Analytical methods in biotechnology
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carbohydrate moieties compared to natural ones. Eur. J . Biochm~.,259 (1989) 679@7. 6. Hancock, W. S., Canova-Davis, E., Chloupek, R. C.. Therapeutic Peptides and Proteins: Assessing the New Technologies, In Bunbury Report 29, 1988, Cold Spring Harbour, pp. 95-107. 7. Bogosian, G., Violand, B . N., Dornward-King, E. J., Workman, W. E., Jung, P. E. & Kane, J. F., Biosynthesis and incorporation into protein of norleucine by Eschrrichia coli. J . Biol. Chrm., 264 (1989) 531-9. 8. Anicetti, V. R., Keyt, B. A. & Hancock, W. S., Purity analysis of protein pharmaceuticals produced by recombinant DNA technology. Trends Biotechnd., 7 (1989) 342-9. 9. Garnick, R. L., Solli, N. J. & Papa, P. A., The role of quality control in biotechnology: an analytical perspective. Anal. Chern.. 60 (1988) 2456-557.
Therapeutic Peptides and Proteins-Challenges for the Regulatory Authorities
S. L. Jeffcoate National Institute for Biological Standards and Control. Potters Bar. UK
This presentation discusses how the recent developments in the manufacturing processes, purification procedures and analytical test methods for the products of biotechnology have created particular challenges for the authorities that control and regulate these products in order to protect public health. Particular reference is made to hormones which currently dominate the ‘Biotech Market’. The National Institute for Biological Standards and Control (NIBSC) (with its new facility which came into operation some two years ago) is responsible to the UK Department of Health for advising on licensing applications and for the on-going control of biological medicinal products. The UK is almost alone in the world in including hormones as ‘biologicals’ and many countries, including the USA and most European countries, make the mistake of classifying them as ‘chemical drugs’. One can now see that in the new era of biotechnology, substances over a wide range of types, from hormones through to vaccines, are made by essentially the same sorts of biotechnological processes and this has revealed the difficulties that can arise from outdated methods of classification. The work of NIBSC places the UK in a strong position in Europe, both for advising on licensing and for the on-going control of medicinal products made by the new biotechnology procedures. Peptide drugs are a challenge for the regulatory authorities. Regulatory authorities around the world are facing major challenges in the 1990s. These challenges are a result of a number of changes. For example, the rate at which the field is changing puts particular pressure on regulatory authorities to respond to changes and to ensure that products of appropriate purity and efficacy reach the market without undue delay. The watchword is ‘to make haste slowly’. The pace of change, the pathway from ‘clone to the clinic’, or from ‘notion to potion’, gets increasingly rapid and short. This is well exemplified by erythropoietin which is discussed below.
118
SCI Biotechnology Group Meeting
These changes, of course, are not only in the manufacturing technologies, but also in the analytical methods which are used to characterise the products. These methods are changing so fast that it is difficult for control laboratories like NIBSC to keep pace with developments in industry and to ensure that its equipment is ‘state-of-the-art’. Important changes, more in the future than current, are taking place in the design of delivery systems for peptide drugs, e.g. of hormones which are not the naturally occurring structure but which have features which may increase their bioavailability, length of action or targeting ability for particular organs. This is an aspect which is likely to be developed strongly over the next decade. The importance of standardisation of rDNA-derived products needs to be emphasised. Standardisation of biological substances, and of hormones in particular, has a long history. In 1926 the League of Nations Health Organization, the forerunner of the World Health Organization, established the first International Standard for insulin, and defined the International Unit for insulin. This was an important step because at that time, when pancreatic insulin was being prepared in a variety of ways, the importance of standardising the dose given to diabetics became recognised. The preparation of this International Standard led to the worldwide adoption of a unit for insulin therapy. The rDNA-derived insulin in use today for diabetics all around the world, is still expressed in terms of this same International Unit, the current International Standards having been crosscalibrated against the previous ones to maintain continuity with this very first International Standard established over 60 years ago. This concept of standardisation remains important today, for example for erythropoietin which is made by several different manufacturers. Because of the high proportion (some 40%) of glycosylation in the molecule, there is batch-to-batch and manufacturer-to-manufacturer variability in glycosylation patterns. The importance of having a biological standard to define the unit for a product like erythropoietin is exactly the same as it was more than 60 years ago for insulin extracted from pancreatic tissue. Regulatory authorities also face politically-derived challenges. In Europe the twelve Member States, which aim to have a free internal market by 1993, have between them nine different languages and a wide range of expertise in regulation, licensing, control and characterising biological products. The problems involved in trying to harmonise the procedure whereby these products are characterised and brought to the market in a way that safeguards public health can be imagined. The aim is to harmonise the scientific principles first and then establish procedural practices in time for the institution of the free market in Europe on 1 January 1993. These principles and procedures are being extensively debated and organised through the Biotechnology Working Party of the EEC Committee on Proprietary Medicinal Products. The majority (eight of the first twelve) of applications that went through the European procedure for market authorisation were for hormones, underlining their current dominance in the field of recombinant DNA medicines. One example of these challenges currently is erythropoietin which is a hormone which stimulates red cell production in the bone marrow. It is produced by the
Analytical methods in hiotechnology
119
kidney and is a large glycoprotein hormone, about 40% of which is composed of carbohydrate moieties. Scientists at the NIBSC have long had an interest in erythropoietin and prepared the first International Standard from urine in the 1960s; this was replaced by the second International Standard in 1972. It is a relatively crude extract of urine but because it was in place as an International Standard, manufacturers of recombinant erythropoietin from different companies worldwide are able to standardise their products against one reference standard. Just as different producers of insulin have been able since the 1920s to standardise doses given to diabetics, so now pharmaceutical manufacturers are able to standardise doses of erythropoietin given to patients with the anaemia of chronic renal failure. The path from cloning to the clinic is now a short one. In 1985 scientists from two organisations (Amgen and Genetics Institute) described the cloning of the erythropoietin gene which was followed within a year or 18 months by two clinical trials of its use in the treatment of anaemia associated with renal failure. Now, in 1990, it is available in hospital pharmacies in many countries. These trials showed that treatment with erythropoietin given intravenously or subcutaneously is able to stimulate red cell production in patients who are otherwise dependent on repeated blood transfusions. This has two benefits; first, in terms of the quality of life of the patients, there is a marked improvement in their sense of well-being and exercise tolerance; and secondly, there is a reduced need for blood transfusion, which is costly and potentially dangerous because of incompatibilities and, perhaps more especially now, because of the dangers of viral contamination with HIV and other retroviruses. Future use of erythropoietin will involve the application of analytical techniques, particularly those for glycoproteins, to ensure quality (purity and potency) on an on-going basis.
Electrophoretic Techniques for Protein Analysis Michael J. Dunn Department of Cardiothoracic Surgery, National Heart and Lung Institute, Dovehouse Street, London SW3 6LY. U K
Proteins are charged at a pH other than their isoelectric point (PI) and will migrate in an electric field in a manner dependent on their charge density. If the sample is initially present as a narrow zone, proteins of different mobilities will travel as discrete zones and separate during electrophoresis. Electrophoresis is an ideal analytical technique for the separation of individual components of protein mixtures. Such separations are best carried out in a support medium to counteract effects of convection and diffusion that occur during electrophoresis and to facilitate the immobilisation of separated proteins. A variety of matrices including starch,
117
carbohydrate moieties compared to natural ones. Eur. J . Biochm~.,259 (1989) 679@7. 6. Hancock, W. S., Canova-Davis, E., Chloupek, R. C.. Therapeutic Peptides and Proteins: Assessing the New Technologies, In Bunbury Report 29, 1988, Cold Spring Harbour, pp. 95-107. 7. Bogosian, G., Violand, B . N., Dornward-King, E. J., Workman, W. E., Jung, P. E. & Kane, J. F., Biosynthesis and incorporation into protein of norleucine by Eschrrichia coli. J . Biol. Chrm., 264 (1989) 531-9. 8. Anicetti, V. R., Keyt, B. A. & Hancock, W. S., Purity analysis of protein pharmaceuticals produced by recombinant DNA technology. Trends Biotechnd., 7 (1989) 342-9. 9. Garnick, R. L., Solli, N. J. & Papa, P. A., The role of quality control in biotechnology: an analytical perspective. Anal. Chern.. 60 (1988) 2456-557.
Therapeutic Peptides and Proteins-Challenges for the Regulatory Authorities
S. L. Jeffcoate National Institute for Biological Standards and Control. Potters Bar. UK
This presentation discusses how the recent developments in the manufacturing processes, purification procedures and analytical test methods for the products of biotechnology have created particular challenges for the authorities that control and regulate these products in order to protect public health. Particular reference is made to hormones which currently dominate the ‘Biotech Market’. The National Institute for Biological Standards and Control (NIBSC) (with its new facility which came into operation some two years ago) is responsible to the UK Department of Health for advising on licensing applications and for the on-going control of biological medicinal products. The UK is almost alone in the world in including hormones as ‘biologicals’ and many countries, including the USA and most European countries, make the mistake of classifying them as ‘chemical drugs’. One can now see that in the new era of biotechnology, substances over a wide range of types, from hormones through to vaccines, are made by essentially the same sorts of biotechnological processes and this has revealed the difficulties that can arise from outdated methods of classification. The work of NIBSC places the UK in a strong position in Europe, both for advising on licensing and for the on-going control of medicinal products made by the new biotechnology procedures. Peptide drugs are a challenge for the regulatory authorities. Regulatory authorities around the world are facing major challenges in the 1990s. These challenges are a result of a number of changes. For example, the rate at which the field is changing puts particular pressure on regulatory authorities to respond to changes and to ensure that products of appropriate purity and efficacy reach the market without undue delay. The watchword is ‘to make haste slowly’. The pace of change, the pathway from ‘clone to the clinic’, or from ‘notion to potion’, gets increasingly rapid and short. This is well exemplified by erythropoietin which is discussed below.
118
SCI Biotechnology Group Meeting
These changes, of course, are not only in the manufacturing technologies, but also in the analytical methods which are used to characterise the products. These methods are changing so fast that it is difficult for control laboratories like NIBSC to keep pace with developments in industry and to ensure that its equipment is ‘state-of-the-art’. Important changes, more in the future than current, are taking place in the design of delivery systems for peptide drugs, e.g. of hormones which are not the naturally occurring structure but which have features which may increase their bioavailability, length of action or targeting ability for particular organs. This is an aspect which is likely to be developed strongly over the next decade. The importance of standardisation of rDNA-derived products needs to be emphasised. Standardisation of biological substances, and of hormones in particular, has a long history. In 1926 the League of Nations Health Organization, the forerunner of the World Health Organization, established the first International Standard for insulin, and defined the International Unit for insulin. This was an important step because at that time, when pancreatic insulin was being prepared in a variety of ways, the importance of standardising the dose given to diabetics became recognised. The preparation of this International Standard led to the worldwide adoption of a unit for insulin therapy. The rDNA-derived insulin in use today for diabetics all around the world, is still expressed in terms of this same International Unit, the current International Standards having been crosscalibrated against the previous ones to maintain continuity with this very first International Standard established over 60 years ago. This concept of standardisation remains important today, for example for erythropoietin which is made by several different manufacturers. Because of the high proportion (some 40%) of glycosylation in the molecule, there is batch-to-batch and manufacturer-to-manufacturer variability in glycosylation patterns. The importance of having a biological standard to define the unit for a product like erythropoietin is exactly the same as it was more than 60 years ago for insulin extracted from pancreatic tissue. Regulatory authorities also face politically-derived challenges. In Europe the twelve Member States, which aim to have a free internal market by 1993, have between them nine different languages and a wide range of expertise in regulation, licensing, control and characterising biological products. The problems involved in trying to harmonise the procedure whereby these products are characterised and brought to the market in a way that safeguards public health can be imagined. The aim is to harmonise the scientific principles first and then establish procedural practices in time for the institution of the free market in Europe on 1 January 1993. These principles and procedures are being extensively debated and organised through the Biotechnology Working Party of the EEC Committee on Proprietary Medicinal Products. The majority (eight of the first twelve) of applications that went through the European procedure for market authorisation were for hormones, underlining their current dominance in the field of recombinant DNA medicines. One example of these challenges currently is erythropoietin which is a hormone which stimulates red cell production in the bone marrow. It is produced by the
Analytical methods in hiotechnology
119
kidney and is a large glycoprotein hormone, about 40% of which is composed of carbohydrate moieties. Scientists at the NIBSC have long had an interest in erythropoietin and prepared the first International Standard from urine in the 1960s; this was replaced by the second International Standard in 1972. It is a relatively crude extract of urine but because it was in place as an International Standard, manufacturers of recombinant erythropoietin from different companies worldwide are able to standardise their products against one reference standard. Just as different producers of insulin have been able since the 1920s to standardise doses given to diabetics, so now pharmaceutical manufacturers are able to standardise doses of erythropoietin given to patients with the anaemia of chronic renal failure. The path from cloning to the clinic is now a short one. In 1985 scientists from two organisations (Amgen and Genetics Institute) described the cloning of the erythropoietin gene which was followed within a year or 18 months by two clinical trials of its use in the treatment of anaemia associated with renal failure. Now, in 1990, it is available in hospital pharmacies in many countries. These trials showed that treatment with erythropoietin given intravenously or subcutaneously is able to stimulate red cell production in patients who are otherwise dependent on repeated blood transfusions. This has two benefits; first, in terms of the quality of life of the patients, there is a marked improvement in their sense of well-being and exercise tolerance; and secondly, there is a reduced need for blood transfusion, which is costly and potentially dangerous because of incompatibilities and, perhaps more especially now, because of the dangers of viral contamination with HIV and other retroviruses. Future use of erythropoietin will involve the application of analytical techniques, particularly those for glycoproteins, to ensure quality (purity and potency) on an on-going basis.
Electrophoretic Techniques for Protein Analysis Michael J. Dunn Department of Cardiothoracic Surgery, National Heart and Lung Institute, Dovehouse Street, London SW3 6LY. U K
Proteins are charged at a pH other than their isoelectric point (PI) and will migrate in an electric field in a manner dependent on their charge density. If the sample is initially present as a narrow zone, proteins of different mobilities will travel as discrete zones and separate during electrophoresis. Electrophoresis is an ideal analytical technique for the separation of individual components of protein mixtures. Such separations are best carried out in a support medium to counteract effects of convection and diffusion that occur during electrophoresis and to facilitate the immobilisation of separated proteins. A variety of matrices including starch,
