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In-process control of insulin production by high-performance liquid chromatography

Gitte Mandrup
Kalundborg, Denmark Awell functioning process monitoring system is necessary in the optimization of purification processes, and to maintain the conditions at the optimal level required to secure production of high purity insulin with maximum yield. In our experience, the desired yield and purity can only be reached through improved and expanded analytical control, where reversedphase high-performance liquid chromatography (RP-HPLC) has a central role. Conditions for the specific RP-HPLC method are described. Chromatograms of actual samples from in-process control of insulin are discussed. Introduction During the past two decades the most important goals of manufacturing plants have been greater productivity and efficiency along with increased attention to quality. Understanding of process parameters and appropriate control measures can improve yield and purity of a product. A critical means of developing the necessary understanding and criteria for control is obtained by continuous process monitoring. In this article the advantages obtained from in-process control of insulin by reversed-phase high-performance liquid chromatography (RP-HPLC) are described. The production of insulin is a rather complicated process, involving several concentration, conversion and purification steps. The power of carrying out a thorough in-process control of samples taken during the progress of the entire process is emphasized. Chemical structure of insulin Insulin is a blood-sugar-lowering hormone used in the treatment and control of diabetes mellitus. Therapeutical preparations of human, bovine and porcine insulin are used. Bovine, porcine and human
0165-993692/$05.00

insulins exhibit minor differences with respect to their amino acid sequence. The difference between human and porcine insulin is confined to a single amino acid; the B30 amino acid of human insulin is threonine, whereas that of porcine insulin is alanine. The structures of human, porcine and bovine insulin are shown in Fig. 1. Insulin production process Insulin has traditionally been produced by isolation from animal pancreas and more recently by genetic engineering. Porcine and bovine insulin are obtained by extraction from the respective pancreatic glands. In Novo Nordisk human insulin has been synthesized from porcine insulin using a single enzymatic step since 1983 [l-4]. Porcine insulin is converted to an ester of human insulin in a transpeptidation reaction, in which the alanine residue No. 30 of the B-chain is replaced by a threonine ester. The enzymatic conversion is catalyzed by trypsin, which is removed by gel filtration at a low pH, where trypsin is inactive. The human insulin ester is separated from any residual unconverted porcine insulin by anionexchange chromatography. As porcine insulin contains an additional negative charge relative to the human insulin ester, the product is the first to emerge from the column [l]. In the next step, the human insulin ester is cleaved, yielding human insulin. The final purification is preparative RP-HPLC. In Novo Nordisk, human insulin has since 1987 also been produced by yeast cells as a single-chain precursor containing the correct disulfide bridges [5,6]. The human insulin precursor is produced from strains of Succharomyces cerevisiae via recombinant DNA technology. After fermentation of the yeast strains, the cells are removed, and the insulin precursor is isolated from the fermentation medium. A tryptic transpeptidation in the presence of a threonine ester converts the single-chain precursor to the double-chain human insulin ester, similarly to the conversion of porcine insulin. From this stage of the process the purification steps and cleavage of the ester bond are analogous to those described pre0 1992 Elsevier Science Publishers B.V. All rights reserved

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Human
A-chain

insulin

GLy-lle-Val-Glu-Gln-Cys-Cys-Thr-Ser-ILe-Cys-Ser-Leu-Tyr-Gln-Leu-Glu-Asn-Tyr-Cys-Asn 123456 7 8 910 11 12 13 14 15 16 17 18 19 20 I I 7 S

I----"-"1

21

Phe-Val-Asn-GLn-His-Leu-Cys-Gly-Ser-His-Leu-Val-G~u-Ala-Leu-Tyr-Leu-Va~-Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr-Thr-Pro-Lys-Thr 1 2 3 4 5 6 i 8. 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 B-chain

30

Fig. 1. Structure of human, porcine and bovine insulin.

viously for human insulin manufactured from pancreatic glands. The flow diagrams from crude porcine insulin and production from continuous fermentation to human insulin are depicted in Fig. 2. Also indicated in Fig. 2 are the main products obtained during the different stages in the process. In the preparation of the above-mentioned species of insulin, the main components of interest for this article are thus: insulin precursor, human insulin ester and human, porcine and bovine insulin. In this article the insulin production process is described for human insulin manufactured from porcine insulin or from a recombinant DNAprocess. As several manufacturers of insulin, including Novo Nordisk, still produce bovine insulin or mixtures of bovine and porcine insulin for therapeutical purposes, bovine insulin is also of interest here. Secondly, for purification reasons it is of relevance to identify the possible presence of bovine insulin in porcine insulin arising from mixed pancreatic glands of porcine and bovine origin. During the isolation of insulin from pancreas, glucagon is also extracted. Glucagon, a polypeptide having 29 amino acids residues, counterbalances the action of insulin [7]. The structure of glucagon is identical whether it is of human, porcine or bovine origin. Since glucagon is present during the first phase of production of insulin from animal pancreas,

and glucagon is a valuable by-product, this component is also considered to be of analytical interest here.

RP-HPLC-method HPLC analysis is essential for monitoring the insulin production process. The conditions of the RPHPLC method used routinely in our in-process control of insulin are described in detail in Table 1. The method is identical to the unified HPLC methodology for insulin purity and potency developed and validated in 1986 in an interlaboratory study in which scientists from Eli Lilly and Company, Nordisk Gentofte A/S and Novo Industri A/S participated. This RP-HPLC method will be published in The United States Pharmacopeia in the near future. The RPHPLC method is very powerful because of its high reproducibility in any laboratory environment. The method has proven to be highly functional and stable in well-equipped laboratories incorporating the most recent analytical HPLC instrumentation as well as in poorly equipped laboratories. As shown in Table 1, the RP-HPLC separation is based on sodium sulphate and acetonitrile eluents in the low pH range on a silica-based Cl8 column, exposed to thermostatting. The reversed-phase col-

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TABLE 1. Conditions control of insulin Column

of RP-HPLC

method

for in-process

Silica based reversed-phase Cw, e.g. Merck LiChrosorb 50333 40-50C 0.2 M Na2S04, 0.04 M H3P04, 10% CH3CN 2.5-3.5 50% CH3CN 1 .O ml/min UV 214 nm or 280 nm

Temperature Eluent A pH in eluent A Eluent B Flow Detection

Cleavage

Of I

ester

bond

Human

lSll

Preparative ,

HPLC

Human

lSll

Biqh purity human

insulin

Fig. 2. Summary flow sheet describing of human insulin.

the production

umn preferred in our laboratory is LiChrosorb RP- 18, which is packed with 5+m particles of silica with pore size of 100 8, (Merck, LiChrosorb Hibar RT 50333). Ethanolamine and phosphoric acid are used to adjust the pH in eluent A. Insulin, glucagon and related substances are either eluted isocratically or by an acetonitrile gradient according to the separation requirements and nature of the sample matrix. Depending on the concentration, the isolated insulin-related components are detected by UV absorption at 214 nm or 280 nm (due to the peptide bond). The response of insulin at 214 nm is approximately 21 times greater than the response at 280 nm (mainly due to the aromatic amino acids). When loading more than 50 pg of insulin on the column, UV-detection at 280 nm is appropriate, otherwise 214 nm is suitable. Comprehensive studies done by B.S. Welinder [8] indicate that insulin is quantitatively recovered from most reversed-phase stationary materials with most commonly used mobile phases. The recovery of insulin with this HPLC analysis is 99% and reproducibility is better than 99% [9]. A chromatogram showing the simultaneous separation of the main components of importance is depicted in Fig. 3. An important feature of this RP-HPLC method is the possibility of changing the order of separation and time of analysis simply by altering the pH of the mobile phase or the temperature of the column. Fig. 4 shows the connection between the retention of the main components and different pH and temperature values. The composition of the mobile phase was maintained at 55% of eluent A and 45% of eluent B

in all experiments. Maintaining the same pH value causes longer retention of the insulin-related compounds with increasing temperature, whereas glucagon is eluted faster with increasing temperature. Note the risk of coelution between glucagon and each of the insulin substances at certain combinations of pH and temperature conditions. Keeping the temperature stable while varying the pH shows an interesting pattern which is similar for all the compounds. The retention time is reduced when the pH is raised from 2.0 to 3.0 and 3.5. There is a noticeable increase in retention for all components at pH 2.5 regardless of the temperature. During the production of insulin from pancreas it is beneficial to follow the content of glucagon along with the yield of insulin. As shown in Fig. 4, the retention of glucagon is highly sensitive to changes in pH and temperature. The resolution between insulin and glucagon often varies when a degraded column is replaced with a new column, even if the columns are from the same supplier as well as the same batch. When it is inconvenient that glucagon is eluted later than insulin then, depending on the sample matrix, one of the following suggestions can save analysis time: use of an acetonitrile gradient, raising the temperature of the column, or raising the pH of

0.12

Fig. 3. Isocratic separation of the primary insulin substances and glucagon at pH 2.5, 40C and 214 nm. Analytical conditions as described in the text and Table 1.

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pH 2.0

pH 2.5

10

10 i0 45 Temperature, 50 C 55 5 I 40 45 Temperature. C

pH 3.0

pH 3.5

45 Temperature,

50 C

45 Temperature,

50 C

t++

Insulin Human

Precusor insulin

b ++-

Bovine insulin Human insulinester

+ +

Porcine Glucagon

insulin

Fig. 4. Influence on retention time with varying pH in mobile phase and column temperature.

the eluent. When altering pH and temperature, the coelution of insulin and glucagon should be avoided.

Analysis of production samples Chromatograms of samples taken at four critical stages in the production of human insulin from a single-chain precursor are shown in Fig. 5. The chromatograms are obtained by RP-HPLC analysis at pH 2.5 and UV-detection at 214 nm. The injected load varies from approximately 5 pg to 50 pg of insulin. The column temperature is 50C for the chromatograms displayed in panels 1-3, while the chromatogram in the lowest panel is acquired at 40C. The upper panel depicts a chromatogram of the cell-free supernatant from a fermentation broth. The relativly high temperature of the column is chosen because the retention of the insulin precursor increases with increasing temperature, whereas the retention of fermentation components decreases [9]. When the HPLC conditions chosen are appropriate, quantification of the expression levels of insulin precursor is achieved without interference from sub-

strate components or proteins stemming from the yeast cells. The RP-HPLC analysis of the yeast fermentation broth was performed by isocratic elution at approximately 60% of eluent A. Panel No. 2 in Fig. 5 shows a chromatogram of a sample taken during the conversion of single-chain precursor to human insulin ester. At this RP-HPLC analysis the composition of the mobile phase is adjusted to a ratio between eluent A and B of approximately 54:46. An increase in the acetonitrile content of the mobile phase results in early elution of the unconverted precursor, while the synthesized human insulin ester is eluted in 25 min. That is, the singlechain insulin precursor is more hydrophilic than the corresponding two-chain molecule, as it is eluted faster in RP-HPLC [lo]. The RP-HPLC analysis of this sample is of major importance in the manufacture of human insulin. It is valuable for obtaining information concerning the conversion process by determination of the purity and yield of the synthesized product. Among the critical factors involved in the transpeptidation process is the amount of trypsin in a mixture of water and organic solvent [5]. Trypsin has a specificity for basic amino acids [ 111. Besides

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Fig. 5. Chromatograms of samples from insulin production process. On the Y-axis in panel 1 and 2, 1000 mV correspond to 1 absorbance unit (AU). Panel 1: fermentation broth; panel 2: conversion to human insulin ester; panel 3: conversion to human insulin: panel 4: final product of human insulin.

the desired reaction at residue B29 (lysine), hydrolysis and transpeptidation at position B22 (arginine) will therefore also occur, which leads to loss of insulin in the form of desoctapeptide-(B23-B30) insulin (DOI)[4]. That is, along with the transpepti-

dation by-products which include DOI, its threonine ester and des-B30 (DAI) insulin are formed. In this way the chromatogram of the synthesized human insulin ester sample is an indication of the degree and success of conversion. After the by-products and the unconverted porcine insulin have been separated from the human insulin ester product by column purification, the ester group is cleaved by hydrolysis to render human insulin. The quality of the second conversion step is determined by RP-HPLC analysis, where the content of any residual human insulin ester will be detected. A chromatogram showing the formation of human insulin is shown in panel 3 in Fig. 5. The composition of the mobile phase is approximately 53% of eluent A. The final purification is by preparative RP-HPLC. The purity of the human insulin end product is between 99.5 and 99.9% as measured by analytical RP-HPLC. The lower panel in Fig. 5 depicts a chromatogram of the final product of human insulin. In this analysis an acetonitrile gradient is needed to detect the late eluted polymers of insulin, which in this case are the small peaks eluted between 30 and 40 min. The maximum load is injected on the column (50 lrg of insulin), which is just below the upper limit of the dynamic range of the method, when detection is at 214 nm. The massive load is necessary to detect the very low concentrations of insulin related compounds. After the preparative chromatography has been done, purity parameters of the resulting product should be controlled after each run. This is to ensure that the purification has proceeded as planned, i.e. to ascertain whether the column material has worn out and should be renewed. However, it is advisable to take samples from all steps involved in the column purification. Quantification of the main component in the application volume of the column compared to the concentration and the purity of the eluate allows one to determine whether the capacity of the column is utilized sufficiently. Often the column purification step is followed by crystallization of the eluate, where the crystals are separated from the mother liquor by centrifugation. RP-HPLC analysis of the crystals allows determination of impurities, whereas the analysis of the mother liquor gives an indication of the efficiency of the crystallization. If samples originate from parts in the process, where there is a considerable amount of impurities, washing of the column during or between the analysis of the samples is recommended. After the peaks of interest are eluted, a change in the composition of the mobile phase to 90% B and a 5 min hold will

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increase the reproducibility of the method. Furthermore, the implementation of column cleaning prevents deterioration of the performance of the column. In order to extend the lifetime of the analytical column, a precolumn should always be inserted, especially when analyzing very impure samples or samples from early steps in the process.

Sample preparation
Some precautions should be considered while handling the insulin samples. Certain guidelines should be followed to minimize degradation of insulin in the samples. For short term storage (days) the samples should be kept refrigerated. If an autosampler is used during HPLC analysis, then a cooling unit is required. For long term storage (weeks and longer) the samples should be stored in a freezer. In the case of samples from fermentation, only the cell-free supernatant should be stored. Insulin is insoluble near to its isoelectric point (pH 5.5) and dissociates at acidic pH, whereas it forms dimers and hexamers at neutral pH. The recommended pH range for insulin in sample solutions is between 2 and 3. Acetic acid is often used for dilution. To avoid formation of acidic desamido insulin, crystals of insulin are usually dissolved in a Tris buffer at pH 9. However, it is important to be aware of the main component in the sample. If the main product in the crystals is human insulin ester, the sample should be dissolved in acetic acid or formic acid, otherwise hydrolysis of the human insulin ester to human insulin will take place during the sample preparation. The decision on the medium to be used for dissolution of sample crystals for HPLC analysis is often a compromise. In the analysis of crystals of the final product, 0.01 M hydrochloric acid is chosen as the solvent. The use of hydrochloric acid causes formation of acidic desamido insulin in the sample. That is, most of the acidic desamido insulin detected in the RP-HPLC analysis is actually formed during the sample preparation step. Selecting a Tris buffer would minimize the formation of desamido insulin, but could cause conversion of any human insulin ester present during the sample preparation, and then the RP-HPLC analysis would give a wrong impression of the purity of the sample. In the sample preparation of crystals from the final product of insulin, the compromise is to dissolve the samples in cooled hydrochloric acid and hold the prepared samples refrigerated in order to minimize deamidation. The

sample shown in Fig. 5, panel 4 is a crystal of the final product, prepared by dissolution in 0.01 M hydrochloric acid. The majority of the acidic desamido insulin shown in this chromatogram is the result of a compromise in the conditions, which were selected so as to optimize the ability to determine unconverted human insulin ester while minimizing the formation of desamido insulin. During the extraction and purification of insulin, alcohol is frequently used. In order to obtain sharp peaks of insulin and avoid differences in affinity to the stationary phase, the alcohol content of the samples injected should be less than 30%.

Additional analytical techniques

HPLC is a vital analytical technique for in-process control of insulin. However, other analytical techniques give valuable complementary information when evaluating the quality of a product. Among the many possibilities, ion-exchange chromatography and capillary electrophoresis are examples of such supplementary separation mechanisms. Recent work has shown applications for separations of insulin-related components by capillary electrophoresis [ 12151.

Conclusions
A properly functioning process control system is an important tool in efforts to optimize and maintain high yield and purity of a product. Advantages of monitoring the process of manufacturing insulin by RP-HPLC have been accentuated. The potentials of the specific RP-HPLC method have been pointed out. Chromatograms from RP-HPLC analysis of real samples collected during the production of insulin have been used to illustrate the importance of information obtained by a thorough in-process control. The final aim is to know the yield, loss and quality of the final product.

References
J. Brange, B. Skelbaek-Pedersen, L. Langkjaer, U. Damgaard, H. Ege, S. Havelund, L. G. Heding, K. H. Joergensen, J. Lykkeberg, J. Markussen, M. Pingel and E. Rasmussen, Galenics of Insulin Preparations, Springer-Verlag, Berlin, Heidelberg. J. Markussen, U. Damgaard, M. Pingel, L. Snel, A. R.

trends in analytical chemistry, vol. 11, no. 70, 1992

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Soerensen and E. Soerensen, Diabetes Care, 6 (1983) 4. J. Markussen, Human Insulin, MTP Press Limited, Lancaster, 1987. J. Markussen, US Pat., 4 343 898, (1982). J. Markussen, U. Damgaard, I. Diers, N. Fiil, M. T. Hansen, P Larsen, E Norris, K. Norris, 0. Schou, L. Snel, L. Thim and H. 0. Voigt, in D. Theodoropoulos (Editor), Peptides 1986, Walter de Gruyter, Berlin, 1987. I. Diers, E. Rasmussen, P. H. Larsen, I. L. Kjaersig, Bioproces. Technol., 13 (1991) 166. A. L. Lehninger, Biochemistry, Worth Publishers, New York, 1975, p. 816. B. S. Welinder, Stationary and Mobile Phase Effects in RP-HPLC of Pancreatic Polypeptides, Denmark, 1992, ISBN 87-9842 19-O-5.

9 L. Snel, U. Damgaard and I. Mollerup, Chromatographia, 24 (1987) 329. 10 J. Markussen, K. H. Joergensen, A. R. Soerensen and L. Thim, Int. J. Peptide Protein Res., 26 (1985) 70. 11 J. Markussen, Dansk Kemi, 10 (1987) 295. 12 G. Mandrup, J. Chromatogr., 604 (1992) 267-281. 13 R. G. Nielsen, G. S. Sittampalam and E. C. Rickard, Anal. Biochem., 177 (1989) 20-26. 14 M. Lookabaugh, M. Biswas and I. S. Krull, J. Chromatogx, 549 (1991) 357-366. 15 P D. Grossmann, J. C. Colbum, H. H. Lauer, R. G. Nielsen, G. S. Sittampalam and E. C. Rickard, Anal. Chem., 61 (1989) 1186-1194.

Dr. G. Mandrup is at Novo Nordisk A/S, Hallas A//6, DK-4400 Kalundborg, Denmark.

Microdialysis sampling
in the Neurosciences (Techniques in the Behavioral and Neural Sciences, Volume 7). edited by T.E. Robinson and J.B. Justice, Jr, Elsevier; Amsterdam, 1991, lJS$ 138.50 (452 pages), ISBN O-444-81194-X; US$ 66SOpaperback, ISBN O-444-89375-X

Microdialysis

Microdialysis sampling has become method in the an important neurosciences and is gaining popularity in other areas requiring in vivo sampling. This book represents the first volume devoted entirely to consideration of microdialysis. It thus fultills a very timely and useful role. The editors have assembled an impressive sampling of some of the leading workers using and developing microdialysis. The area of microdialysis sampling is well covered by these authors, although the focus is predominantly on sampling in the CNS. The first two chapters of this book should be required reading for anyone considering using microdialysis. Chapter 1 provides an excellent general introduction to microdialysis. Chapter 2 then clearly positions microdialysis utility relative to other in vivo techniques in the neurosciences. The next chapters deal with some of

the practical aspects of the microdialysis experiment. Chapter 3 is a detailed discussion of mass transport phenomena important to microdialysis sampling. This chapter is essentially a discussion of mathematical modeling of these phenomena and is likely of interest mainly to true afticionados of microdialysis theory. Chapter 4 presents a more practical discussion of considerations in using the technique. A better organization would have been to switch the order of these two chapters. Chapters 5 and 6 are discussions of the requirements of the analytical methods to be coupled to microdialysis sampling. It must be remembered that microdialysis is a sampling method and its ultimate utility often depends on combination with an appropriate analvtical method. The second half of the book is devoted to chapters dealing with application of microdialysis to specific neurochemical problems or systems. It begins with a brief chapter (Chapter 7) outlining the utility to pharmacokinetic determinations. Chapters 8 through 11 discuss sampling of the monoamine neurotransmitters. Chapters 12 and 13 discuss the challenge of sampling neuropeptides. While all of the previous chapters have focused on rodent experiments, Chapter 14 describes the use of microdialysis in

large animals, with the specific example of sheep. Chapter 15 moves the focus from neurotransmitters to metabolic monitoring by measurement of lactate, glucose, ethanol and choline. The last chapters describe new developments of coupling chemical measurements from microdialysis to behavioral studies (Chapter 16) or moving the technique into the clinic (Chapters 17 through 19). Chapter 19 in particular describes the exciting development of in vivo microdialysis in man. This book is generally well written with useful information in each chapter. With over half of the book devoted to specific applications in the neurosciences, as should be expected from the title, it may appear to be useful only for a neuroscientist. However, anyone interested in microdialysis sampling, regardless of the specific application, would do well to peruse this volume. In conclusion, this book is essential for anyone using or considering the use of microdialysis sampling. C.E. LUNTE
Craig E. Lunte is assistant professor at the Department of Chemistry, University of Kansas, Lawrence, KS 660450046, USA.