Regulatory Issues

For the production of biopharmaceuticals regulatory guidelines affecting the design and operation of bioreactors have to be considered. Issuing authorities are the Food and Drug Authority in the United States (417), the EMEA in Europe (418), and national regulatory authorities. The international conference of harmonization (419) aims to provide one single guiding document, which is independent of the country. These guidelines cover equipment construction, installation and operational...

Conclusion

A wide range of reactor types has been suggested in the literature for animal cell culture including stirred tanks, airlift reactors, fluidized bed reactors, packed bed and solid bed reactors, hollow fiber and membrane reactors. The number of different types is even increased for small-scale simple systems such as spinner, roller, and shake flasks. Typical operation modes are batch, fed-batch, and perfusion although the suitability is strongly dependent on the application, the cell line, and...

Nadh

Figure 6 Interaction of the metabolism of the main amino acid compounds with the central metabolism of the cells. groups of the different amino acid molecules are released by aminotransferases or transaminases. This group of enzymes is substrate specific, but has pyrodoxal phosphate as a prosthetic group. The acceptor for the released ammonium is always a-ketoglutarate, allowing the different amino groups to be funneled into L-glutamate. In turn, L-glutamate will be used by the cell for the...

Production Of Biologically Active Substances By Serumfree Cultures

Animal cell technology has allowed the production of a great diversity of biological substances including monoclonal antibodies, vaccines, hormones, and other regulatory molecules. The development of serum-free media has improved these processes by conferring many advantages as discussed previously. The vaccine industry has seen the emergence of new processes for production utilizing serum-free culture systems. In many cases the traditional method of production has led to many problems...

Progress in Cell Line Acceptability

As mentioned before, cells used for early vaccine production were primary cells such as monkey kidney cells and chicken embryo. These cells are attachment-dependent cells requiring a surface on which to grow. In addition the primary cells have stringent growth requirements (growth factors, serum) and their expansion requires a lot of manipulation (trypsinization). These requirements inhibit the use of attachment-dependent cells for large-scale production. The shift from attachment cells to...

Electrophoresis Methods

Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis Electrophoresis-based methods separate the material of interest by electric potential. For SDS-PAGE, the SDS coats a protein with a uniform negative charge, such that the proteins are separated by molecular weight when a voltage is applied. SDS-PAGE is a method accepted by FDA for determining the purity and quantity of therapeutic MAbs, and it is used extensively to compare material from different batches and to monitor quality during a...

Strategies For The Development Of Serumfree Media

There are many analytical approaches for designing serum-free formulations. The objective is to identify growth-promoting components that may be present in serum and include them in a completely synthetic medium. This method is a systematic approach to the development of serum-free media by the gradual reduction in the concentration of undefined supplements in the culture (142). It is a time-consuming but successful method to develop a single formulation for a given cell line. For nutrients in...

Progress in Large Scale Cell Culture Technology

While small-scale cell culture provided sufficient products for investigational and diagnostic purposes, the demand for vaccines and therapeutic products required large-scale production. Some of the cell culture technology-based products such as monoclonal antibodies need to be manufactured at 1000 kg year. This can only be done in large bioreactors (up to 20,000 L) by efficient processes that produce several kilograms a day (1,7). Early cell lines used for vaccines were attachment-dependent...

The Metabolism of Other Amino Acids

Aspartate Oxalo

Unlike prokaryotic cells, animal cells cannot synthesize the branched carbon chains found in branched-chain amino acids or the ring systems found in phenylalanine and the aromatic amino acids. Neither can they incorporate sulfur into covalently bonded structures. For this reason, amino acids are grouped as essential and nonessential. The nonessential amino acids are alanine, asparagines, aspartate, cysteine, glutamate, glutamine, glycine, proline, serine, and tyrosine. Glutamine, however, is...

Microcarrier Culture in Stirred Tank Reactors

The basic idea of microcarrier culture was to develop a unit operation system with similar scale-up and environmental control potential as suspension cell culture in stirred tanks. Van Wezel 240 introduced the use of dextran carriers for the growth of human fibroblast like cells in 1967. A historical review of the development of microcarrier technology is presented in Ref. 244 . In principle, this technique comprises the cultivation of anchorage dependent cells on small solid particles...

Bioreactors For Suspension Cell Cultures Small Scale Culture Systems

Small-scale culture systems are characterized by a relatively simple design and low level of instrumentation and control. Traditionally, roller bottles and spinner flasks have been used for small-scale suspension culture although even T-flasks, Petri dishes, multiwell plates, and other stationary culture systems are applicable for suspension cell propagation in small-scale. Spinner flasks Techne, Integra Biosciences, etc. are available from 125 mL to 5L working volume for operation in...

Vectors for Regulated Expression of Genes in Mammalian Cells

Mammalian Cell Transplantation

The promoters described in the preceding text are employed to drive constitutive transcription of cDNA, leading to continuous mRNA and protein synthesis. However, there are situations where regulated or induced gene expression is desired for instance, when an expressed protein is cytotoxic or cytostatic to host cells. To address these situations there have been several inducible mammalian expression systems developed. Early systems relied on inducers that had pleiotropic effects on host cells,...

Mammalian Cell Culture

Species that are more closely related to humans may be better at producing glycopro-teins that are similar to human glycoproteins. Rodent cell lines the most common host cell lines have been shown to have many differences in glycosylation compared to humans 8 . For a very comprehensive list of recombinant protein hosts and their glycosylation characteristics, Jenkins et al. 8 provides a good summary. Some highlights will be presented here. Most mammalian-derived glycoproteins contain...

Mass Spectrometry

MS can be used to determine the primary structure of a glycoprotein. Structural characteristics that can be obtained are branching, linkages, configuration, and identification of isomer sugars. MS requires that the sample be volatilized. Electron impact and chemical ionization are both used. Permethylation chemical ionization can be used to release the oligosaccharides by b-elimination. Soft ionization can be obtained by electron impact, which then allows for the direct ionization and...

Stirred Tank Reactors

Structure Stirred Tank Reactors

Stirred tank bioreactors STR are the most widely used bioreactor type to cultivate suspension cells, mainly due to the broad experience obtained in microbial Inflated pla forms a dis cultivation cl Inflated pla forms a dis cultivation cl Figure 1 Schematic representation of the Wave bioreactor with wave-induced agitation. Reprinted from Ref. 32 , Copyright 1999 with kind permission from Kluwer Academic Publishers. angle 5-10 deg rate 5-40 rpm air 0.01 -0.1 wm Figure 1 Schematic representation...

Ldh

Figure 1 Overview of the glycolysis pathway and the PPC. The enzymes involved in the pathway are indicated in italics and the corresponding full names are as follows AC, aconitase AlaTA, alanine transaminase aspTA, aspartate transaminase ALD, aldolase ATP, adenosine triphosphate CS, citrate synthase D, dilution rate in continuous cultures h_1 ENO, enolase FU, fumarase GAPDH, glyceraldehyde 3-phosphate dehydrogenase GDH, glutamate dehydrogenase GS, glutamine synthetase G6PDH, glucose 6-phosphate...

Effects of Extracellular Enzymes

Extracellular enzymes, namely proteases and glycosidases, are believed to play an important role in protein glycosylation 8,109 . A properly glycosylated recombinant protein is thought to be more resistant to protease degradation 18 . Since media protein components are also substrates for extracellular proteases, industry's switch to serum-free and protein-free medias could potentially increase the probability of proteolytic degradation of the recombinant glycoprotein. There are five identified...

Nutrient Limitations and Feeding Strategies

Many literature studies have investigated glycosylation variations of recombinant proteins with respect to nutrient requirements including sugar feeding, nucleotide sugar feeding, oxygen levels, amino acid additions, and serum components 3,7278 . Serum is known to contain growth factors, which improve cell growth, and lipids, which improve shear resistance, but serum also contains waste products and proteases, which can be detrimental to the cell and glycoprotein products. Gaw-litzek et al. 74...

Glucose Metabolism

Once incorporated into the cytosol, glucose is further converted by two main pathways glycolysis and the pentose phosphate cycle PPC , as represented in Fig. 1. In the main pathway, glycolysis, glucose is converted to pyruvate, generating a number of intermediates for biosynthesis, two ATP molecules, and two NADH molecules. Glucose 6-phosphate is used for glycogen synthesis alternatively it can be converted into 6-phosphogluconate and further to ribose 5-phosphate, through the PPC. The PPC...

Top Down vs BottomUp Approaches to Serum Free Media Development

There are two basic approaches to developing serum-free media formulations. The first top-down involves taking an existing formulation for a similar cell line, supplemented with serum, and selecting constituents that are stimulatory for growth. This is followed by the slow reduction of serum in the medium. The second approach bottom-up involves the selection of a basal medium, analyzing individual components for their effects on growth, and combining them to yield a serum-free formulation....

Malate-aspartate Shuttle

Citrate Malate Pyruvate Shuttle

Figure 4 The glutaminolysis pathway and malate shunt, indicating alternative routes and main transport systems around the mitochondria. The enzymes involved in the pathway are indicated in italics and the corresponding full names are given in the list of abbreviations in Fig. 1. Figure 4 The glutaminolysis pathway and malate shunt, indicating alternative routes and main transport systems around the mitochondria. The enzymes involved in the pathway are indicated in italics and the corresponding...

Transient Expression

In transient expression systems or ''transient transfections,'' DNA is introduced into the cell and maintained replicated as an extrachromasomal unit. Besides the strength of the promoter driving expression of the product gene, transfection efficiency percentage of cells taking-up and expressing DNA is the most important factor in determining the overall expression level, and several methods have been recently developed which yield reproducibly high transfection efficiencies 50 , see following...

References

Proc Soc Exp Biol Med 1950 73 1-8. 2. Eagle H. Science 1955 122 501. 3. Dulbecco R, Freeman G. Virology 1959 8 396-397. 4. Ham R. Clonal growth of mammalian cells in a chemically defined synthetic medium. Proc Natl Acad Sci 1965 53 288. 5. Moore GE, Gerner RE, Franklin HA. Culture of normal human leukocytes. J Am Acad Sci 1967 199 519-524. 6. Eagle H. Science 1959 130 432. 7. Stoker MGP, MacPherson I. Virology 1961 14 359. 8. Stoker MGP, MacPherson I. Nature...

Fluorescence Activated Cell Sorting Based Methods

The number of clones that need to be screened in order to identify members of the population with acceptably high productivity will be determined by the ability of the chemical selection method to find high producers within the transfected population and or the availability of a rapid high throughput screen for cell line evaluation. Fluorescence-activated cell sorting FACS methods represent the potentially most elegant means of high-throughput screening. Most methods described below can be used...

The Role of Mitochondria The TCA Cycle Respiration and ATP Generation

Pyruvate Through Tca Cycle

The TCA cycle Fig. 2 is a major pathway in the central metabolism, having two main functions It provides different compounds that are precursors for the cell ana-bolism and generates most of the metabolic energy. Regarding metabolism compart-mentalization, this pathway takes place inside the mitochondria. It is also important to highlight its central role as an internal mechanism to balance the rate of glycolysis and glutaminolysis, as discussed later. The connection between TCA and glycolysis...

Selection Vector Design

When selectable markers are used to identify cells that have stably integrated the vector sequences into their genome, they are essentially being used as surrogates to identify cells that express the product. However, the linked integration of the marker gene and the product gene is not always guaranteed, and it is possible that cells which exhibit drug-resistant phenotypes, or survive in nutrient deficient media may not express the product. Initially, genes for the product and the selectable...

High Efficiency Selection Of Stable Cell Lines Selectable Markers

Folate Metabolism Dhfr

In order to differentiate cells that have integrated the plasmid into the host genome, a selectable marker that allows identification and selection of those cells is required. Moreover, by physically linking the expression of the marker gene and the product gene, and using selection strategies that impair the ability to express the marker gene product relative to product gene, it is possible to stringently select for cells producing high levels of product gene. There are several types of...

The Flexibility of Animal Cell Metabolism Interaction Between Glutamine and Glucose Metabolic Pathways

Up to this point the main characteristics of glucose and glutamine metabolism have been described, giving special emphasis to the apparent deregulation of the glucose and glutamine uptake rates, which takes place at higher rates than those strictly necessary for the cell functions when the cells are exposed to a nonlimiting concentration of these main nutrients. This analysis will now be completed by including account the interaction between glucose and glutamine metabolism, together with the...

Genetic Engineering Of Host Cell Lines To Improve Production Characteristics

One of the most notable advances in recent years has been the application of genetic engineering approaches to rationally modify specific features of mammalian host cells to improve their utility in recombinant protein expression applications. One such example is in the area of glycosylation control. A breadth of work in the last decade has demonstrated how various reactions in the glycosylation pathway can be influenced by cell culture factors, host cell selection, and protein specific...

Cell Source And Host Cell Line Selection

Terminal Glcnac Residue Biopharma

There are several mammalian systems capable of yielding high-level expression of recombinant proteins that are suitable for use in the manufacture of protein pharmaceuticals. Cell lines derived from rodents such as Chinese hamster ovary CHO cells, baby hamster kidney BHK cells, and lymphoma cell lines such as NSO, SP2, and YB2 0 are the most commonly used. However, there are no restrictions on the type of host cell that can be used for commercial manufacturing provided the cells 1 support...

Early Days of Cell Culture Technology First Products

Utilization of cultured cells for the production of viral vaccines was the first application of cell culture technology. Viruses for vaccine production need living cells to propagate. Embryonic chicken in eggs traditionally was used for vaccine production. Due to increased demand, alternative methods were sought and cell culture technology was the answer. The production of polio vaccine using cells grown in culture started in 1954 4 . The cells were primary monkey kidney cells grown on surfaces...

Lipofection and Polyfection

Polyfection and lipofection are the most recently developed and probably simplest transfection methodologies for gene delivery in a variety of cell types. Lipofection refers to cationic lipid-mediated gene transfer into the cells. The cationic lipids containing a quaternary amine form positively charged liposomes that interact with negatively charged DNA to form a complex. Various models have been proposed to describe how DNA and lipids interact. The most popular model, supported by in situ...