A novel method for the synthesis of peptides

A novel method for the synthesis of peptides in solution DioRaSSP (Diosynth Rapid Solution Synthesis of Peptides) offers substantial benefits for the large-scale synthesis of peptides meeting all the specifications required of peptide manufacturing in the 21st century. Ivo Eggen and Paul Ten Kortenaar, Diosynth BV Ever-increasing pressure is being imposed upon the pharmaceutical industry to reduce time-tomarket for new drugs. From the point-of-view of the API (active pharmaceutical ingredient) manufacturer, time-to-market comprises the development of synthesis routes for new compounds, the scale-up of the ensuing processes, and their subsequent validation and registration. An additional challenge lies in the eventual manufacturing of APIs of increasingly higher and reproducible purity in a commercially competitive way one that is environment-friendly and compatible with evermore stringent guidelines regarding cgmp. ese incentives prompted Diosynth s R&D Peptides Department to perform a thorough re-evaluation of the two classical approaches to peptide synthesis that is, classical solution-phase peptide synthesis (CSPS) and solid-phase peptide synthesis (SPPS), taking into account the extensive knowledge of impurity profiles built up by the company over the course of 50 years of peptide manufacturing. Speed of development During the development of a synthesis route, speed is firstly achieved through the application of a generic protocol; this is an important prerequisite for the eventual automation of a synthesis. A generic protocol has never been achieved for CSPS, since functional side-chains of amino acid residues are often not protected in this approach thus accounting for intermediates (that is, the growing peptide) with strongly varying chemical and, more importantly, physical properties. Intermediates of such syntheses are usually isolated by precipitation and filtration. Especially during these isolations when a transition from a homogeneous to a heterogeneous system occurs a great variation in the protocols applied is inevitable. Heterogeneity is also the major cause of complications during the scale-up of a synthesis with both the CSPS and SPPS approaches. Finally, the heterogeneic nature of SPPS inhibits the proper characterisation of intermediates, and therefore complicates validation of the process. It is obvious that integral homogeneity during a synthesis expedites its development, as well as its scale-up and validation. Manufacturing efficiency In the last decade, there has been a shift from CSPS towards SPPS for the manufacturing of peptides on In the last decade, there has been a shift from CSPS towards SPPS for the manufacturing of peptides on a large scale. Innovations in Pharmaceutical Technology 123

As well as commercial competitiveness, the robustness of a manufacturing process and hence quality assurance is a highly important requirement in API manufacturing. a large scale. Two intrinsic properties of the SPPS approach contribute to its commercial competitiveness. First, no intermittent isolations occur during synthesis on a solid support. Second, SPPS follows a generic protocol and is therefore automatable. Both of these aspects increase the manufacturing efficiency and are not applicable to CSPS. Despite these benefits of the SPPS approach, manufacturing efficiency is compromised somewhat by the cost of starting materials. ese are relatively high due to the use of expensive non-reusable resins and amino acid derivatives. e intrinsic heterogeneity of SPPS is often reflected in retarded coupling rates, thus necessitating the application of large molar excesses of reagents and amino acid derivatives during SPPS couplings. Moreover, all functional side-chains must be protected in SPPS in order to make the hydrophobic resin accessible to the otherwise polar amino acids, and avoid the accumulation of sequences containing modified side-chains. Product quality assurance As well as commercial competitiveness, the robustness of a manufacturing process and hence quality assurance is a highly important requirement in API manufacturing. Impurities in the final product of a peptide synthesis may be roughly divided into five different categories: epimers, insertion sequences, deletion sequences, truncated sequences and impurities arising from modifications of functional side-chains on the actual peptide. Epimers originate from racemisation of amino acid derivatives during their coupling, and are essentially independent of the synthesis approach being determined rather by the conditions and reagents applied during coupling. Modifications of functional side-chains on a peptide are often introduced in the later stages of a synthesis hence after the assembly of the actual sequence when protecting groups on the constituting functional side-chains are being or have been removed. However, in CSPS, these side-chains are often not protected, and modifications may thus also occur during the assembly of the actual sequence. Insertion, deletion and truncated sequences, on the other hand, always originate from assembly of the (protected or semi-protected) sequence. Insertion sequences containing one or more additional amino acid residues are mainly encountered in products originating from CSPS. In order to impede their formation, all residual unactivated carboxylic compound should be removed before the coupling step of the next cycle of the synthesis, while all residual activated carboxylic compound should be removed even before the following deprotection step. It is generally believed that residual activated carboxylic compound is destroyed during the aqueous work-up after coupling and, as such, is removed by aqueous work-up or precipitation before the coupling step of the next cycle of the synthesis. However, the detection of substantial quantities of insertion sequences in peptide products originating from CSPS shows this assumption to be incorrect. In classical synthesis approaches, quenching of residual activated carboxylic compound sometimes occurs with a polyamine. is type of quenching generates basic quenched compounds which are depending on their hydrophobicity only partly removed prior to the following deprotection step, and which cannot be actively removed (that is by means of acidic aqueous extraction) before the coupling step of the next cycle of the synthesis due to the risk of loss of peptide material. is approach therefore necessarily results in the formation of C- terminally truncated sequences. Deletion sequences lacking one or more amino acid residues are primarily encountered in peptide products originating from SPPS, since thoroughly quantitative in-process analysis of single synthesis steps is not practicable due to the heterogeneous character of the synthesis. Reproducibility of the impurity profile of a peptide product is dependent on the reproducibility of the production process and its parameters. In CSPS, reproducibility is compromised during the isolations, whereas in SPPS, reproducibility is difficult to achieve in terms of swelling properties and loadings of the applied solid supports. Consequently, an adaptation of the classical methods for peptide synthesis is required to prevent the formation of these various impurities and assure peptide products of reproducible high quality. A final aspect of CSPS and, especially, SPPS that demands re-evaluation pertains to the reduction of organic waste streams originating from the application of these syntheses on a manufacturing scale. The DioRaSSP approach Based on the above considerations, Diosynth s R&D Peptides Department has developed a new and patented method for the preparation of peptides in solution called DioRaSSP, Diosynth Rapid Solution Synthesis of Peptides. e characteristics of this and the two classical methods for peptide synthesis are summarised in Table 1. With the DioRaSSP approach, the growing peptide is essentially anchored in a permanent organic phase (generally ethyl acetate) by means of its hydrophobic C-terminal and side-chain protecting groups. A synthesis performed according to the DioRaSSP protocol is completely homogeneous and its intermediates are not isolated. Excess reagents and by-products are intermittently removed by aqueous extractions, and no organic waste streams are generated during performance of the synthesis. 124 Innovations in Pharmaceutical Technology

Table 1. Comparison of methods for peptide syntheses. Aspect Determined by CSPS SPPS DioRaSSP Time-to-market Route development Generic protocol + + Scale-up & Validation Homogeneous synthesis +/ + Manufacturing efficiency Cycle times No isolations + + Automation + + Materials Small excess reagents + + No solid support + + Minimal side-chain protection + +/ Product quality assurance High purity No insertion sequences + + No deletion sequences + + No side-chain reactions + + Reproducibility Reproducible isolations n.a. n.a. Reproducible supports n.a. n.a. Environmental demands Organic waste streams No solvent changes + + No organic washings + + a member of the Solvay group PEPTISYNTHA s activities focus on the production of large volumes of therapeutic peptides (peptide APIs). Our experts have decades of experience in producing c-gmp peptides in accordance with the most stringent FDA and EMEA requirements. Both solution phase and solid phase technologies are available. Applying a proprietary solution phase technology, PEPTISYNTHA is able to scale up the production of peptides up to hundreds of kilograms, including HPLC purification and lyophilisation. PEPTISYNTHA : the right solution for bulk peptides! Peptisyntha S.A. 310 rue de Ransbeek - B-1120 Brussels, Belgium Tel. : +32 2 264 2213 - Fax. : +32 2 264 3470 Email : pierre.barthelemy@solvay.com Peptisyntha Inc. Higgins Court 20910 - Torrance, California 90501 Tel. : +1 310 782 7524 - Fax. : +1 310 782 7352 Email : satish.joshi@solvay.com www.peptisyntha.com Innovations in Pharmaceutical Technology 125

Figure 1. DioRaSSP synthesis cycle. one cycle of the DioRaSSP protocol consists of a coupling step, quenching of residual activated carboxylic compound, aqueous extractive work-up, deprotection of the N-terminal amino function, and finally another aqueous extractive work-up. As shown in Figure 1, one cycle of the DioRaSSP protocol consists of a coupling step, quenching of residual activated carboxylic compound, aqueous extractive work-up, deprotection of the N-terminal amino function, and finally another aqueous extractive work-up. e benzyloxycarbonyl (Z) function is applied for temporary amino protection, whereas tert-butyl-type functions or functions of similar lability are applied for the semi-permanent protection of certain functional side chains. e former is removed by hydrogenolysis in each cycle of the process. Side-chains are only protected if this is required from a chemical point-of-view, or to ensure solubility of the growing peptide in the organic phase. e applied protection scheme in the DioRaSSP protocol justifies the commercial viability of its application on a manufacturing scale. Moreover, on account of its homogeneous character, reagents and amino acid derivatives may be applied in low molar excess. After completion of a coupling, residual activated carboxylic compound if hydrophobic is quenched using an anion-forming amine such as benzyl β-alaninate. is approach allows the completely quantitative removal of quenched compounds before the coupling step of the next cycle of the synthesis by basic aqueous (that is active) extraction. e application of an anion-forming amine in the quenching step of the DioRaSSP protocol, accompanied by the appropriate work-up procedures, enables absolute prevention of the formation of insertion and/or truncated sequences. 126 Innovations in Pharmaceutical Technology

Deletion sequences are avoided in the DioRaSSP protocol, since all reactions can be closely monitored. Functional side-chains on the growing peptide, being shielded by protecting groups, are not modified during assembly of the sequence. The state-of-the-art Syntheses performed according to DioRaSSP proceed by a generic and fast protocol. In the last three years, a considerable number of protected peptides have thus been synthesised at Diosynth varying from tripeptides to a dodecapeptide. Purities and yields are generally high, as exemplified by the synthesis of a protected human insulin octapeptide fragment, which was obtained in 98% purity and 85% yield (corresponding to an average yield of 99% per chemical conversion) in the first seven days trial. Several DioRaSSP processes have been directly scaled up after a preliminary feasibility study at the laboratory scale, achieving reproducible results in terms of both yield and purity. e application of DioRaSSP implies the same process and impurity profile throughout all stages of development that is, from the first laboratory sample to production batches. Accordingly, Diosynth aims at a process time of 2-4 days per residue for the synthesis of a first 10 gram-scale sample of a protected peptide, 3-6 days per residue for route optimisation, 3-6 days per residue for the synthesis of a 1kg scale development batch, and 2-4 days per residue for the synthesis of a multi-kg scale validation batch. Moreover, Diosynth is currently handling implementation of the first fully automated solution-phase synthesiser, which will increase the potential of the DioRaSSP method even further. It may be concluded that DioRaSSP offers substantial benefits concerning time-to-market, manufacturing efficiency, quality assurance and the environment, and thus meets all specifications for peptide manufacturing of the 21st century. Dr Ivo Franci Eggen is Head of the Research Department for Peptide Synthesis at Diosynth BV, Oss, the Netherlands. He received his PhD in organic chemistry at the University of Nijmegen, the Netherlands, in 1999; his PhD investigation which was supervised by renowned peptide chemists Zahn, Tesser and Brandenburg was focused on the synthesis of peptides by solid-phase peptide synthesis. In the following years at Diosynth, he gained much experience in solution-phase peptide synthesis and, in his role as Head of the Research Department for Peptide Synthesis, conducted the investigations towards development of the DioRaSSP method. Dr Paul BW Ten Kortenaar is Section Manager of the R&D Peptides & Chemicals Department at Diosynth BV, Oss, the Netherlands. He received his PhD in Organic Chemistry at the University of Nijmegen, the Netherlands, in 1983. After postdoctoral positions at the University of North Carolina, Chapel Hill, USA, and at the University of Wageningen, the Netherlands, he took up the position of Head of the Large-scale Custom Synthesis Laboratory at the University of Nijmegen. Subsequently, he spent several years at Organon BV as a Senior Scientist before taking up his present position at Diosynth, where he is responsible for all R&D activities in the fields of peptides, carbohydrates, alkaloids and heterocyclic compounds. Several DioRaSSP processes have been directly scaled up after a preliminary feasibility study at the laboratory scale, achieving reproducible results in terms of both yield and purity. Innovations in Pharmaceutical Technology 127