
Vogel Pharmopex24
Cuxhaven, Germany

ISSN 2197-6708     ISSN 2197-6716 (electronic)

essentials
ISBN 978-3-658-38912-3    ISBN 978-3-658-38913-0 (eBook)
[Link]

© The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Fachmedien
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What You Can Find in this Essential

• An introduction to the validation of bioanalytical methods

• The presentation of the different test categories
• An overview of method properties that are checked during validation
• A description of the requirements that must be fulfilled for a method validation
• Examples of companies neglecting their duty to validate and the consequences
thereof

v
Abstract

This book describes the validation of bioanalytical methods. This is an important

element in the quality control of drugs, especially biological drugs. During the
manufacture of medicinal products, samples are analysed for compliance with
quality requirements at many points in the very complex manufacturing process.
Depending on the nature of the drug, bioanalytical methods are also used. These
must provide trustworthy results so that no false conclusions are drawn when
evaluating the results. The trustworthiness is checked by validation.

vii
Contents

1 Introduction ����������������������������������������������������������������������������������������������� 1
2 Method Categories������������������������������������������������������������������������������������� 5
3 Validation Parameters�������������������������������������������������������������������������������13
3.1 Accuracy���������������������������������������������������������������������������������������������13
3.2 Precision���������������������������������������������������������������������������������������������16
3.3 Further Validation Parameters�������������������������������������������������������������18
4 
Validation Environment and Validation Planning�����������������������������������23
4.1 Validation Environment: The Big Difference Between Academic
and GMP Laboratories �����������������������������������������������������������������������23
4.2 Validation Planning�����������������������������������������������������������������������������26
5 
Validation of Bioanalytical Methods���������������������������������������������������������29
5.1 Identity: Validation of a PCR Test�������������������������������������������������������29
5.2 Determination of Content: Validation of a Virus Titration�����������������33
5.3 Qualitative Impurities: Validation of a Test for Absence of
Microorganisms (Sterility) �����������������������������������������������������������������36
5.4 Quantitative Impurity: Validation of an Endotoxin Assay �����������������37
6 
Errors, Problems and Risks Associated with Insufficient Method
Validation ���������������������������������������������������������������������������������������������������39

ix
x Contents

7 Summary�����������������������������������������������������������������������������������������������������43

What the Reader Can Take Away from this Essential�������������������������������������45

References �����������������������������������������������������������������������������������������������������������47
Introduction
1

Pharmaceuticals represent a heterogeneous group of products that are used,

among other things, to treat or prevent diseases. The production of medicinal prod-
ucts takes place in specialised companies under consideration of Good
Manufacturing Practice (GMP). The most important European set of rules in this
regard, the EU GMP Guide, is a comprehensive work that lays down the “rules of
the game” for all quality-relevant processes. This covers everything from the drug
manufacturer’s suppliers, the quality of the materials used, the production pro-
cesses, quality control and other important areas such as how changes are to be
made and how activities are to be recorded. A key aspect of drugs is that they must
pass extensive laboratory analytical examination before they are released for use
and shipped. Many drugs are based on small active substances that consist of a
certain number of atoms bonded together and whose composition can be repre-
sented relatively easily as a chemical structural formula. These so-called low-­
molecular active substances include, for example, typical antipyretics such as
paracetamol or ibuprofen. Many of these drugs are mainly analysed using chemical-­
physical analytical methods. This can be, for example, chromatography, a method
for separating and, if necessary, determining the quantity of substances.
A comprehensive presentation of bioanalytical methods would require more
than 1000 pages, as for example in the excellent textbook Bioanalytik by Lottspeich
and Engels, which can be regarded as the bible for students of the life sciences in
the field of methodology (Lottspeich and Engels 2012). Nevertheless, we will try
to make the essence of bioanalytics understandable in this book. Bioanalytical
methods in the narrow sense are used to study biomolecules, i.e. molecules of liv-
ing organisms. Biomolecules are, for example, proteins, DNA, RNA, carbohy-
drates or lipids. Bioanalytical in a broader sense means analyzing certain ­properties

© The Author(s), under exclusive license to Springer Fachmedien 1

Wiesbaden GmbH, part of Springer Nature 2023
P. U. B. Vogel, Validation of bioanalytical methods, essentials,
[Link]
2 1 Introduction

of biological material, i.e. biomolecules, viruses and living cells (see Fig. 1.1).
Biological drugs include genetically engineered proteins, DNA molecules, stem
cells, antibodies or vaccines based on viruses and bacteria. Depending on the na-
ture of the product, various methods are used for quality control of biological
products, such as cell cultures, polymerase chain reaction (PCR), protein bio-
chemical methods, microbiological methods such as nutrient media for the detec-
tion of specific microorganisms, etc. However, it would be wrong to think in abso-
lute categories, such as biological medicinal product = bioanalytical methods and
chemical medicinal product = chemical analytical methods. Depending on the
product, classical chemical-physical methods are also used in the quality control of
biological medicinal products, e.g. pH-value determinations or determinations of
the moisture content, but in most cases bioanalytical methods predominate in the
case of biological medicinal products. The entirety of the analytical methods of a
medicinal product should provide evidence that the product fulfils all the quality
requirements specified in the marketing authorisation and is thus effective and safe.

Proteins

Viruses,
cells DNA

Bioanalytics

Lipids RNA

Carbon-
hydrates

Fig. 1.1 Objects of investigation in bioanalysis. (Source: Created by Patric Vogel)

1 Introduction 3

In order to be able to make this statement at all, one must be able to trust the
results of these bioanalytical methods. Many trust results or displays intuitively
and do not even question whether what they see really corresponds to the truth.
There are many situations in everyday life where we trust information. This may be
the time on the illuminated sign of a pharmacy, the speedometer display of our car
while driving, the receipt of a letter from the doctor with the results of laboratory
tests or graphics on the current development of unemployment figures in the news.
We trust that the information is accurate. But how do we know? The answer is
simple: we don’t. Some of the data can be confirmed by doing our own research or
comparing it to other measurements. Other data is not so easy to check. Errors can
always occur, with graphics but also with electronic displays, and do not always
have to have serious consequences. An incorrect time, e.g. 14.45 instead of 15.00
on the pharmacy clock may have no effect at all when we are on our way to the
beach. But the same mistake can have significant consequences if, for example, we
are on our way to a job interview and, based on the wrong time, decide to have a
leisurely coffee at the bakery next door before presenting at the company and thus
unfortunately arrive too late. In the worst case scenario, the job slips through our
fingers as we are deemed unreliable or unpunctual.
A similar scenario arises with bioanalytical methods used in the GMP area. If
we want to make statements on the basis of results from bioanalytical methods as
to whether a drug meets the quality requirements, we must first be able to trust the
results and for this we need validation. Validation is the process of proving that the
method is fit for its intended purpose, i.e. that it produces reliable results. The vali-
dation of analytical methods is required in the European area by the Guide to Good
Manufacturing Practice of the European Union (Chap. 6 of the EU GMP Guide).
This chapter deals with the tasks of quality control, which also includes the per-
formance of laboratory analytical checks (EudraLex 2014).
While the GMP guideline only requires validation in one sentence, without
formulating how this is to be done, there are other international or national guide-
lines that describe how method validation has to be done, including which proper-
ties are to be checked. These general guidelines are not formulated down to the last
detail and allow room for interpretation or different approaches. For certain long-­
established methods, there are even pharmacopoeial monographs that describe in
detail how the suitability should be checked in one’s own laboratory. Compliance
with the requirements for validations, like any other area of Good Manufacturing
Practice, is checked at regular intervals by the competent authorities as part of
GMP regulatory inspections. It is not uncommon for deficiencies to be identified
which relate to the lack of or inadequate validation of analytical methods (see
Chap. 6). This makes it clear how important the validation of methods is for GMP-­
compliant quality control.
4 1 Introduction

This book describes the validation of bioanalytical methods. In addition to

general (quality of reagents, training of staff, equipment qualification) and formal
aspects (work instructions, validation protocols), validation is illustrated for each
test category using an example.
Two important terms are the method category and so-called validation pa-
rameters. There are a large number of different bioanalytical methods with which
properties, or quality attributes, are determined. In most cases, the methods can
be assigned to one of a few method categories. For example, if the amount of insu-
lin is determined, this method falls into the category of content. In addition, it could
also be analyzed whether certain undesirable substances are contained in the insu-
lin preparation. Such methods fall into the category of impurities. In Chap. 2 the
method categories are explained with examples. The method categories are based
on the properties of the analysed sample. In addition, validation parameters are
properties of the method, e.g. how accurately it can measure, how much individual
measurements vary, what minimum amount of the analysed biomolecule can still
be measured, etc. In Chap. 3 we will get to know all relevant method properties.
The examples chosen in this book include:

• Content: Validation of a virus titration of a live vaccine

• Identity: Validation of a polymerase chain reaction
• Contamination: Validation of a test for the absence of microorganisms (sterility)
• Contamination: Validation of an endotoxin test
Method Categories
2

Validation means that a certain sequence of experiments or laboratory tests veri-

fies whether results of the method used are reliable. These tests are different and
serve to examine certain aspects. It also depends on the purpose of a method which
properties, so-called validation parameters, are to be checked. The important
thing is that there are only a handful of method categories and validation parame-
ters. These are listed in the ICH Q2 (R1) guideline along with recommendations on
how to conduct the tests (number of measurements, etc.) (ICH 2005). There are
four designated method categories: Content, Identity, Impurities qualitative and
Impurities quantitative. This represents a rough classification that can be applied to
many analytical methods. If, for example, 12 different analytical methods are used
on a biological medicinal product for release, most of the methods used can be
classified in one of the four broad categories. However, there are also individual
methods that cannot be classified in one of these categories (see Fig. 2.1).
The content is the quantity determination of the active substance in the drug,
that is, the component that produces the actual effect. The active substance can be
almost anything, from small chemical substances such as paracetamol, to proteins
or DNA, to living organisms. While chemical methods are used for paracetamol, as
mentioned before, the others would fall into the field of bioanalytics.
For example, insulin is a protein (growth hormone) whose amount can be de-
termined using a chromatographic method called high-performance liquid chro-
matography (HPLC) (Moses et al. 2019). This HPLC is a complex system consist-
ing of several components (see Fig. 2.2A). In this example, the analysis involves
adding the insulin fluid to a closed system. The insulin is transported with a liquid
buffer through the tubing or metal lines until it hits a column. This column contains
a surface to which insulin can attach, i.e. bind. However, this binding is changeable

© The Author(s), under exclusive license to Springer Fachmedien 5

Wiesbaden GmbH, part of Springer Nature 2023
P. U. B. Vogel, Validation of bioanalytical methods, essentials,
[Link]
6 2 Method Categories

Content

Other
methods Identity

Category

Contamination Contamination
(quantitative) (qualitative)

Fig. 2.1 Method categories. (Source: Created by Patric Vogel)

(reversible) and depends on environmental factors. Other molecules present in the

sample migrate through the column. By changing the properties (e.g. concentra-
tion, pH) of the liquid constantly pumped through the system, one achieves that the
insulin at a certain point passes back into the liquid, i.e. detaches from the column
surface. As this happens quite abruptly, all the insulin is released within a short
time and is carried along with the liquid flow. At the end of the system, the fluid
passes through a detector that constantly measures what is passing through the line.
The more insulin that was in the sample, the stronger the signal. This signal is plot-
ted as a curve (the amount passing the detector increases until it reaches a maxi-
mum and then decreases again). Since different substances have different binding
strengths to the column surface, they dissolve at different times (= substance sepa-
ration) and can be represented as different curves, so-called peaks (see Fig. 2.2B).
The results of a test run are also called a chromatogram (see Fig. 2.2). Depending
on the drug, additional signals can be generated, e.g. by other components (also
impurities) contained in the test sample, which, however, can be distinguished
from the active substance if the method is well adjusted (see Fig. 2.2C). The signal
is converted into the actual unit, e.g. amount/millilitre, by means of a so-called
2 Method Categories 7

Fig. 2.2 Chromatography system with theoretical representation of substance separation

and an example chromatogram for a substance separation. (Source: Image A (left): Adobe
Stock, file no.: 5773410; Image B (top right): Adobe Stock, file no.: 181689009; Image C
(bottom right): Adobe Stock, file no.: 82895709, modified afterwards; Images licensed by
Patric Vogel)

standard curve (different amounts of a known insulin preparation). In principle,

there are usually several methodical possibilities to determine the content of
­biomolecules or cells. In the case of insulin, for example, the HPLC described is
even a substitute that has only become established in recent years. Before (and still
today), an in vivo assay in live rabbits was/is the standard test to measure the bio-
logical activity of insulin for batch release (Hamza 2018). In recent years, as regu-
latory agencies have increasingly pushed to replace these ethically questionable
live animal drug tests with in vitro laboratory methods, an HPLC method was de-
veloped in this example (Hack et al. 2016).
DNA is a biomolecule whose quantity can be determined, for example, by
means of absorption measurement. For this purpose, a solution containing DNA is
placed in a so-called photometer. This device sends light of a certain wavelength
through the liquid. DNA has the property to absorb this light, i.e. to intercept it.
Behind the liquid there is a detector that catches the light that passes through. The
more DNA there is in the solution, the more light is absorbed and the less light
8 2 Method Categories

reaches the detector. The photometer then calculates for itself (using a stored stan-
dard curve) how much DNA is present in the solution. This method is very simple,
but is not always, at least not alone, suitable as a true content determination, as it
does not capture the state form of the DNA, which is important for the activity of
many biological DNA-based products.
Whole organisms or living cells are perhaps the most complex form of an ac-
tive substance. These are used, for example, in vaccines (e.g. the vaccine against
tuberculosis called Bacille-Calmette-Guérin (BCG)). The content of this live vac-
cine, which is based on attenuated live tuberculosis bacteria, can be determined by
microbiological methods (Milstien and Gibson 1990). This involves diluting the
vaccine suspension and placing the liquid on special culture media (round plastic
dishes with a high rim containing a fairly solid jelly-like substance containing all
the nutrients that bacteria need to grow). Since bacteria multiply continuously by
dividing into two, a bacterial colony visible to the naked eye forms at each site
where a bacteria was present after a few weeks (other bacteria form a visible col-
ony after only one day) (Fig. 2.3). The colonies are counted and, taking into ac-
count the dilution, it is calculated back how many bacteria there are in the vaccine
solution, e.g. per ml.
However, biological drugs are now more versatile. For example, stem cells are
whole, viable cells that are the active substance. But there are also various thera-
peutic products under development, such as attenuated bacteria that have been ge-
netically modified and are to be used to fight certain tumours. There are different
modes of action, ranging from bacteria that damage tumour cells themselves, to
bacteria that produce tumour-associated antigens, to bacteria that carry only DNA
molecules. In the latter case, the genetic information is translated into proteins by
the immune cells themselves after they have “eaten” the bacteria (Xiong et al.
2010; Sedighi et al. 2019). These products are thought to damage the tumor or
stimulate a strong immune response that targets the tumor. These examples show
how the methodology used is highly dependent on the nature of the test sample
being analyzed. In some cases, a simple count of these bacteria as a content deter-
mination would no longer be sufficient to measure the content or biological activ-
ity, as the bacteria are in some cases only a vehicle to smuggle the active substance
(recombinant DNA molecule) into body cells.
Another category is identity. Here it is examined whether it is really the drug
that is supposed to be contained according to the package insert and label. Almost
the entire range of bioanalytical methods can be used as identity tests, depending
on the properties of the drug. For example, a drug containing a protein as active