Manufacturing of Pharmaceutical Proteins E-Book

Stefan Behme

• Herausgeber: Wiley-VCH GmbH
• Kategorie: Wissenschaft und neue Technologien
• Sprache: Englisch

Seitenzahl: 711

Veröffentlichungsjahr: 2021

Beschreibung

Now in its third edition, the text covers all aspects of biopharmaceutical manufacturing. Structured like a textbook, it is aimed at a wide audience in industry and academia and can be used as a reference as well as for training purposes.

Leseprobe

Table of Contents

Cover

Title Page

Copyright

Medical disclaimer

Preface to Third Edition

Preface to First Edition

List of Abbreviations

Part I: Introduction

1 Biopharmaceutical Production: Value Creation, Product Types, and Biological Basics Introduction

1.1 Role of Production in Pharmaceutical Biotechnology

1.2 Product Groups

1.3 Basics of Biology

Part II: Technology

2 Manufacturing Process

2.1 Role of the Manufacturing Process in Biotechnology

2.2 Process Schematic and Evaluation

2.3 Cell Bank

2.4 Fermentation

2.5 Purification

2.6 Formulation and Filling

2.7 Labeling and Packaging

3 Analytics

3.1 Role of Analytics in Biotechnology

3.2 Product Analytics

3.3 Process Analytics

3.4 Environmental Monitoring

3.5 Raw Material Testing

3.6 Product Comparability

Part III: Pharmacy

4 Pharmacology and Drug Safety

4.1 Action of Drugs in Humans

4.2 Routes and Forms of Administration

4.3 Drug Study

4.4 Path of the Drug from the Manufacturer to Patients

4.5 Drug Safety

Part IV: Quality Assurance

5 Fundamentals of Quality Assurance

5.1 Basic Principles

5.2 Benefit of Quality Assurance Activities

5.3 Quality Management According to ISO 9000

5.4 Structure of Quality Management Systems

5.5 Quality Management System Components in the Pharmaceutical Area

5.6 Quality Assurance in Development

6 Quality Assurance in Manufacturing

6.1 GMP

6.2 Operative Workflows under GMP Conditions

6.3 Production of Investigational Drugs

Appendix A: Case Study Part IV: Warning Letters by FDA

Part V: Pharmaceutical Law

7 Pharmaceutical Law and Regulatory Authorities

7.1 Fields of Pharmaceutical Law

7.2 Bindingness of Regulations

7.3 Authorities, Institutions, and Their Regulations

7.4 Official Enforcement of Regulations

7.5 Drug Approval

Appendix B: Case Study Part V: Clinical Trials for Protein Products

B.1. Mabthera®/Rituxan®

B.2. Enbrel®

B.3. Remicade® Infliximab

B.4. Humira® 40 mg

B.5. Lucentis®

B.6. Zaltrap®

Part VI: Production Facilities

8 Facility Design

8.1 Basic Principles

8.2 GMP-Compliant Plant Design

8.3 Basic Concepts for Production Plants

8.4 Clean and Plant Utilities

8.5 Equipment Cleaning

8.6 Clean Rooms

8.7 Automation

8.8 QC Laboratories

8.9 Location Factors

9 Planning, Construction, and Commissioning of a Manufacturing Plant

9.1 Steps of the Engineering Project

9.2 Project Schedules

9.3 Cost Estimates

9.4 Organization of an Engineering Project

9.5 Successful Execution of an Engineering Project

9.6 Legal Aspects of Facility Engineering

Part VII: Economy

10 Production Costs

10.1 Drug Life Cycle

10.2 Position of the Manufacturing Costs in the Overall Cost Framework

10.3 Basic Principles of Cost Calculation

10.4 Costs of Biotechnological Manufacturing Processes

10.5 Accounting Methods

11 Investments

11.1 Basic Principles

11.2 Value–Benefit Analysis

11.3 Investment Appraisal

11.4 Dynamic Payback Time

12 Production Concept

12.1 Capacity Planning

12.2 Dilemma of In-House Manufacturing

12.3 Aspects of Manufacturing Outsourcing

12.4 Make-or-Buy Analysis

12.5 Process Optimization

12.6 Supply-Chain Management

Appendix C: Examples Part VII: Manufacturing Cost Calculation

C.1. Introduction

C.2. Basic Assumptions for Both Production Processes

C.3. Step 1: Production of Product 1 in Dedicated Facility

C.4. Step 2: Addition of a Second Product

Part VIII: Production Organization and Digitalization

13 Organization of a Manufacturing Facility

13.1 Functional Setup of a Manufacturing Plant

13.2 Development of a Plant Organization

13.3 Organizational Charts and Cooperation Pathways

13.4 Cultural Aspects: The Human Factor

14 Digitalization

14.1 Operational and Digital Perspective

14.2 Digital Maturity

14.3 Integration and IT Architecture

14.4 Digital Transformation

14.5 Digital Applications in the GMP Environment

References

Further Reading

Fermentation

Purification

Aseptic Filling and Lyophilization

Bioanalytics

Regulatory

Pharmacy and Clinical Development

Quality and Validation

Good Manufacturing Practice

Facility Design

Clean Rooms

Project Management

Engineering

Economy

Weblinks

Index

End User License Agreement

List of Tables

Chapter 1

Table 1.1 Examples for recombinant proteins.

Chapter 2

Table 2.1 Comparison between batch and continuous culture.

Table 2.2 Differences between suspension fermenters for cell culture and mic...

Table 2.3 Examples for matrix materials and their brand names.

Table 2.4 Labeling of chromatography gels.

Chapter 3

Table 3.1 Methods for structural analysis of proteins.

Table 3.2 Methods for physical data of protein solutions.

Table 3.3 Methods for identification of impurities.

Table 3.4 Methods for identification of contaminants.

Chapter 4

Table 4.1 Phases of clinical testing.

Table 4.2 Sources for post-marketing data collection of safety-relevant info...

Chapter 5

Table 5.1 Examples of “Good Practice” rules.

Table 5.2 Categories of quality costs (Schneppe and Müller 2003).

Table 5.3 Examples of documentation in the pharmaceutical area.

Chapter 6

Table 6.1 Structure of the EU GMP Guideline (EudraLex, Vol. 4).

Table 6.2 Clean-room gowning for different clean-room classes.

Table 6.3 Different storage areas have to be set up for different substance ...

Table 6.4 Regulatory classification of changes to the manufacturing process.

Chapter 7

Table 7.1 EudraLex – the European comprehensive body of legislation for the ...

Table 7.2 List of the ICH Q series (quality documents).

Table 7.3 ISPE baseline pharmaceutical engineering guides.

Chapter 8

Table 8.1 Chances and risks for realization of high technical complexity and...

Table 8.2 Impact of basic design concepts (rows) on optimization parameters ...

Table 8.3 Water qualities and their areas of use.

Table 8.4 Phases of the qualification of a WFI system.

Table 8.5 Utility users and suppliers.

Table 8.6 Generation of utilities.

Table 8.7 Overview of types of waste and usual disposal routes.

Chapter 9

Table 9.1 Phases of facility engineering.

Chapter 10

Table 10.1 Cost positions of fermentation.

Table 10.2 Cost positions of purification.

Table 10.3 Cost positions of filling and packaging.

Table 10.4 Types of calculations.

Chapter 11

Table 11.1 Classification of investments according to the investment object.

Table 11.2 Effect of investments.

Table 11.3 Other features of investments.

Chapter 12

Table 12.1 Calculation scheme and examples for processing times in fermentat...

Table 12.2 Types and features of cooperation.

Table 12.3 Items to be regulated in manufacturing and supply agreements.

Table 12.4 Overview of in-house (“make”) and external (“buy”) manufacturing.

Appendix C

Table C.1 Cost structure of example step 1.

Table C.2 Cost structure of example step 2.

Table C.3 Costs of products.

Table C.4 Costs comparison between purely internal and mixed option.

Chapter 13

Table 13.1 Typical functional split in a manufacturing facility.

Chapter 14

Table 14.1 Typical IT systems in a pharmaceutical manufacturing plant with m...

List of Illustrations

Chapter 1

Figure 1.1 Role and tasks of production.

Figure 1.2 Subject areas in production. Inner circle...

Figure 1.3 Relationship between production and development.

Figure 1.4 Relationship between production and marketing.

Figure 1.5 Schematic production workflows of important product groups. Produ...

Figure 1.6 Schematic comparison of traditional therapy and cell and gene the...

Figure 1.7 Schematic of a prokaryotic cell.

Figure 1.8 Schematic of a eukaryotic cell.

Figure 1.9 Schematic of energy metabolism.

Figure 1.10 Schematic of cell division.

Figure 1.11 Intentional and unintentional addition of viruses (or phages) in...

Figure 1.12 Basic principles of protein biosynthesis.

Figure 1.13 Protein structures.

Figure 1.14 Molecular structure of human insulin. S–S disulfide bridges betw...

Figure 1.15 Schematic of antibody IgG.

Figure 1.16 Amphoteric behavior of proteins. The ambient pH value determines...

Figure 1.17 Schematic of a cytoplasmic membrane with lipids.

Chapter 2

Figure 2.1 Variability of the process generates variable product quality.

Figure 2.2 Basic terms of manufacturing of biopharmaceutical products.

Figure 2.3 Schematic of the manufacturing process of the drug substance. Chr...

Figure 2.4 Schematic of the manufacturing process of the final product (phar...

Figure 2.5 Typical path for the generation and use of the cell bank.

Figure 2.6 Development of the cell mass and the target protein concentration...

Figure 2.7 Schematic of fermentation modes: batch, fed-batch, and continuous...

Figure 2.8 Sterilization kinetics. The achievable germ reduction depends on ...

Figure 2.9 Typical fermenter for suspension culture with microorganisms or a...

Figure 2.10 Principles of reactors for adherently growing cell cultures....

Figure 2.11 Typical installation for a batch or fed-batch process for mammal...

Figure 2.12 The path from harvest to purified target protein. (a) Host cell ...

Figure 2.13 Interaction between overall yield, step yield, and number of sep...

Figure 2.14 Product purity and volume from harvest through active agent. The...

Figure 2.15 Sources of process-related impurities.

Figure 2.16 Methods for cell separation (centrifuge, chamber-filter press) a...

Figure 2.17 Primary treatment of proteins expressed intracellularly.

Figure 2.18 Adsorption and desorption mechanism.

Figure 2.19 Adsorption in batch and expanded-bed mode.

Figure 2.20 Normal-flow filtration and TFF. Both can be operated as surface ...

Figure 2.21 Schematic of micro- and ultrafiltration (UF) in the TFF mode and...

Figure 2.22 Membrane processes and their potential applications for the sepa...

Figure 2.23 Schematic of the chromatography operation in the binding mode (I...

Figure 2.24 Schematic of chromatography operation in the flow-through mode....

Figure 2.25 Mode of operation of an IEC (here anion exchanger).

Figure 2.26 Mode of operation of affinity chromatography.

Figure 2.27 Mode of operation of SEC and HIC.

Figure 2.28 Design of a chromatography installation. Enlargement shows the g...

Figure 2.29 Principles of precipitation and extraction.

Figure 2.30 Separation characteristic of an ultrafiltration membrane (Rauten...

Figure 2.31 Typical separation process for an extracellularly expressed prot...

Figure 2.32 Typical separation process for an extracellularly expressed prot...

Figure 2.33 Schematic of formulation and filling.

DS

 = 

drug substance

.

Figure 2.34 Position of freeze-drying in the formulation process.

Figure 2.35 Labeling and secondary packaging.

Chapter 3

Figure 3.1 The four areas of quality control.

Figure 3.2 Concept of measure for activity. The effect achieved with a defin...

Figure 3.3 Schematic of amino acid analysis.

AA

 = 

amino acid

.

Figure 3.4 Schematic of Edman protein sequencing. AA = amino acid.

Figure 3.5 Schematic of peptide mapping.

m

/

z

 = mass-to-charge ratio.

Figure 3.6 Principle of SDS–PAGE. Left...

Figure 3.7 Isoelectric focusing.

Figure 3.8 Schematic of capillary gel electrophoresis.

Figure 3.9 Western blot (immunoelectroblot). GE = gel electrophoresis.

Figure 3.10 Schematic of a HCP ELISA.

AB

 = 

antibody

; FERM...

Figure 3.11 Typical chromatogram.

Figure 3.12 The essential elements of a mass spectrometer.

m

/

z

...

Figure 3.13 Principle of nucleic acid sequencing. A, C, G, T...

Figure 3.14 Typical plot of DSC.

Figure 3.15 Analytics around the fermenter.

Figure 3.16 Process analytics in purification.

Figure 3.17 Process analytics in formulation and packaging.

Figure 3.18 Sources of contamination of the process.

Figure 3.19 Workflow for evaluating comparability. Comparison of product qua...

Chapter 4

Figure 4.1 Correlation between drug quantity, drug effect, mechanism of acti...

Figure 4.2 Pathways of drugs in the body at systemic application.

Figure 4.3 Time–concentration curve for single dosing.

C

0

...

Figure 4.4 Time–concentration curve for multiple dosing.

Figure 4.5 (a) Quantification of PD parameters by analog data collection and...

Figure 4.6 Times, costs, and risks during drug development.

Figure 4.7 Path of the drug from the manufacturer to the patient (example fo...

Figure 4.8 Avoidable and non-avoidable causes for detrimental side-effects (...

Chapter 5

Figure 5.1 Fields of activity of quality assurance according to ISO 9001.

Figure 5.2 Documentation inside a

quality management

(

QM

) system (Modified f...

Figure 5.3 Flow diagram for the control of defective goods before delivery. ...

Figure 5.4 Sources of errors and preventive measures in manufacturing.

Chapter 6

Figure 6.1 GMP requirements increase with increasing market supply proximity...

Figure 6.2 Clean rooms in the process chain of biotechnological manufacturin...

Figure 6.3 Prevention of cross-contamination in different plant concepts. Ty...

Figure 6.4 V-model of qualification.

Figure 6.5 Documentation in manufacturing and testing.

MBR

 = 

master batch re

...

Figure 6.6 Process of product release under GMP. QC = quality control;

QA

 = 

Figure 6.7 Management of changes to the manufacturing process. QC...

Chapter 7

Figure 7.1 Bindingness of regulations. See text for abbreviations.

Figure 7.2 Organization of the FDA (excerpt).

Chapter 8

Figure 8.1 Functional areas of a biotechnological production facility.

Figure 8.2 Principal functional areas and flows of the process core and supp...

Figure 8.3 Process flow diagram for purification. Chrom...

Figure 8.4 Example of a conceptual design for a plant with mobile equipment....

Figure 8.5 (a) Product flow in the plant with mobile equipment. (b) Personne...

Figure 8.6 Room classifications in the production plant. White...

Figure 8.7 Air pressure steps and mapping of ventilation systems. Colored fr...

Figure 8.8 Conflicts of interest when optimizing a production plant for the ...

Figure 8.9 Integrated and fractal construction. Expansion areas are dashed....

Figure 8.10 Different configurations of flexible piping. (a) Transfer panel ...

Figure 8.11 Typical process steps for generating pharma-grade water from dri...

Figure 8.12 Elements of a water system, AP system, CIP system, WFI system, a...

Figure 8.13 Heating and cooling loops in a production plant.

Figure 8.14 Typical configuration of a CIP/SIP system. NaOH...

Figure 8.15 (a) Typical personnel air lock (side view) and (b) procedure for...

Figure 8.16 Example of a room-in-room concept.

Figure 8.17 Typical configuration of an HVAC system for clean rooms.

Chapter 9

Figure 9.1 Planning costs and failure cost risk in the planning phases.

Figure 9.2 Typical project schedule for the planning and construction of a p...

Figure 9.3 Recommended agreement structure (Modified from Braganz (2001).)

Chapter 10

Figure 10.1 Life cycle curve of a drug. Dashed line...

Figure 10.2 Overview over the profit/loss situation of a drug during develop...

Figure 10.3 Relations between investment, manufacturing costs, sales, and pr...

Figure 10.4 Basic scheme of costs (overhead calculation).

Figure 10.5 Steps of the decision-making process.

Figure 10.6 Influence of plant utilization on fixed, variable, and unit cost...

Figure 10.7 The areas of cost accounting.

Figure 10.8 Types of manufacturing costs.

Figure 10.9 Cost allocation in overhead calculation.

Figure 10.10 Options for parameters and calculation segments for the calcula...

Figure 10.11 Left = full costing; right = direct costing for the calculation...

Chapter 11

Figure 11.1 Life cycle of capital investment.

Figure 11.2 Decision process for investments with example for a facility con...

Figure 11.3 Different scaling methods in evaluation matrices of value–benefi...

Figure 11.4 Overview of static methods of investment appraisal. Comparison o...

Figure 11.5 Calculation of the NPV from the series of payments of an investm...

Chapter 12

Figure 12.1 Typical schedule for the implementation of market supply for a b...

Figure 12.2 Typical time line for technology transfer. Small-scale model...

Figure 12.3 Typical schedule for the implementation of market supply for a b...

Figure 12.4 DMAIC cycle.

Figure 12.5 Process capability. (a) Statistical process control. (b) Definit...

Figure 12.6 Process capability. (a) Process potential

C

p.

(b) Process capabi...

Figure 12.7 Supply chain for the market supply of a drug and typical values ...

Figure 12.8 Risk matrix with three risk examples, likely to trigger differen...

Appendix C

Figure C.1 Product unit costs dependency on facility usage (34 batches equal...

Figure C.2 Average costs per grams for two products plotted over external of...

Chapter 13

Figure 13.1 Production management and value flow. Up-/downstream: fermentati...

Figure 13.2 Swim lanes and exemplary RACI chart for the process: material de...

Figure 13.3 Typical organizational chart of a manufacturing plant. Up-/downs...

Chapter 14

Figure 14.1 Digital plant maturity model (Anttonen et al. 2020). ERP: enterp...

Figure 14.2 Horizontal and vertical integration of IT-based systems and comp...

Figure 14.3 IT-architecture models for integrating systems.

DSS

:

domain-spec

...

Figure 14.4 Digital strategy and functional layers connecting business and i...

Guide

Cover Page

Table of Contents

Title Page

Copyright

Medical disclaimer

Preface to Third Edition

Preface to First Edition

List of Abbreviations

Begin Reading

References

Index

End User License Agreement

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Stefan Behme

Manufacturing of Pharmaceutical Proteins

From Technology to Economy

Third Edition

Author

Dr.-Ing. Stefan Behme

Berlin, Germany

Chlumer Str. 3

12203 Berlin

Germany

Cover Design: Wiley

Cover Images: © Artisticco/Shutterstock;Reptile8488/Getty Images

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Medical disclaimer

The contents of this work are intended to further general scientific research, understanding, and discussion only and are not intended and should not be relied upon as recommending or promoting a specific method, diagnosis, or treatment by medical care providers for any particular patient. The publisher and the author make no representations or warranties with respect to the accuracy or completeness of the contents of this work and specifically disclaim all warranties, including without limitation, any implied warranties of fitness for a particular purpose. In view of ongoing research, equipment modifications, changes in governmental regulations, and the constant flow of information relating to the use of medicines, equipment, and devices, the reader is urged to review and evaluate the information provided in the package insert or instructions for each medicine, equipment, or device for, among other things, any changes in the instructions or indication of usage and for added warnings and precautions. Readers should consult with a specialist where appropriate. The fact that an organization or Website is referred to in this work as a citation and/or a potential source of further information does not mean that the author or the publisher endorses the information the organization or Website may provide or recommendations it may make. Further, readers should be aware that Internet Websites listed in this work may have changed or disappeared between when this work was written and when it is read. No warranty may be created or extended by any promotional statements of this work. Neither the publisher nor the author shall be liable for any damages arising herefrom.

Preface to Third Edition

What started out over ten years ago as being a book focused on protein manufacturing, in the meantime has evolved into a broad introduction of many different aspects of pharmaceutical operations. All but two sections – technology and regulatory – can be applied to any pharma production, which also gives testimony to the maturity and standardization that pharmaceutical protein manufacturing has achieved by now. The third edition of this book has been amended by two new chapters about plant organization and digitalization, broadly applicable to pharma. The general, conceptual, and simplifying approach makes the book a valuable source also for those working on transferring advanced pharmaceutical technologies like cell and gene therapies into economically feasible, large-scale solutions.

The original concept of the book – keep it simple and speak through pictures and examples – has been kept alive. The concept of strong simplification has been well received by many readers over the last years. Dipping into specific chapters of interest and quickly getting familiar with basic concepts and terminology has obviously addressed the needs of readers both in industry and academia. So, I am very happy to present the third edition of this book now and thank my editor Wiley-VCH for the ongoing support.

Berlin, 23 May 2021

Stefan Behme

Preface to First Edition

This book introduces the basic knowledge of industrial manufacturing of biopharmaceuticals. It is written for those wanting to understand the landscape, interfaces, and interactions between the different disciplines relevant for production as such; aspects of technology and analytics, pharmacy, quality assurance, regulatory affairs, facility technology, and economic efficiency are illustrated. The work shall serve as a textbook and reference at the same time, and is directed toward students as well as industry-experienced engineers, pharmacists, scientists, or economists wanting to acquire a basic knowledge of biotechnological production.

My daily industrial practice has inspired this book. Manufacturing advanced drugs under good manufacturing practice conditions can indeed be a critical factor for drug development and marketing. Being part of multidisciplinary teams, it became obvious to me that the technological and economic challenges of biopharmaceutical manufacturing and its interdependencies with adjacent disciplines are not understood everywhere. Decision making in interdisciplinary teams requires communication and appreciation of the constraints on the various counterparts in order to address them efficiently in the overall program. In contrast to this, particular disciplines become more and more specialized, using their language on a level difficult to understand for the counterparts foreign to the field, sometimes flavoring modern project work with a taste of the tale of the Tower of Babel.

Facilitating communication about manufacturing issues is the goal of this book. It does so by using numerous illustrations and simplifications, making the book easy to read. Correlations between disciplines are highlighted by cross-references, and a detailed keyword index facilitates the search for special topics. After having read this book, the reader should have a high-level understanding of the roles, correlations between terminologies of the different disciplines engaged in the production of biopharmaceutical proteins. For those wanting to dig deeper into the topics, literature recommendations and web links are provided for further reading.

I would like to thank Andrea Rothmaler and Andreas Janssen for their valuable input into the manuscript, my students at the Technical University of Dortmund for their instructive questions, and my company Bayer Schering Pharma AG for providing the opportunity to participate in exciting biotechnological projects.

I hope that my readers will enjoy reading this book as much as I have enjoyed writing it.

Berlin, October 2008

Stefan Behme

List of Abbreviations

AA

Amino Acid (= AS)

ADR

Adverse Drug Reaction

AE

Adverse Event

AIEX

Anion Exchanger

AMG

Arzneimittelgesetz

AMWHV

Drug and drug manufacturing Regulation

AP

Aqua Purificata

API

Active Pharmaceutical Ingredient

APR

Annual Product Review

AR

Adverse Reaction (= ADR)

AR

Annual Report

ATP

Adenosine Triphosphate

AUC

Area Under the Curve

AVP

Aqua Valde Purificata

BAS

Building Automation System

BDS

Bulk Drug Substance

BLA

Biological License Application

BOD

Basis of Design

BP

Basen Pair

BPMN

Business process model and notation

BR

Batch Record

BRR

Batch Record Review

BSE

Bovine Spongiforme Encephalopathie

CAPA

Corrective Action Preventive Action

CBE30

Changes Being Effected in 30 days

CDW

Cell Dry Weight

CFR

Code of Federal Regulations

CFU

Colony Forming Unit

cGMP

Current Good Manufacturing Practice

CI

Chemical Ionization

CIEX

Cation Exchanger

CIP

Cleaning in Place

CJD

Creutzfeldt–Jakob Disease

CMC

Chemistry, Manufacturing, and Control

CMO

Contract Manufacturing Organization

CoA

Certificate of Analysis

CoC

Certificate of Compliance

COP

Cleaning out of Place

CRF

Case Report Form

CSV

Computerized system Validation

CTA

Clinical Trials Authorization

CTD

Common Technical Document, Clinical Trials Directive

CVMP

Committee for Medicinal Products for Veterinary Use

DIN

Deutsches Institut für Normung

DNA

Desoxyribonucleic Acid

DPPM

Digital plant maturity model

DQ

Design Qualification

DSC

Differential Scanning Calorimetry

EBR

Electronic Batch Record

ED

Effective Dose

EDQM

European Directorate for the Quality of Medicines

EIS

Electron Impact Spectroscopy

ELISA

Enzyme Linked Immunosorbent Assay

EMA

European Medicines Agency

EP

European Pharmacopoeia (PharmEur)

EPO

Erythropoietin

ERM

Enterprise recipe management

ERP

Enterprise resource planning

ETL

Extract–transform–load

FaaS

Function as a service

FAB

Fast Atom Bombardment

FBS

Fetal Bovine Serum

FCS

Fetal Calf Serum

FDA

Food and Drug Administration

FMEA

Failure Mode and Effect Analysis

FP

Final Product, Finished Product

HMI

Human machine interface

GAMP

Good Automated Manufacturing Practice

GCP

Good Clinical Practice

G-CSF

Granulocyte Colony Stimulating Factor

GEP

Good Engineering Practice

GFC

Gel Filtration Chromatography

GLP

Good Laboratory Practice

GM-CSF

Granulocyte Macrophage Colony Stimulating Factor

GMO

Genetically Modified Organism

GMP

Good Manufacturing Practice

GPC

Gel Permeation Chromatography

GSP

Good Storage Practice

GSS

Gerstmann–Sträussler Syndrom

GTP

Good Tissue Practice

HCP

Host Cell Protein

HIC

Hydrophobic Interaction Chromatography

HIV

Human Immunodeficiency Virus

HPLC

High Pressure Liquid Chromatography (also High Performance LC)

HPMC

Hydroxypropylmethyl-cellulose

HSA

Human Serum-Albumin

HVAC

Heat Ventilation Air Conditioning

IaaS

Infrastructure as a service

ICH

International Conference on Harmonization

IEF

Isoelectric Focusing

JEC

Jon Exchange Chromatography

IEX

Ion Exchanger

IF

Interferon

IGG

Immunoglobulin G

IIoT

Industrial internet of things

IL

Interleukin

IMP

Investigational Medicinal Product

IMPD

Investigational Medicinal Product Dossier

IND

Investigational New Drug

IOM

Investigations Operations Manual

IPC

In-Process Control

IQ

Installation Qualification

IR

Infrared

ISO

International Organization of Standardization

ISPE

International Society for Pharmaceutical Engineering

JP

Japanese Pharmacopoeia

KPI

Key Performance Indicator

LADME

Liberation, Absorption, Distribution, Metabolism, Excretion

LAL

Limulus Amebocyte Lysate

LD

Lethal Dose

LES

Laboratory execution system

LFH

Laminar Flow Hood

LIMS

Laboratory Information Management System

LOD

Limit of Detection

LOQ

Limit of Quantification

MALDI

Matrix Assisted Laser Desorption Ionization

MBR

Master Batch Record

MCB

Master Cell Bank

MCO

Molecular Cut Off (MWCO)

MES

Manufacturing execution system

MF

Microfiltration

MHLW

Ministry of Health, Labor, and Welfare

MSA

Manufacturing and Supply Agreement

MTD

Maximal Tolerated Dose

MWCO

Molecular Weight Cut Off

NDA

New Drug Application

NIST

National Institute of Standards and Technology

NPV

Net Present Value

OOS

Out of Specification (QC Context) or Out of Stock (Logistical Context)

OQ

Operational Qualification

PAB

Pharmaceutical Affairs Bureau

PAGE

Polyacrylamid Gel Elektrophoresis

PAS

Prior Approval Supplement

PAT

Process analytical technology

PCR

Polymerase Chain Reaction

PD

Pharmacodynamics

PD

Plasma Desorption

PDA

Parenteral Drug Association

PEG

Polyethylene glycol

PFBS

Pharmaceutical and Food Safety Bureau

PharmEur

European Pharmacopoeia

PIC/S

Pharmaceutical Inspection Convention/Scheme

PK

Pharmacokinetics

PLC

Programmable logic controller

PM

Posttranslational Modification

PMDA

Pharmaceutical and Medical Devices Agency (KIKO)

PoC

Proof of Concept (PoP)

PoP

Proof of Principle (PoC)

PQR

Product Quality Review

QA

Quality Assurance

QAA

Quality Assurance Agreement

QC

Quality Control

QM

Quality Management

rFVIII

Recombinant Factor VIII

RNA

Ribonucleic Acid

ROI

Return on Investment

RPC

Reversed Phase Chromatography

RP-HPLC

Reversed Phase HPLC

RPM

Regulatory Procedures Manual

SCADA

Supervisory control and data acquisition

SDS

Sodiumdodecylsulfate

SEC

Size Exclusion Chromatography

SIP

Sterilization in Place (also Steaming in Place)

SKU

Stock Keeping Unit

SOP

Standard Operating Procedure

SPC

Statistical Process Control

SPC

Supplementary Protection Certificate

TEM

Transmission Electron Microskopy

TFF

Tangential Flow Filtration

TOC

Total Organic Carbon

TOF

Time of Flight

TSE

Transmissible Spongiform Encephalopathie

UF

Ultrafiltration

UML

Unified modeling language

URS

User Requirements Specification

USP

United States Pharmacopoeia

UV

Ultra Violet

WCB

Working Cell Bank

WFI

Water for Injection

WHO

World Health Organization

ZLG

Zentralstelle fur Gesundheitsschutz bei Arzneimitteln und Medizinprodukten

Part IIntroduction

1Biopharmaceutical Production: Value Creation, Product Types, and Biological Basics Introduction

1.1 Role of Production in Pharmaceutical Biotechnology

Over recent years, pharmaceutical biotechnology has developed very dynamically. An important driver for this success has been the enormous increase of scientific know-how in the areas of genetics and immunology, which has created huge expectations for the development of innovative medicinal treatments.

The scientific pioneer spirit has been fueled by public and private sponsorship, resulting in a biotechnological landscape that has long been dominated by highly innovative, venture capital-based, small- and mid-size companies. However, before patients can benefit from scientific achievements, it is necessary that the identified molecule is transformed into a medicine – fit for achieving the therapeutic target – and tested in comprehensive trials in the field. The production of such a medicine has to be carried out in officially licensed, often tailor-made technical manufacturing facilities.

From project to product

This path from project to product usually lasts several years and is associated with enormous costs and risks. On average, the development costs of a new compound are in the region of US$ 500–1000 million and only 10% of all projects that enter clinical trials find their way into the market.

Owing to these immense investments in drug development, the costs of drug manufacturing often seem acceptable, particularly as the costs are absorbed by sales of the marketed drug in the same accounting period; however, safe and efficient product supply is the cornerstone of a company's success. In biotechnology, the overlap between development and market launch is particularly intensive, motivating companies to take care of manufacturing early on:

Many targets of process development result from requirements of large-scale manufacturing.

The classical separation of development (pre-marketing) and production (post-marketing) does not work for biologics, as both the manufacturing process and plant are factors that determine the quality of the final medicinal product.

Production is the basis for long-term market supply. Decisions about capital investment or outsourcing of manufacturing mostly have to be taken long before the market launch of the product.

Biotechnological processes are much more difficult to control than small-molecule preparations. The limited ability to monitor and characterize the product results in increased manufacturing risks.

Significance of production in the value chain

The main target of production is to supply the product safely and cost-efficiently. It is positioned between the development and marketing of a product. Figure 1.1 illustrates its significance in the value chain.

The chain starts with research that has a clear focus on the identification of targets, which involves analyzing the interaction between the biochemical molecule and its potential therapeutic functionality. In the subsequent development phase, a process for the scale-up and more consistent manufacturing of the molecule is designed. Here, the target structure is developed into a pharmaceutical form and tested in animals and humans as to its safety and efficacy. Once this is achieved, production kicks in, taking care of a high-quality and profitable product supply, addressing the following main tasks:

When, where, and in what quantities should the drug be produced? (

Production concept

)

Figure 1.1 Role and tasks of production.

How should market supply be organized? (

Supply-chain organization

)

How should the quality of the product and

Good Manufacturing Practice

(

GMP

) compliance be assured? (

Quality assurance

)

What are costs of manufacturing and how can these costs be controlled? (

Manufacturing costs

)

How attractive is an investment in one's own facilities? (

Investment decision

)

The marketing of the product stands at the end of the value chain; from this position, essential goals are formulated for production: supply safety and cost efficiency.

Production is interdisciplinary

The integrated position of production in the value chain results in interdisciplinary tasks that are best treated by multilateral teams managed by experts in different disciplines such as biology, engineering, chemistry, economics, law, pharmacy, and medicine.

Figure 1.2 shows the subject areas that are important for the understanding and control of production processes and workflows. This volume provides an overview of these subject areas, while special emphasis is given to the interaction between these areas.

Figure 1.2 Subject areas in production. Inner circle = sections of this book; outer circle = subject areas treated in the sections.

Following this introductory part, Part II, “Technology,” focuses on processes and analytics. This section illustrates why the manufacturing process plays such a large role in biotechnology, and to what extent product quality is determined by processes and analytics. Moreover, essential technologies for industrial manufacturing as well as methods and areas of application of analytical testing are described.

Part III, “Pharmacy,” briefly elaborates on the basic principles of drug effects on humans and the essential steps of pre-clinical and clinical drug studies. The successful end of the clinical test marks the starting point of commercialization.

Product quality plays a crucial role in pharmaceutical manufacturing. Part IV, “Quality Assurance,” elucidates the organizational and operative workflows for quality assurance, including the rules of GMP.

Almost all activities of commercial production happen in the framework of legal regulations. Part V, “Pharmaceutical Law,” describes drug regulations and laws as well as institutions and enforcing official authorities.

The translation of process technology into large-scale manufacturing capacities is described in Part VI, “Production Facilities.” Basic principles of the design of GMP-compliant manufacturing facilities are given and different building concepts compared. The planning process that leads to industrial plants is illustrated. Here, we include a brief look at the regulations regarding health, safety, environment, and construction that form the legal framework of industrial production facilities.

Commercial thinking is the spine of efficient production. Part VII, “Economy,” introduces essential principles around product sales and cost of goods accounting. It compares concepts of in-house manufacturing with outsourcing strategies and elucidates the decision factors leading to capital investments in biotechnological plants.

The book closes with a Bibliography providing literature and web references and an appendix providing a list of abbreviations and an alphabetical index of keywords.

1.1.1 Relationship Between Production and Development

It is widely understood that production starts when development provides a marketable product and a commercially feasible manufacturing process. Ongoing market supply is secured by process optimization or the provision of additional manufacturing capacities, depending on how market demand develops. For biotechnological pharmaceuticals, the flexibility to react to demand changes is reduced due to the following reasons.

Drug application and the manufacturing process are described and fixed in the regulatory license. As the biotechnological manufacturing process is a quality-determining factor, it has to be finally defined at the time point of regulatory submission and can thereafter be changed only with relatively high effort. The market application contains proof of the safety and efficacy of the drug; it adds to the complexity that in biotechnology, this proof has to be made – at least partly – with material from the commercial process and manufacturing site. Changes to the process or site require comparability exercises that can be more or less complex depending on the risk associated with the change. All of this means that the manufacturing process is fixed at a relatively early time point during development and can only be changed with quite some effort.

Clinical and process development

This coherence is illustrated in Figure 1.3. Product development consists of clinical development, on the one hand, and development of the manufacturing process and the analytical methods, on the other hand. The clinical development renders proof of safe and efficacious use of the drug in humans. Ideally, this proof is generated with material from the process and the site designated for commercial supply. There is a challenge with this ideal approach: if the process would be finally established and only after that clinical development be initiated, the timelines of development would add up unacceptably. Therefore, the different branches in the development workflow occur in parallel; different stages of the clinical development are supplied with different development stages of the manufacturing process.

Figure 1.3 Relationship between production and development.

From lab to large-scale process

Validation and critical parameters

Coming from the laboratory scale, the process is evolved step by step into the final and mature manufacturing process. The scale and maturation a technical process achieves until first used for commercial supply depend on the characteristics of the process, on the requested product demand, and, often, on the available time for development. At the endpoint of development, the process is implemented in the designated commercial supply facility. Product generated in these so-called full-scale runs must be used in representative amounts in the clinical trial. Process validation shows that the drug can be manufactured reproducibly and in good quality under consideration of applicable operating procedures. Product generated in these so-called “validation runs” must usually be used in representative amounts in the clinical trial.

An “easy-to-validate” process means that product quality is essentially independent of fluctuations of the critical process and equipment parameters. Critical parameters as well as measures to control them should be identified in the lab-scale process. These links result in interactions between production and development long before the actual supply to the commercial market.

Role of analytics

Owing to the heterogeneous composition of biological pharmaceuticals, analytical methods play a special role. Just like the process, the developed methods find their way into the regulatory license documentation. Concurrently – while being optimized itself – analytics has to support process development from very early on. Production requirements such as speed, robustness, and simplicity of testing methods have to be taken into account. Moreover, it has to be decided which method should support processing, which method is necessary for product characterization and process validation, and which method should be used only in the development phase.

This short outline illustrates how deeply the aspects of production reach into the development phase. An early recognition of production aspects can help to avoid detours and project delays.

Production facilities

An important interface, that is, not shown in Figure 1.3 is the one to the facility in which the manufacturing process is carried out. The capital investment in a manufacturing plant, and also the alternative contractual obligation with an outside source, means an additional financial risk of considerable size. This issue is further discussed in Part VII.

1.1.2 Relationship Between Production and Marketing

Production makes the final and packed product available for marketing (Figure 1.4). The packaging provides product protection and a possibility for attracting customers; especially in the pharmaceutical arena, the packaging contains a considerable amount of user information. The coordination and distribution of the country-specific final products is done by production logistics, which has to react flexibly to requirements from sales and marketing.

Figure 1.4 Relationship between production and marketing.

Life-cycle projects support the development of the project in the market place and usually affect not only the pharmaceutical dosage and form but also the indication of the product. In these cases, production has to adapt to changes in demand, packaging materials, or formulation processes.

Cost aspects

The acceptable manufacturing costs are determined by the achievable price in the marketplace, which is often regulated by country-specific reimbursement systems. The construction or maintenance of manufacturing plants has to be justified by adequate profitability calculations that are based on estimates and expectations of the market situation, and the desired profit margin, on the one hand, and the operating and capital expenses, on the other hand. These projections often reach far into the future (more than 10 years) and leave large room for variations.

Specialties of the pharma market

While the aforementioned characteristics also apply to other goods, there are indeed pharma-specific features, for example, the governmental monitoring system, the exceptionally high ethical responsibility of pharmaceutical companies, and the official regulation of drug reimbursement. Safety of patients is guaranteed by instruments used for pharmacovigilance and intensive product quality assurance. Pharmacovigilance systems serve to register unforeseen adverse effects of drugs and route them to the supervisory body. To achieve this goal, the pharmaceutical company collects and evaluates blinded patient data; in case of an unforeseen adverse event, a root-cause analysis has to be performed. To perform this analysis, it is necessary that the specific medicament used by the patient can be traced back to the manufacturing site and batch. It is the specific batch documentation that then provides insight into whether deviations have occurred during the operation that might have influenced the quality of the product. If yes, it needs to be clarified in a second step whether such a quality variation could have triggered the adverse reaction. Thus, the requirements of pharmacovigilance lead to a comprehensive documentation obligation of the entire manufacturing process.

The target of pharmacovigilance is to recognize risks retrospectively. As a complement to the framework of drug safety, there are intensive measures for prospective quality assurance. This has a significant impact on the operational workflows as will be shown in Part IV.

1.2 Product Groups

Pharmaceutical biotechnological products can be classified into:

Vaccines derived from non-genetically modified organisms or blood.

Therapeutics from blood or animal organs (e.g., Factor VIII and insulin).

Antibiotics manufactured traditionally in biological processes. Usually this is done with non-genetically modified organisms.

Recombinant proteins (i.e., active ingredients) derived from cultivation of genetically modified cells. Including monoclonal antibodies, these represent the biggest sector of current pharmaceutical biotechnology.

A new branch of therapy opens up with the possibilities of cell and gene therapeutics. These complex interventions into the human body require the reassessment of the pharmaceutical safety concept and demand special precautions from production technology and engineering.

Manufacturing technologies of different product groups can be similar

The focus of the present work lies in the production of therapeutic recombinant proteins; however, the principles described can be applied to the other product groups as well. A closer look at the groups reveals interesting therapeutic and technological overlaps. For example, innovative gene therapy can learn from experiences in virus production gathered in the conventional vaccine field. Also, vaccines will face a new era due to the possibility to produce monoclonal antibodies (Section 1.3.2.1). In the following text, the product groups – with the exception of antibiotics – will be covered in more depth.

Figure 1.5 Schematic production workflows of important product groups. Product groups are shown on the right. Attenuation = elimination of reproducibility, but retention of infectivity; inactivation = elimination of reproducibility and infectivity.

Figure 1.5 schematically shows the production workflows for different product groups. There are differences regarding the genetic modification of the starting material. Genetically modified organisms are mainly deployed for recombinant proteins and gene therapeutics, but cell therapy can also use this technology. The products can be proteins, viruses, bacterial fragments, cells, or intact viruses for gene therapy.

1.2.1 Vaccines

There are two principles of vaccination:

Passive vaccination

: Antibodies against the pathogen are administered.

Active vaccination

: The immune system is confronted with alleviated pathogens and builds up its own immune defense against the causative organism.

Antibodies for passive vaccination are prepared by injecting the pathogen into animals. The immune system of the animals pours out so-called polyclonal antibodies into the blood system. Blood is collected from the animals, and the antibodies isolated and purified, so that they can be administered to humans.

Active vaccination uses inactivated germs that are no longer pathogenic, but still immunogenic. Activation allows the immune system to recognize the real pathogenic germs much faster and therefore fight them before they can spread out and cause the illness. It suffices to present only a moiety instead of the whole pathogen to enable the immune system to recognize the substance foreign to the body. This moiety can be the hull protein of a virus, whole inactivated cells, or pathogen-specific deoxyribonucleic acid (DNA). The general term for these immune response-inducing agents is “antigen.” Active vaccines like the influenza vaccine can be proliferated in chicken eggs and reworked to vaccines.

1.2.2 Pharmaceuticals from Blood and Organs

Many diseases can be attributed to the lack of certain proteins in the blood. In part, these proteins can be extracted from animal or human blood or organs, such as insulin against diabetes or Factor VIII against bleeding disorders. Biotechnology has made it possible for these proteins to be obtained without being tied to these expensive and – under aspects of safety – questionable raw materials from natural sources. In some cases, blood-derived products still play a role as it has not yet been possible to successfully replace them completely by recombinant proteins.

Plasma fractionation

Risks of protein extraction from blood

To isolate the proteins from the blood, it is first separated into its two main components: plasma and cells. The plasma is further fractionated to obtain the proteins. It is associated with considerable analytical and organizational effort to guarantee the safety of the raw material blood, especially the absence of viral contamination and transmissible spongiform encephalopathy (TSE)-inducing components. Despite the intensive surveillance of blood donors, the danger of safety-relevant incidents persists. It can be expected that the production of proteins will be more and more shifted to recombinant technologies, while whole-blood donations will remain irreplaceable for patient treatment in hospitals.

1.2.3 Recombinant Therapeutic Proteins

Recombinant proteins, including monoclonal antibodies, by far, make up the largest group of biotechnological pharmaceutical products. Table 1.1 shows some examples; in addition to the medical indication and the functionality in the human organism, it provides details regarding the size and type of the molecule. Section 1.3 gives further insight into the structure of proteins and the terms of amino acids and glycosylation.

A huge growth potential is expected for monoclonal antibodies and antibody fragments.

The starting point for all protein production is the genetic modification of the host cell in which the protein should be expressed. The endpoint usually is a parenterally (per injection) administered product in liquid or solid form.

1.2.4 Cell and Gene Therapeutics

Cell therapy: implantation of intact cells

Therapeutic proteins are administered to compensate for the lack of the respective natural protein in the organism. As the molecule is eliminated either by degradation or excretion, the administration has to be repeated to achieve a constant active agent level. In contrast, cell and gene therapy is based on the idea of fighting the disease at its source and enabling body cells to express the missing proteins by themselves. Figure 1.6 schematically illustrates the differences between the philosophies of protein versus cell and gene therapy treatment. The starting point is a disease caused by a lack of the example protein X. In conventional therapy, the protein is produced ex vivo and injected into the patient. Owing to elimination processes, the protein disappears after a while. In gene therapy, a genetic sequence is introduced into the body, which contains the construction plan for the desired protein as well as the capability to infect suitable target cells. This combined capability is generated by means of biotechnological methods: the protein-encoding gene is linked to a molecular “ferry” (or vector) that carries the gene into the designated target cells. This ferry is a virus that has been modified in such a way that it retains its infectivity but lacks its ability to replicate. After having been infected the cells start to produce the desired protein. In the ideal case – if the construct is genetically very stable and the expression rate high – this process has to be carried out only once. If the modified viruses are injected directly into the body, it is called in vivo gene therapy, but it is also possible that the cells are extracted from the patient and re-implanted after being infected in the lab (ex vivo gene therapy). The latter is basically a crossover between cell and gene therapy.

Table 1.1 Examples for recombinant proteins.

Name

Indication

Functional group

Number of amino acids; glycosylation, and fraction of sugars of molecular weight; molecular weight

Insulin

Diabetes

Hormone

AA 51; Gly no; 5.8 kDa

Human growth hormone

Dwarfism

Hormone

AA 191; Gly no; 22.1 kDa

Factor VIII

Bleeding disorder

Clotting factor

AA 2332; Gly to 35%; 300 kDa

Lepirudin

Thrombosis

Anticoagulant

AA 64; Gly no; 7 kDa

Tissue plasminogen activator

Thrombosis

Thrombolytic agent

AA 72; Gly to 25%; 72 kDa

Interferon

s (

IFN

s)

Diverse: multiple sclerosis, hepatitis, arthritis, and so on

Immune modulator

IFN-β: AA 166; Gly yes and no; 18.5 kDa

Interleukin

s (

IL

s) (13 different types)

Diverse: asthma, HIV, cancer, mucositis, and so on

Immune modulator, signal agent between immune cells

IL-2: AA 133; Gly yes; 15.5 kDa

Erythropoietin

Anemia

Growth factor

AA 165; Gly to 40%; 34 kDa

Granulocyte colony-stimulating factor

(

G-CSF

),

granulocyte macrophage colony-stimulating factor

(

GM-CSF

)

Infections, cancer

Growth factor

G-CSF: AA 174–180; Gly yes; 19.6 kDa; GM-CSF: AA 127; Gly yes, 15.5, 16.8; and 19.5 kDa

Monoclonal antibodies

Cancer, transplantation, and so on

Antibodies

IgG: AA about 1300; Gly yes; about 150 kDa

Gly = glycosylation; AA = amino acids; Gly to 30% = molecular weight fraction of glycosylation can reach up to 30%.

Figure 1.6 Schematic comparison of traditional therapy and cell and gene therapies.

The basic principle of cell therapy is to convey cells to the body that have the desired functionality; thus, cell therapy does not aim at repairing dysfunctional cells, but rather replacing them. These cells can originate from animals (xenogenic) or humans, either patient proprietary (autologous) or non-patient proprietary (allogeneic). The allogeneic and xenogenic approaches raise questions regarding immunogenic responses, comparable to tissue rejection in organ transplantation. If the cells are genetically modified, they belong to transformed cell lines; cells that have not experienced any genetic modifications are called primary cells. Thus, “allogeneic cell therapy with primary cells” denotes a therapy in which cells of a foreign donor are implanted without genetic modification into the patient. The functionality of cells is not restricted to protein production – other molecules can also be expressed. A prominent area of cell therapy is the replacement of tissue or organs (tissue engineering). Consequently, the widely discussed therapy with human stem cells is only one form of cell therapy, which is characterized by the adoption of non-differentiated stem cells.

Cell production for therapy

Cell therapy production starts with isolating the cells intended for implantation. This is clearly a crucial issue of cell therapy, as the starting material is limited and the subsequent expansion of the cells is restricted by the natural limit of generation numbers. The implantable cells are available for surgical implantation after an additional manipulation step such as washing or buffer exchange.

Gene production for therapy

The genetic construct for gene therapy is produced by proliferating the DNA in a host organism that can be first expanded and subsequently transfected with a modified virus. This infected cell produces the desired virus. To control infectivity, the virus can be weakened (attenuated); at the end of this production process, the product consists of the attenuated, modified virus.

Safety questions around cell and therapy

Cell and gene therapies are at the advent of their development. Despite the fact that the approaches seem plausible, complex questions around drug safety arise. The administration of generally replicable and propagatable substances is very different compared to conventional protein therapy. The scarce source and the handling of the living “cell” system impose new challenges on production and distribution processes.

1.2.5 Antibiotics

Antibiotics have been produced biologically for many decades. A penicillin-producing yeast strain is cultivated in a biological fermentation step, and the expressed penicillin is further purified into a pharmaceutical product. In particular, fermentation resembles that with recombinant microbial expression systems (Section 2.3.1). In contrast, purification is different because penicillin is a relatively small and robust molecule compared to proteins.

1.3 Basics of Biology

This section is dedicated to some basic principles of biology and biochemistry that are relevant for the understanding of production processes on the level of this book. After a short outline of cell biology and microbiology, the four basic molecular entities of biochemistry are introduced: proteins, nucleic acids, polysaccharides, and lipids.

1.3.1 Cells and Microorganisms

Each form of life – plants, animals, or microorganisms – consists of biological cells. While plants and animals constitute themselves as enormous networks of different cell types, microorganisms are predominantly single celled.

Microorganisms can survive in their selected habitat independently, while cells of higher organisms depend on their united cell structure. Cells are characterized by some general features:

Cells contain a carrier of genetic information (double-helical DNA) and a single-strand

ribonucleic acid

(

RNA

) derived thereof. During replication – in the process of propagation – its genetic information is prone to erratic variations (mutation).

Cells exchange nutrients and waste products with the environment (metabolism) for the purpose of energy recovery (catabolism) or substance construction (anabolism).

They are confined by a membrane that allows for controlled substance exchange with the environment.

They communicate via so-called receptors with the environment and can react to changes in external conditions.

They are capable of replicating themselves and a number of higher cells differentiate into other cell types.

In addition to genotypic characterization of cells and organisms, based on gene technology, there is a phenotypic characterization based on the differences in shape, movement pattern, staining (Gram staining), metabolic pattern, and preferred habitat.

Mycoplasmas are special bacteria. They do not possess a cell wall and are exceptionally small. Obviously, they are resistant against types of antibiotics that attack cell walls. As with viruses, they are not retained by filters of a pore size of 0.22 μm. Some members of this family are pathogenic (e.g., Mycoplasma pneumoniae, Mycoplasma genitalium).

Viruses are not cells but consist of encapsulated genetic information in the form of DNA or RNA. They do not have their own metabolism and depend on other biological cells for their replication. Many representatives of this group are pathogenic [e.g., human immunodeficiency virus (HIV), hepatitis, and herpes].

1.3.1.1 Structure and Types of Cells

There are two types of cells: the simple prokaryotic cells and the more complicated eukaryotic cells.

Chromosome and plasmids

Structure of Prokaryotic Cells  Prokaryotic cells consist of a cytoplasm surrounded by a membrane, which is itself surrounded by a stabilizing cell wall. The most important functional units embedded in the cytoplasm are the ribosomes, the chromosome, and the plasmids (Figure 1.7).

Figure 1.7 Schematic of a prokaryotic cell.

Inclusion bodies and secretion

The chromosome is a single-stranded DNA double helix and contains the genetic information for the construction and replication of the cell. Protein biosynthesis from the DNA happens at the ribosomes after the DNA information has been transcribed into RNA. In addition to the chromosome, prokaryotes often carry further genetic information in the so-called plasmids. These are ring-shaped DNA molecules located in the cytoplasm. They usually encode secondary functional proteins such as the substances enabling penicillin resistance. The inclusion bodies shown in Figure 1.7 are storage locations for substances that for the time being are not required.

Gene expression

Inclusion bodies