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Animal Sera, Animal Sera Derivatives and Substitutes Used in the Manufacture of Pharmaceuticals
BIOLÐMS 0163 --- 26/4 issue --- MB 23/4/99 Biologicals (1998) 26, 365±368 Article No. bg980165
MEETING REPORT
Animal Sera, Animal Sera Derivatives and Substitutes Used in the Manufacture of Pharmaceuticals 5–6 May 1998, Strasbourg, France Peter Castle1 and James S. Robertson2 European Pharmacopoeia Commission, F-67029 Strasbourg, France; 2NIBSC, Potters Bar, EN6 3QG, U.K.
1
Many biological medicinal and veterinary products including vaccines are produced from cell cultures whose growth depends on the presence of animal serum, mainly bovine, in the growth medium. Whilst very safe and effective biologicals have been produced in this way for decades, the issue of adventitious viral contamination by animal serum and its derivatives has prompted an examination by experts because of the widespread use of such serum. A conference on animal sera, jointly organized by the European Department for the Quality of Medicines of the Council of Europe and the International Association of Biological Standardization, examined these issues. It was attended by more than 200 delegates and the high proportion present on the last afternoon demonstrated the wide interest in the subject. Twenty-four speakers presented papers in six plenary sessions: , Benefits and risks of serum use , Practicalities of serum production , Minimizing the risk at source , Virus removal and inactivation procedures , Elimination of serum from culture media , Future developments and round-table discussion A summary of the information presented at the conference and recommendations formulated are given below. The full proceedings will be published at a later date in the Developments in Biological Standardization series (Karger, Basel).
cultures to which serum is added can be contaminated with viruses potentially present in the serum. Stringent testing of such products is required, and in place, to assure safety and quality. Many manufacturers of recombinant DNA products, where continuous cell lines are generally used, have been successful in adapting their cells to serum-free medium, but for many biological products, such as live viral vaccines, the replacement of serum by ingredients of non-animal origin remains difficult and can lead to changes in the product which may have unforeseen consequences. What does serum contribute? The ability of serum to promote the growth of a wide range of cells in culture arises from the presence of a complex range of substances which include growth factors, albumin, transferrin, anti-proteases, attachment factors, minerals, nutrients, hormones and inhibitors. Thus, in addition to issues such as cost, logistics of supply, need for downstream processing to remove serum components and the possibility of contamination, the complexity of animal serum has consequences for batch-to-batch variation. It also makes the replacement of serum a difficult operation since an alternative source, preferably of non-animal origin, has to be found possibly for each essential component. The risks involved in the use of serum
The need for serum Bovine serum has long been used as an essential growth supplement for cell cultures which produce biological medicinal and veterinary products. However, biological products derived from cell 1045±1056/98/040365 + 04 $30.00/0
Contamination of serum with viruses is a well known and established risk associated with the use of animal serum. For veterinary products, there is a risk of direct transmission of pathogens and for products used in humans, zoonotic agents are a 7 1998 The International Association of Biological Standardization
BIOLÐMS 0163 --- 26/4 issue --- MB 23/4/99
366
P. Castle and J. S. Robertson
concern and need to be excluded. However, in principle, non-zoonotic agents also should be excluded from human products. Despite testing of batches of serum, agents can escape detection and contaminate production cell cultures. Contamination with mycoplasmas is also a continuing problem, particularly in cell banks, but are more easily detected. Agents of transmissible spongiform encephalopathies (TSEs), and especially bovine spongiform encephalopathy (BSE), remain a theoretical risk which, according to available evidence, can be effectively excluded by appropriate geographic sourcing of the animal material from countries with approved surveillance and where BSE has not been detected. Furthermore, repeated experimentation has so far failed to detect infectivity in serum. The cells routinely used for production of biological medicines do not support the replication or amplification of TSE agents; the only cell lines known to do so are of neuronal origin and use of such cells for production requires special justification. Bovine viral diarrhoea virus (BVDV) has been identified as the most common contaminant of bovine serum. Other possible contaminants include reovirus, infectious bovine rhinotracheitis virus, parainfluenza virus 3, bovine leukaemia virus and bovine polyoma virus. Porcine parvovirus is a common contaminant in preparations of porcine trypsin. To avoid the use of a contaminated serum, testing of the serum by both the supplier and the user is routinely applied. Bovine polyoma virus (BPyV) is a viral infection of cattle which has been reported as a contaminant of serum and of veterinary vaccines derived from cells grown in the presence of bovine serum. Antibody status is not a reliable indicator of the presence of the BPyV in a herd and the use of nucleic acid amplification technologies (NAT) may provide a superior method of detection. Although it is not yet recognised as a pathogen, measures to avoid its presence, for example by nucleic acid amplification (e.g. PCR) testing of serum, should be undertaken. Reducing the risk When manufacturers use serum during production, a number of measures are or may be available for reducing the associated risks: , selection of the source animals, , testing for contaminants in the serum batch to be used,
, treatment of the serum to inactivate or remove extraneous agents, , inclusion of virus removal or inactivation steps in the downstream processing of the biological product. No single method can give a complete guarantee and the application of several of the above methods is the best strategy to ensure the absence of adventitious agents. Selection of the source Geographical source is an important factor in assessing risk although no source can be assumed to be free from contaminants. For veterinary products, sourcing serum from areas in which the indigenous pathogens are non-indigenous to the region of use of the end product, should be avoided. In this respect, ``home-produced'' serum will therefore often be preferred. For countries that import serum for the production of biologicals, certain geographical areas, e.g. New Zealand, have become established as a major source country because their animal population is free from many major diseases including those in the Organisation Internationale des Epizooties, list A. In addition, in some areas, continuous, thorough surveillance of all possible means of entry of undesirable pathogens into the country is carried out to maintain the disease-free status for many pathogens. For transmissible spongiform encephalopathies, in the absence of a test for the agents, the most effective measure is to obtain serum from countries that have no recorded cases and an adequate system of reporting of TSE in animals.1 In addition to the selection of the geographic source, there will be an increasing possibility of choice between serum obtained from abattoirs and serum obtained from donor herds. Testing of serum The established method for testing for viruses by inoculation onto cell cultures and subsequent passaging has limitations. The usual detection techniques are cytopathic effect, haemadsorption, cytological staining and/or specific fluorescent staining. Many field strains of BVDV do not produce a cytopathic effect and a specific detection method is needed. Batches of serum can be produced as a large pool and a single contaminated donation may
BIOLÐMS 0163 --- 26/4 issue --- MB 23/4/99
Meeting Report
be diluted sufficiently that it may not be detected by culture methods. A number of speakers at the conference emphasised this point and gave details of ways of increasing the effectiveness of the testing procedure. Novel or improved assays to detect virus contamination are important. For example, for several common pathogens the use of nucleic acid amplification technologies is now possible. These techniques have a very low limit of detection and their application will no doubt increase. Results may be obtained within a few days compared with several weeks for culture methods. The presence of viral nucleic acid is not necessarily indicative of infectivity, but several speakers presented data showing close association between the two for untreated sera. Treatment of serum Gamma irradiation is well established as a treatment method but even at the usual absorbed dose (025 kGy) some viruses may survive; dose rate as well as total dose may be of importance in terms of effectiveness and the quality of the serum after irradiation. Irradiated serum does not have the same properties as native serum for promotion of cell growth and can lead to an unacceptably high occurrence of abnormal cells. Alternatives to gamma irradiation are now available. Nanofiltration has been shown to be effective in removing all but the smallest viruses (Q35 nm) and with proper performance checks these filters can add to the security of a production process. However, they can have the disadvantage in some cases of removing the larger proteins along with the viruses. Ultraviolet radiation in the C region (220±290 nm, notably 254 nm) has been used successfully to produce very high clearance rates for viruses of concern and mycoplasmas with minimal changes to the serum. The effectiveness of this method against parvovirus is particularly noteworthy. Inactivation of viruses with imine derivatives has also been shown to be very effective. Downstream virus removal or inactivation This option is available for some products, particularly recombinant proteins, and when high clearance values can be achieved it contributes significantly to reduction of risk. A clear identification of the process parameters that contribute to
367
virus removal or inactivation is necessary as is an evaluation of the kinetics of virus inactivation in order to obtain a realistic assessment of the capacity of the process. Model viruses for such studies are cited in regulatory guidelines. Species source of serum The principal source is bovine with some use of equine and occasionally other species. Fetal calf serum is usually collected in abattoirs and a rigorous approach to collection procedures and documentation is needed to ensure suitable quality. Bovine serum is increasingly obtained from donor herds kept under defined conditions with standard operating procedures including continuous monitoring of the herds. These herds clearly have many advantages for the end users in terms of regularity and in the transparency of the procedures used. They can also be maintained as specific-pathogenfree (SPF) herds with clear benefit both for risk reduction and regularity of production. Alternatives to serum Some manufacturers have developed serum-free growth medium. Often such media still contain ingredients of animal origin but these can sometimes be autoclaved thus removing the risk from any but the most resistant agents. Considerable ingenuity has been applied in identifying the various ingredients of serum and the role they play in cell growth so that substitutes can be found. Synthetic chemicals and substances of plant origin are increasingly being evaluated as replacements of substances of animal origin. Serum-free medium has to be developed for cell lines on an individual basis. This increases development work for manufacturers and makes the manufacturing process more complex if a range of media has to be used rather than the generalpurpose media that are common when serum is included. The replacement process has been more successful with continuous cell cultures. Changing an existing cell manufacturing process to serum-free medium requires appropriate validation work, as well as a variation in the product licence. Regulatory authorities should consider expediting such licence variations. Other substances of animal origin Trypsin, usually obtained from pigs, is frequently used for the preparation of cell cultures. The risk of
BIOLÐMS 0163 --- 26/4 issue --- MB 23/4/99
368
P. Castle and J. S. Robertson
contamination with viruses and mycoplasmas exists as for serum, especially porcine parvovirus, and effective steps for inactivation or elimination of contaminants must be applied. Peptone and similar meat digests are common ingredients of culture media and stringent inactivation procedures should be included. Human albumin is an ingredient of some cell-culture media. The introduction of substances obtained from transgenic animals can also be expected in the future and appropriate precautions will be required.
guidelines than in terms of firm requirements. Although manufacturers of biological medicinal and veterinary products are encouraged to reduce or eliminate the use of substances of animal origin in their manufacturing process, the regulatory obstacles in terms of revalidation of the product and the manufacturing process can be formidable. Manufacturers asked for a review of this situation in light of present knowledge to facilitate progress.
References Regulatory provisions Apart from overall requirements for freedom from contaminants in finished products, current regulatory provisions are more in the form of
1. Bovine Spongiform Encephalopathy (BSE). WHO Consultation on public health issues related to BSE and the emergence of a new variant of CreutzfeldtJakob disease. WHO Weekly Epidemiological Record, 12 April 1996, 71, No. 15, 113±115.
MEETING REPORT
Animal Sera, Animal Sera Derivatives and Substitutes Used in the Manufacture of Pharmaceuticals 5–6 May 1998, Strasbourg, France Peter Castle1 and James S. Robertson2 European Pharmacopoeia Commission, F-67029 Strasbourg, France; 2NIBSC, Potters Bar, EN6 3QG, U.K.
1
Many biological medicinal and veterinary products including vaccines are produced from cell cultures whose growth depends on the presence of animal serum, mainly bovine, in the growth medium. Whilst very safe and effective biologicals have been produced in this way for decades, the issue of adventitious viral contamination by animal serum and its derivatives has prompted an examination by experts because of the widespread use of such serum. A conference on animal sera, jointly organized by the European Department for the Quality of Medicines of the Council of Europe and the International Association of Biological Standardization, examined these issues. It was attended by more than 200 delegates and the high proportion present on the last afternoon demonstrated the wide interest in the subject. Twenty-four speakers presented papers in six plenary sessions: , Benefits and risks of serum use , Practicalities of serum production , Minimizing the risk at source , Virus removal and inactivation procedures , Elimination of serum from culture media , Future developments and round-table discussion A summary of the information presented at the conference and recommendations formulated are given below. The full proceedings will be published at a later date in the Developments in Biological Standardization series (Karger, Basel).
cultures to which serum is added can be contaminated with viruses potentially present in the serum. Stringent testing of such products is required, and in place, to assure safety and quality. Many manufacturers of recombinant DNA products, where continuous cell lines are generally used, have been successful in adapting their cells to serum-free medium, but for many biological products, such as live viral vaccines, the replacement of serum by ingredients of non-animal origin remains difficult and can lead to changes in the product which may have unforeseen consequences. What does serum contribute? The ability of serum to promote the growth of a wide range of cells in culture arises from the presence of a complex range of substances which include growth factors, albumin, transferrin, anti-proteases, attachment factors, minerals, nutrients, hormones and inhibitors. Thus, in addition to issues such as cost, logistics of supply, need for downstream processing to remove serum components and the possibility of contamination, the complexity of animal serum has consequences for batch-to-batch variation. It also makes the replacement of serum a difficult operation since an alternative source, preferably of non-animal origin, has to be found possibly for each essential component. The risks involved in the use of serum
The need for serum Bovine serum has long been used as an essential growth supplement for cell cultures which produce biological medicinal and veterinary products. However, biological products derived from cell 1045±1056/98/040365 + 04 $30.00/0
Contamination of serum with viruses is a well known and established risk associated with the use of animal serum. For veterinary products, there is a risk of direct transmission of pathogens and for products used in humans, zoonotic agents are a 7 1998 The International Association of Biological Standardization
BIOLÐMS 0163 --- 26/4 issue --- MB 23/4/99
366
P. Castle and J. S. Robertson
concern and need to be excluded. However, in principle, non-zoonotic agents also should be excluded from human products. Despite testing of batches of serum, agents can escape detection and contaminate production cell cultures. Contamination with mycoplasmas is also a continuing problem, particularly in cell banks, but are more easily detected. Agents of transmissible spongiform encephalopathies (TSEs), and especially bovine spongiform encephalopathy (BSE), remain a theoretical risk which, according to available evidence, can be effectively excluded by appropriate geographic sourcing of the animal material from countries with approved surveillance and where BSE has not been detected. Furthermore, repeated experimentation has so far failed to detect infectivity in serum. The cells routinely used for production of biological medicines do not support the replication or amplification of TSE agents; the only cell lines known to do so are of neuronal origin and use of such cells for production requires special justification. Bovine viral diarrhoea virus (BVDV) has been identified as the most common contaminant of bovine serum. Other possible contaminants include reovirus, infectious bovine rhinotracheitis virus, parainfluenza virus 3, bovine leukaemia virus and bovine polyoma virus. Porcine parvovirus is a common contaminant in preparations of porcine trypsin. To avoid the use of a contaminated serum, testing of the serum by both the supplier and the user is routinely applied. Bovine polyoma virus (BPyV) is a viral infection of cattle which has been reported as a contaminant of serum and of veterinary vaccines derived from cells grown in the presence of bovine serum. Antibody status is not a reliable indicator of the presence of the BPyV in a herd and the use of nucleic acid amplification technologies (NAT) may provide a superior method of detection. Although it is not yet recognised as a pathogen, measures to avoid its presence, for example by nucleic acid amplification (e.g. PCR) testing of serum, should be undertaken. Reducing the risk When manufacturers use serum during production, a number of measures are or may be available for reducing the associated risks: , selection of the source animals, , testing for contaminants in the serum batch to be used,
, treatment of the serum to inactivate or remove extraneous agents, , inclusion of virus removal or inactivation steps in the downstream processing of the biological product. No single method can give a complete guarantee and the application of several of the above methods is the best strategy to ensure the absence of adventitious agents. Selection of the source Geographical source is an important factor in assessing risk although no source can be assumed to be free from contaminants. For veterinary products, sourcing serum from areas in which the indigenous pathogens are non-indigenous to the region of use of the end product, should be avoided. In this respect, ``home-produced'' serum will therefore often be preferred. For countries that import serum for the production of biologicals, certain geographical areas, e.g. New Zealand, have become established as a major source country because their animal population is free from many major diseases including those in the Organisation Internationale des Epizooties, list A. In addition, in some areas, continuous, thorough surveillance of all possible means of entry of undesirable pathogens into the country is carried out to maintain the disease-free status for many pathogens. For transmissible spongiform encephalopathies, in the absence of a test for the agents, the most effective measure is to obtain serum from countries that have no recorded cases and an adequate system of reporting of TSE in animals.1 In addition to the selection of the geographic source, there will be an increasing possibility of choice between serum obtained from abattoirs and serum obtained from donor herds. Testing of serum The established method for testing for viruses by inoculation onto cell cultures and subsequent passaging has limitations. The usual detection techniques are cytopathic effect, haemadsorption, cytological staining and/or specific fluorescent staining. Many field strains of BVDV do not produce a cytopathic effect and a specific detection method is needed. Batches of serum can be produced as a large pool and a single contaminated donation may
BIOLÐMS 0163 --- 26/4 issue --- MB 23/4/99
Meeting Report
be diluted sufficiently that it may not be detected by culture methods. A number of speakers at the conference emphasised this point and gave details of ways of increasing the effectiveness of the testing procedure. Novel or improved assays to detect virus contamination are important. For example, for several common pathogens the use of nucleic acid amplification technologies is now possible. These techniques have a very low limit of detection and their application will no doubt increase. Results may be obtained within a few days compared with several weeks for culture methods. The presence of viral nucleic acid is not necessarily indicative of infectivity, but several speakers presented data showing close association between the two for untreated sera. Treatment of serum Gamma irradiation is well established as a treatment method but even at the usual absorbed dose (025 kGy) some viruses may survive; dose rate as well as total dose may be of importance in terms of effectiveness and the quality of the serum after irradiation. Irradiated serum does not have the same properties as native serum for promotion of cell growth and can lead to an unacceptably high occurrence of abnormal cells. Alternatives to gamma irradiation are now available. Nanofiltration has been shown to be effective in removing all but the smallest viruses (Q35 nm) and with proper performance checks these filters can add to the security of a production process. However, they can have the disadvantage in some cases of removing the larger proteins along with the viruses. Ultraviolet radiation in the C region (220±290 nm, notably 254 nm) has been used successfully to produce very high clearance rates for viruses of concern and mycoplasmas with minimal changes to the serum. The effectiveness of this method against parvovirus is particularly noteworthy. Inactivation of viruses with imine derivatives has also been shown to be very effective. Downstream virus removal or inactivation This option is available for some products, particularly recombinant proteins, and when high clearance values can be achieved it contributes significantly to reduction of risk. A clear identification of the process parameters that contribute to
367
virus removal or inactivation is necessary as is an evaluation of the kinetics of virus inactivation in order to obtain a realistic assessment of the capacity of the process. Model viruses for such studies are cited in regulatory guidelines. Species source of serum The principal source is bovine with some use of equine and occasionally other species. Fetal calf serum is usually collected in abattoirs and a rigorous approach to collection procedures and documentation is needed to ensure suitable quality. Bovine serum is increasingly obtained from donor herds kept under defined conditions with standard operating procedures including continuous monitoring of the herds. These herds clearly have many advantages for the end users in terms of regularity and in the transparency of the procedures used. They can also be maintained as specific-pathogenfree (SPF) herds with clear benefit both for risk reduction and regularity of production. Alternatives to serum Some manufacturers have developed serum-free growth medium. Often such media still contain ingredients of animal origin but these can sometimes be autoclaved thus removing the risk from any but the most resistant agents. Considerable ingenuity has been applied in identifying the various ingredients of serum and the role they play in cell growth so that substitutes can be found. Synthetic chemicals and substances of plant origin are increasingly being evaluated as replacements of substances of animal origin. Serum-free medium has to be developed for cell lines on an individual basis. This increases development work for manufacturers and makes the manufacturing process more complex if a range of media has to be used rather than the generalpurpose media that are common when serum is included. The replacement process has been more successful with continuous cell cultures. Changing an existing cell manufacturing process to serum-free medium requires appropriate validation work, as well as a variation in the product licence. Regulatory authorities should consider expediting such licence variations. Other substances of animal origin Trypsin, usually obtained from pigs, is frequently used for the preparation of cell cultures. The risk of
BIOLÐMS 0163 --- 26/4 issue --- MB 23/4/99
368
P. Castle and J. S. Robertson
contamination with viruses and mycoplasmas exists as for serum, especially porcine parvovirus, and effective steps for inactivation or elimination of contaminants must be applied. Peptone and similar meat digests are common ingredients of culture media and stringent inactivation procedures should be included. Human albumin is an ingredient of some cell-culture media. The introduction of substances obtained from transgenic animals can also be expected in the future and appropriate precautions will be required.
guidelines than in terms of firm requirements. Although manufacturers of biological medicinal and veterinary products are encouraged to reduce or eliminate the use of substances of animal origin in their manufacturing process, the regulatory obstacles in terms of revalidation of the product and the manufacturing process can be formidable. Manufacturers asked for a review of this situation in light of present knowledge to facilitate progress.
References Regulatory provisions Apart from overall requirements for freedom from contaminants in finished products, current regulatory provisions are more in the form of
1. Bovine Spongiform Encephalopathy (BSE). WHO Consultation on public health issues related to BSE and the emergence of a new variant of CreutzfeldtJakob disease. WHO Weekly Epidemiological Record, 12 April 1996, 71, No. 15, 113±115.