Gel Filtration and Size Exclusion

It is useful to use the term size exclusion to describe the type of gel filtration used to separate proteins by molecular weight. Size exclusion chromatography can be a useful purification tool in separating by size two proteins that may have similar charges and so co-purify on ion exchange. However, for this technique to be efficient, it is reliant on a concentrated feedstock, and a very large column may be required as the load volume is often only about 1-2 of the column volume. This...

Validation of filtration

The primary goal of validation of a filtration process is to prove its ability to remove contaminants this is performed in different steps (Docksey et al. 1999 PDA 1998a). First the performance of the filter per se should be validated via bacterial-retention testing (usually using B. diminuta, as discussed in Section 14.2.2.1) as well as extractable, compatibility and adsorption studies it is common to have the tests conducted by the filter manufacturer at laboratory scale. Furthermore, the...

References

Akhnoukh R, Kretzmer G, Sch gerl K (1996) Enzyme Microb. Technol. 18 220-228. Bailey JE, Ollis DF (1986) Biochemical Engineering Fundamentals. McGraw-Hill Book Company, New York Second edition 661-666. Baker KN, Rendall MH, Patel A et al. (2002) TIBTECH 20(4) 149-156. Behrendt U, Koch S, Gooch DD, Steegmans U, Comer MJ (1994) Cytotechnol. 14 157-165. Bestetti C (2002) Pharmaceutical Eng. 22(6) 90-98. Beyeler W, DaPra E, Schneider K (2000) Adv. Biochem. Engin. Biotechnol. 70 139-164. Bilitewski...

Key Metabolites 13241 Introduction

D-Glucose, L-lactate, ammonia and L-glutamine, as well as several other L-amino acids, have long been recognized as being key metabolites in animal cell cultivation and have thus been routinely monitored, first off line, using enzymatic and HPLC methods, and now increasingly using on-line techniques. There are of course numerous other chemical species that play a role in the metabolism of animal cells, such as vitamins, lipids, peptides, various growth factors, etc. These species are rarely, if...

The Validation Master Plan VMP

In the opinion of the author, this document is frequently made far too long. The Rules Governing Medicinal Products in the European Union Volume 4, under Annex 15, Planning for Validation, state that 'The VMP should be a summary document, which is brief, concise and clear.' (European Commission 2001a) Too often, VMPs contain large amounts of information and a great deal of repetition, little of which facilitates the intended function of the document, namely to lay out the requirements and...

Formulation design optimization

The elevated cell densities and extended bioreactor campaigns required to improve the spacetime utilization efficiency of a manufacturing suite for cost-effective production of human and veterinary biomedicines necessitate both quantitative and qualitative changes in composition of the nutrient medium (Jayme et al. 1998, 1999). Constituent levels adequate for short-term incubation or at low cell inoculation density may provide insufficient buffering capacity or metabolic substrates under pilot-...

Overview Of The Aims Of Purification

The use of biological medicines in humans requires that they are purified to standards determined by regulatory authorities primarily to ensure patient safety, but also to ensure that product is consistent and effective. The standards relating to purification are used to determine the permissible levels of impurities in the administered product, and hence the specifications that are set for the product during development and manufacture (Seamon 1998). In beginning to devise a purification...

Batch versus Fed Batch and Perfusion Culture

Since many early animal cell culture practitioners had transitioned from bacterial fermentation experience, it was logical that early bioproduction protocols mimicked bacterial batch fermentation processes (Kadouri & Spier 1997). The batch production process facilitates materials handling and regulatory definition of what constitutes a 'batch' of product, since all of the raw materials including cells are placed into a single bioreactor and the target product is harvested following an...

Ultrafiltration Control Strategies

Ultrafiltration processes offer some choices in control strategies, depending on the type of process control and the level of automation desired. One of the simplest strategies involving the least amount of automation is to operate at a constant retentate pressure or constant transmembrane pressure. Constant retentate pressure processes maintain selected feed flow rate and retentate pressures throughout the process while the filtrate rate and filtrate pressures are uncontrolled. Similarly,...

Final Product Testing

Serum suppliers test the final product against written specifications and provide certificates of analysis (CoA) for each lot of serum produced. As no pharmacopoeial monograph exists for serum, specific tests performed and reported on the CoA may follow existing test protocols found in the United States Code of Federal Regulations, or the United States, European or other pharmacopoeias, e.g. tests for sterility and mycoplasma. Where no guidance exists, such as for growth promotion testing,...

Oncoviral Vectors

MuLV) vectors, all viral genes are removed from the viral genome between the long terminal repeat (LTR) sequences and replaced by a transgene and its regulatory sequences (insert) of up to 6-8 kb in size (Naviaux & Verma 1992). Oncoviral vectors (OV) based on MuLV are produced as follows (1) a murine fibroblast cell line is first transfected with a 'helper-retrovirus' genome lacking the packaging signal psi (y), which is necessary to pack retroviral RNA into viral...

Design Purification Protocol

The purification protocol is central to establishing the safety profile of the final product. The choice of specific purification processes should be based not only on product purification, but also with the elimination or reduction of process residuals and adventitious agents in mind. (see Chapters 18 and 19) Preliminary purification schemes may be designed to reduce process residuals in order to meet Phase I clinical timelines, but adventitious agent reduction is a requirement. Common process...

Multiproduct Cultivation Equipment

To contain costs and to respond quickly to the ever-changing needs of the market, it is desirable to build multi-product facilities, where two or more products can be produced with the same, or shared manufacturing equipment. The main concern of regulatory authorities is the risk of cross-contamination between the different products. The important issues to address with multi-product manufacturing facilities are therefore the engineering design, the procedural, temporal or spatial Figure 14.11...

Adenoassociated Viral Vectors

One of the most effective ways to deliver genes into cells is to enclose plasmids within viral capsids that efficiently bind to target cells. Certain non-enveloped viruses with relatively simple genomes offer the means for manufacture of 'capsid plus plasmid' viral vectors. Among such viruses, par-voviruses have been those most frequently exploited for this purpose. Adeno-associated viruses (AAV) are small single-stranded DNA parvoviruses of which there are six known human sero-types. AAV-2,...

Integration of upstream and downstream processes

Misalignment of upstream and downstream processes can result in costly delays and yield losses. Development and optimization of the nutrient medium should not proceed independent of subsequent processes that may also be impacted. As suggested above, a nutrient formulation optimized in tissue culture flasks or other small-scale, relatively uncontrolled, environments may be sub-optimal within a bioreactor. Homogeneous bulk-phase bioreactors, such as a stirred tank reactor, will demand different...

Physical containment barriers for mammalian cell culture

In the US, mammalian cells used for the production of recombinant proteins, such as CHO cells, normally require a BL1 level in the laboratory and a GLSP level at large-scale. In the EU, containment level 1 normally applies. Human and other primate cells, on the other hand, should be handled in a BL2 BL2-LS facility. For both the US BL1 level and the EU containment level 1 at small scale, all primary and most secondary containment barriers are optional. Standard microbiological practice should...

Impurities In Water Sources

Purified water is usually produced by the multi-stage treatment of a potable water supply. Potable water is sourced from a combination of surface water, river water and underground aquifer. Impurities originally present can be divided into dissolved ionic matter, organic compounds, particulates, colloids, and a range of bacteria and other life forms. Dissolved salts are leached into the water from rocks or soil - calcium, sodium, bicarbonate, chloride and sulphate are the most common ions...

Design of the steam circuit

During sterilization, a large number of valves have to be opened and closed in complex specific sequences. To ensure that this is performed reproducibly, automation should (as for CIP), be an essential part of a modern SIP system. For the sterilization of vessels, the steam inlet should be located at the top of the bioreactor, whereas condensates should be eliminated from the bottom through a drain valve and diverted to a stream trap, or flow orifice, to regulate the flow (Oakley 1994). To...

User Requirement Specification

Ultimately, validation is a service, normally performed for an end-user or users who actually require the functioning system - usually to a timescale that does not take into account the actual time required to create, execute, review and report the validation requirements One way to get buy-in from the end-users, and also ensure that they get the desired equipment facility, is to involve them in the creation of the user requirement specification. This document should be prepared once the VMP...

For Transfection Of Mammalian Cells Gene Therapy And Cell Culture Applications

The use of baculoviruses as a method of gene delivery to mammalian cells was first evaluated for its potential in gene therapy as a safer alternative to adenovirus, retroviruses, and other viral vectors. Hofmann et al. (1995) and Boyce and Butcher (1996) reported that genes incorporated into baculovirus under the control of a strong mammalian promoter (Rous sarcoma virus, RSV, promoter and cytomegalovirus immediate early, CMVie, promoter) were efficiently expressed in liver cells (primary...

Removal of viruses

Several cases of viral contamination of bioreactor contents by adventitious viruses have been reported in the past few years. The sources may be raw materials, even process gases, GMP failures by operators, or equipment barrier breaches, and are, in any case, very difficult to identify (Garnick 1998). Viral contamination can often spread to several tanks before it can be detected, unlike bacterial contamination. Liu et al. (2000) estimated an overall cost of 6-8 million US for a viral...

Sterilization of Culture Media and Supplement Solutions 14221 Sterilization by filtration

Most culture media and supplement solutions for mammalian cell culture are heat-sensitive and must therefore be sterilized by filtration, as described below. Autoclaving and steam sterilization in place are also discussed briefly in Section 14.2.2.4, for the few cases where this method is applied to solutions in mammalian cell culture. A sterilizing filter is defined as a filter that, when challenged with the microorganism Brevundi-monas diminuta, formerly called Pseudomonas diminuta (ATCC...

Membrane Adsorbers

Some suppliers, such as Sartorius, Pall and Millipore, now sell membranes that have been given surface properties like those of chromatography beads. These membrane adsorbers (Gottschalk Figure 18.8 The Pall Centrasette system can be directly scaled from a 0.01m2 scale-down model to industrial production scale with membrane areas of 80m2 or more, and capable of processing around 25,000L. (Above) (a) Pilot scale ultrafiltration equipment used in the concentration and diafiltration of a live...

Higher Cim Levels 1341 Supervision Level

In complex manufacturing facilities, the coordination of PLCs, to automate operations involving several units (e.g. tanks) at the same time, may become extremely complex. At the limit, each PLC would have to mirror the status of all other involved PLCs for a proper synchronization. This challenge has lead to the development of distributed control systems (DCS), where a supervision tool (often termed supervisory control and data acquisition (SCADA) application) controls a group of separated...

Inoculum Expansion and Production Steps

Very limited information is available in the public domain regarding the general design and sizing of an upstream processing line for pilot- or large-scale production. Chu and Robinson (2001) present an overview of the technologies (bioreactor type and mode, medium type) for the various commercial products recently approved (recombinant proteins, vaccines, diagnostics and tissue culture), but few details on the scale of the cultivation vessels are actually available. In the following, typical...

FDA Biological License Approvals 19962003

Table 5.2 lists Biological License Approvals (BLAs) granted by the United States Food and Drug Administration (FDA) for biotechnology products derived using recombinant DNA technology and mammalian cell lines over the period January 1996 to July 2003 (derived from Chu & Robinson 2001, and updates from the FDA website). There are two categories Recombinant Therapeutics and Diagnostic Products products such as inactivated or live viral vaccines and natural products derived from cells or blood...

Design of a CIP unit

A CIP system is typically made of one or several CIP units or skids, each being dedicated to a set of process equipment parts (e.g. tanks and associated piping) to be cleaned in place. The number of units in a plant should be based on the maximum rate of utilization of the process equipment and the estimated duration of one CIP cycle. Usually the units are independent although they are fed with concentrated cleaning and rinse solutions from common storage tanks. A CIP unit consists of one or...

Expression Of Hiv Envelope Proteins An Hivaids Vaccine

It is no exaggeration to state that a greater effort has been committed to developing an effective vaccine against HIV than any other infectious agent. It is perhaps fortunate that the emergence of AIDS coincided with the blossoming of recombinant DNA technology and the promise of unlimited supplies of exquisitely characterised antigens, carefully honed to potentiate the components of the human immune system associated with HIV neutralisation or clearance. Unfortunately, despite 20 years of...

Analysis of Thermal Properties

The thermal properties of a formulation to be used for freeze-drying may be determined by a number of analytical methods. Several common methodologies are reviewed here but this list is not exhaustive. 20.4.1.1 Differential scanning calorimetry Differential scanning calorimetry (DSC) is a powerful method for the detection of eutectic points and some glass transition temperatures. Although the quantity of sample that can be tested is small (typically less than 100 il), and hence the thermal...

Ultrafiltration Principles And Theory

Protein Ultrafiltration

During UF, pressure (retentate pressure) is applied on the upstream side of the membrane that forces the filtrate to flow through the membrane. The flow rate per unit area, or flux, J, is Figure 17.5 Industrial-scale ultrafiltration systems for concentration of proteins produced in cell culture fluid. (Reproduced by permission of Millipore Corporation.) Figure 17.5 Industrial-scale ultrafiltration systems for concentration of proteins produced in cell culture fluid. (Reproduced by permission of...

Inactivation of biological wastes

Mixing Tank

At laboratory scale, manual autoclaving of biological solid and liquid waste is the simplest and most common method alternatively, chemical sterilization using, for instance, iodine- or chlorine-containing agents can be performed. This second method is recommended for small-scale cultivation vessels, since heat treatment before washing may cause accumulation of cell debris as solid residues on surfaces. In a facility where a central liquid waste decontamination system is built for large-scale...

Depth Filtration

Filtration Depth

One of the more readily implemented cell separation techniques is normal-flow depth filtration. Depth filters employ the conventional filtration technique of separating cellular solids from the cell culture fluid by forcing the liquid through a porous medium in dead-end or normal-flow mode. The medium retains solids and the liquid flows through to a collection vessel. Especially well suited for smaller batch volumes (> 2000 l), depth filtration can be an economical and easy-to-use primary...

Ultrafiltration Operation And Process Development

Diafiltration

Since the concentration of proteins in biological systems involves fairly complex fluids with unknown fluid dynamic behaviour, process development depends almost entirely on empirical data collection. Although several models do exist to help the process scientist, it is typically too risky to rely exclusively on them when implementing a UF DF process for high-value products. Therefore, process development involves conducting small-scale experiments with representative Diafiltration Retentate...

Normalflow Versus Tangentialflow Filtration

Membrane separations are termed either normal-flow or tangential-flow, referring to the direction of feed flow relative to the membrane surface. Figure 17.1 illustrates the flow paths for normal (a) and tangential (b) filtration. In normal-flow operation, the feed and filtrate flow perpendicular to the surface where the retained molecules are deposited. In tangential-flow operation, the feed flow is parallel, or tangential, to the membrane surface and the filtrate flow is perpendicular to the...

Process Range Justification

One key role during development is to provide sufficient data to allow an appropriate selection of the critical process parameters that will be used in process validation and to justify the range of specifications for each of the process parameters, which we refer to here as process range justification. Traditional examples of range justification are described by Schenerman et al. (1999) and Moran et al. (2000). Schenerman et al. (1999) performed studies to examine three critical process...

Validation of CIP

All cleaning procedures need to be validated to demonstrate their efficacy in maintaining a plant in a hygienic condition and preventing cross-contamination. The validation of CIP implies both the qualification of the cleaning equipment and the validation of the cleaning cycle. Prior to starting the CIP validation, however, the qualification of the associated utilities (WFI, compressed air, etc.) should be completed. The discussion below focuses on the steps that are specific to CIP. The first...

Freeze Drying Principles

Lyophilization is the sublimation of ice from a frozen material to leave behind a stable dessicated product. For practical purposes most of the lyophilization process is performed at sub-ambient temperatures under vacuum, to ensure that the material remains completely frozen and to enhance the sublimation rate while protecting the biological material from thermal damage. The lyophilization process can be seen as comprised of three basic steps (Figure 20.1) freezing, under conditions that will...

Operational Qualification

On completion of the IQ, the operational qualification (OQ) can commence. OQ tests the operation of the equipment against predetermined acceptance criteria. The PIC S guide offers the following requirements for OQ (PIC S 2004b) 'Studies on the critical variables (parameters) of the operation of the equipment or systems will define the critical characteristics for operation of the system or sub-system'. Put simply, the OQ protocol should provide evidence to demonstrate that the system is capable...

Design of the CIP circuit

Bioreactor With Sip

In traditional CIP systems, solutions were circulated via numerous supply and return lines, separate from process lines however, as previously mentioned (Section 14.3.2.6), it is desirable, for economic and practical reasons, that a major portion of the tubing, fitting and valves be shared between the CIP, SIP and process circuits. CIP circuits have thus evolved towards a higher integration level with that of the process, as reviewed in Seiberling (1992) manual and ring transfer panels are...

Transfer systems

Flow Transfer Panel

For efficient plant utilization, it should be possible to perform cleaning and sterilization operations at the same time on adjacent pieces of equipment, without any risk of cross-mixing. This is actually part of the 3-A Accepted Practices for Permanently Installed Sanitary Pipelines and Cleaning Systems (Stewart & Seiberling 1996). For instance it should be possible to clean a bioreactor together with its transfer line immediately after use, and then to sterilize it in preparation for the...

Types Of Serum

A listing and the descriptions of commonly available sera is shown in Table 4.1. FBS is considered the serum of choice as it has the strongest growth-promoting capacity along with the lowest immunoglubulin (IgG) level. It takes between one and three bovine foetuses to yield a single litre of raw serum. In drought years, ranchers take larger numbers of cattle to market for slaughter to save on feed costs, therefore more material becomes available for processing. Conversely, as herds are...

Equipment and Scaleup

Column Packing

The availability of hardware at the required scale, or the complexity of its deployment, may influence which techniques are used and how they are implemented. Large-scale engineering, for example of flow distributors, can be a critical issue. The difficulties of large-scale operation can be balanced against the cost of running multiple cycles at a Figure 18.3 Pilot-scale columns used in Phase II clinical manufacture. A 30-litre size exclusion column and a 2-litre ion exchange column used for...

Formulation Choices in Freeze Drying 20321 Cryoprotectants

Damage during lyophilization can be of two fundamental types, damage during the freezing process and damage either during the dehydration process or on reconstitution. Freezing introduces a concentration of the biological and the excipients surrounding it as the available water is preferentially compartmentalized into ice crystals. Eventually a glassy state is achieved where the biological is maximally concentrated. This may result in marked changes in the local concentration of excipients (for...

Regulatory And Manufacturing Strategy

Prior to starting process development, one should review the regulatory and manufacturing strategy, as these considerations may dictate the course of process development. Primary considerations Table 11.1 Resources for regulatory guidelines. Regulatory agency FDA Center for Biologies Evaluation and Research (CBER) FDA Center for Drug Evaluation and Research (CDER) European Medicines Agency (EMEA) World Health Organization International Conference on Harmonization (ICH) safety...

Principles of biosafety regulations

There are numerous regulations and guidelines on biosafety and containment requirements for the cultivation of mammalian cells, with some differences between the US, the EU and other countries. Their analysis is further complicated by the fact that they are regularly updated, reflecting the continuously expanding knowledge and experience with recombinant DNA technologies. In the US, some seminal guidelines were published by the National Institutes of Health (NIH, 1976) to provide a framework...

Trends In Biopharmaceutical Facility Design

The biotechnological revolution is bringing in new pharmaceutical products including those for gene therapy, skin and cartilage from cultured tissue, and novel vaccines. There is also the possibility of tailoring drugs to suit small groups of people. However, there are major challenges in the processing of these materials and making them profitably. By contrast the manufacture of enzymes, monoclonal antibodies and therapeutic proteins is well established. Processes are being developed to be...

Principles Of Chromatography

The fundamental principle behind the development of purification schemes for biological medicines should be to develop a robust process suitable for use at the scale required for manufacturing. Processes in manufacture should work the same each time, within predefined limits, and produce material meeting predetermined specifications. Yields need to be predictable and as high as possible, so that the scale can be set long before implementing transfer into manufacture. The key to ensuring...

Preparation and Storage of Culture Media and Supplement Solutions

Culture media and supplement solutions are normally prepared on site from dry powder materials, concentrated solutions and high-quality water. Water for injection WFI , as defined in the US or EU Pharmacopeia, is normally used, although a quality such as USP purified water is also acceptable at this stage of the process see Chapter 2 . Culture media and supplement solutions in small amounts and up to about 100 litres are typically prepared in mobile open containers, then sterilized and stored...

Medium optimization

Given the accelerated cycle times associated with process development from gene isolation to pilot production of Phase I clinical trial materials, a decision must be made rapidly regarding the preferred cultivation including optimal nutrient medium and purification schemes. While pro-liferative rates are clearly important to the economics of biomass expansion, it is possible to lose valuable time if a scale-down system that truly mimics the pilot- or production-scale bioreactor is not employed,...

Tangential Flow Microfiltration

Tangentially Flow

One of the most broadly used harvest technologies for animal cell cultures is cross-flow or tangential-flow filtration TFF . Unlike normal-flow filtration where the feed and filtrate flows are perpendicular to the filter media, in TFF operations the feed flows tangentially along the filter surface. The filtrate is forced through the membrane by applying a pressure drop across the surface while the cells and cellular debris are retained by the membrane retentate and flow back to the bioreactor....

Design Cell Expansion Protocol

Recombinant protein therapies often require significant quantities of product of the order of 100 mg dose . In order to produce sufficient quantities of product, it is often necessary to design a cell expansion process that will maximize productivity, maintain reliability, and minimize production costs. The prevailing large-scale model is the stirred tank suspension culture batch or fed-batch where the principles of scaling parameters and process control are well understood. Another variation...

Main Steps of a SIP Cycle

The main steps for the steam sterilization of empty equipment are summarized below Wood 1999 . Following the completion of a CIP cycle, a few manual operations are first required before a SIP cycle can be initiated i installation of hydrophilic and hydrophobic filter cartridges in their housings ii connection of jumpers on transfer panels, according to the desired process route to be sterilized iii installation of peripheral equipment, such as probes and connections for sampling. The typical...

Site Location And Layout

At the beginning of a project decisions will be made on site location and permit requirements the optimum number of storeys, for example to allow the location of utilities above and below the manufacturing level as appropriate the list of rooms and their functions the provision of utilities generated within the facility or taken from the site the use of 'grey space' to minimize clean room volume any provision in the design for future expansion the extent of prefabrication and pre-testing of...

List of Abbreviations

Liquid ingredients concentrated by fifty-fold adult bovine serum, from animals 12-72 months old Autographa californica multiple nucleopolyhedrosis virus N-acetylethyleneimine, chemical inactivant for virus L-asparagine H2N-CH CH2CONH2 -CO2H beta-propiolactone, chemical inactivant for virus FDA Center for Biologics Evaluation and Research FDA Center for Drug Evaluation and Research contour-clamped homogeneous electric field Committee for Medicinal Products for Human use cytomegalovirus immediate...

Cell Line Development

Not only is cell line development the first challenge addressed by a process development team, it forms the foundation of process productivity. A process that is developed with a low-producing cell line will be limited in reaching maximum productivity level. Medium improvements and additional processing improvements cannot overcome a poor cell line choice. In addition post-translational modifications such as glycosylation are largely dictated by the host cell line. Finally, the downstream...

Industrially Relevant Examples

Limited published literature exists for industrially relevant processes. However, the few that are available provide a nice overview of the evolution of processes that have been successfully implemented at manufacturing scale. One of the earlier therapeutic protein processes, the development of a process for tissue plasminogen activator tPA production, is described by Builder et al. 1988 . This process was initially produced in E. coli, but inadequate glycosylation and inaccurate...

Evaluating Virus Reduction Steps

The ability of specific steps in the purification process to remove or activate viruses can be evaluated or validated by virus 'spiking' studies. This involves the deliberate addition of virus to a small-scale version of the purification process, and following the virus through the process. Such studies are a regulatory requirement for any purification process for a new medicinal product. Guidance on the approach to be taken has been published Committee for Proprietary Medicinal Products 1996 ....

Standard Bioreactor Sensors 13221 Introduction

Bioreactor Sensors

Temperature, pH and pO2 sensors or 'probes' are essentially always present on bioreactors for monitoring and control purposes. They are installed in situ and are steam-sterilizable in place. They are covered with a housing that provides pressure balance during sterilization and other pressurization operations, as well as protection from contamination. Additionally, pilot- and production-scale bioreactors are commonly equipped with on-line devices to measure the liquid level in, or weight of,...

THE HeLa Scandal

During the 1940s and 1950s all attempts at establishing human cell lines failed until 1952, when the HeLa cell line was derived by Gey and coworkers 1952 from a cervical cancer. Everyone was waiting for such an event in order to initiate studies of human cancer, and the cell line was distributed to hundreds of laboratories worldwide. There then followed a proliferation of reports of new cell lines established from all sorts of human tumours. However, with the advent of karyotype analysis,...

Survey of Recombinant Viral Vaccines Currently under Development 6211 Herpes viruses

Genital herpes is caused by the herpes simplex virus type 1 or 2 HSV-1, HSV-2 , or human herpes virus 1 and 2 HHV-1, HHV-2 . Subunit HSV vaccines are based upon two envelope glycopro-teins, gB and gD which have been shown to be strongly immunogenic and protective in animal studies Stanberry 1991 . Four separate formulations have been evaluated to date, all derived from CHO expression systems. Three vaccines developed by Chiron contained truncated HSV-2 gD absorbed to alum, gD with a muramyl...

Systems for Either Suspension or Attached Cells not on microcarriers

These systems can be broadly classified on the basis of whether the medium is mixed within a single compartment, or is intentionally segregated into two compartments separated by a semipermeable membrane. The CelliGen is a small to medium-sized, round-bottomed, stirred-tank bioreactor from New Brunswick Scientific that is best characterized by the mixing principle of its cell lift impeller. The rotation of the ports of this specially designed impeller through the upper level of the culture...

Systems for Suspension Cells or attached cells on microcarriers

The Gas Exchange System Insects

These are cylindrical, agitated vessels, varying in capacity from around 100 ml to 36 litres. They represent an intermediate level of scale-up between flasks or dishes and fermenters, but are far cheaper to purchase and use than a small fermenter of equivalent size. However, far less instrumentation and control is available than on a fermenter, and thus they are not generally of great use as scaled-down models for fermenter process development. Where they may be useful is in the early stages of...

Methods Used for Virus Reduction

The various methods used for virus inactivation or removal are considered below. Their effectiveness may vary and can only be fully determined for a particular product by virus spiking studies. In general, the methods are of limited effectiveness against small, resistant, non-enveloped viruses. Many of the purification steps used during the product purification process may provide some degree of virus reduction see Table 19.3 . For instance, ultrafiltration steps used to concentrate protein...

Room Air Quality Classification And Air Conditioning

The quality of the local environment that is needed for reliable production depends on the degree to which the raw materials, API and components are exposed while processing occurs. The air conditioning system has to achieve several objectives Segregation of the environment by ensuring that air and its associated particles cannot flow from one area to another. Examples are the segregation of the areas where there are live and dead cells. Providing a suitable environment for processing exposed...

Systems for Attached Cells 10311 Roller bottles

Roller Bottles Cell Culture

The use of rotating bottles for large-scale cell culture was first described in 1933 Gey 1933 , since which time roller systems have been used for culturing a great many different types of attached cell. Clean glass bottles, e.g. 2.5-litre Winchester bottles, were used for many years, but purpose-designed reusable glass bottles or disposable plastic vessels are now available. The inner surface of the bottle or vessel is used as a cylindrical growth surface. Cells are introduced in a limited...

Expression Vectors 5221 Plasmid vectors

Cho Protein Production

A wide selection of plasmid-based expression vectors is now available. For many studies of gene expression, small quantities of protein are sufficient and can be obtained using transient assay systems. When larger amounts of protein are required, it is necessary to identify clonal cell lines in which the vector sequences are retained during cellular proliferation 'stable' cell lines . This can be achieved either by episomal plasmid replication or by the integration of the vector into the host...

History Of Viral Vaccines

The first vaccine Table 1.1 , Jenner's smallpox 1798 , was produced on the skin of living animals and was a very 'dirty' preparation. The next vaccine, rabies 1885 produced in spinal cord preparations, was equally contaminated with host proteins and caused severe anaphylactic shock and other side effects. The need for cleaner and safer vaccines led to the use of embryonated chicken eggs yellow fever, 1935 influenza, 1936 and although an improvement, these preparations were still often...