多肽疗法已成为现代药物发现与开发中日益重要的治疗模式。从胰岛素和GLP-1受体激动剂，到用于肿瘤、代谢性疾病、感染性疾病和罕见病的疗法，多肽药物已在广泛适应症中展现出重要临床价值。如今，全球已有超过100款多肽药物上市，还有更多项目正在临床前和临床管线中推进。

药物开发中的订书肽：更佳稳定性、更强靶点结合与CMC挑战

多肽疗法介于小分子药物与生物制品之间，兼具高靶点特异性、灵活的分子设计空间和较强的生物活性。然而，许多天然多肽的成药性有限，可能限制其进一步治疗应用。

为改善多肽药物的成药性，化学修饰已成为一项关键策略。其中，大环化修饰，包括多肽订书技术，可以限制多肽分子的构象，并增强其蛋白水解稳定性。通过稳定常参与蛋白-蛋白相互作用的α-螺旋结构，订书肽（stapled peptide）有望进一步拓展多肽药物的治疗潜力。

环合烯烃复分解反应（RCM）是制备订书肽最成熟的方法之一。Helen Blackwell和诺贝尔奖得主Robert H. Grubbs的开创性研究证明，RCM可用于生成共价交联的多肽螺旋结构，从而推动全烃订书成为订书肽设计中的重要策略。

然而，正是这些赋予订书肽重要科学价值的结构特征，也可能增加其生产难度。从实验室合成放大至公斤级临床试验用物料，并不仅仅意味着验证反应可行性。临床规模的订书肽生产可能面临化学、生产和控制（CMC）挑战，包括非天然氨基酸（UAA）来源、RCM转化率以及金属残留控制。对于处于临床阶段的生物技术公司而言，这些问题并非孤立的化学难题，而是可能影响开发周期、成本、质量和临床供应的开发风险。

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案例研究：一个面临低收率、高催化剂用量和临床试验物料（CTM）时间压力的2期订书肽项目

订书肽临床生产面临的多重挑战，在一家生物技术公司的2期三重订书肽（triple stapled peptide）项目中集中显现。该项目的既有生产工艺导致产品纯度低、收率低且不稳定、催化剂用量高以及催化剂残留控制困难等问题。与此同时，项目还需要开发超高浓度注射制剂，并及时交付CTM，项目时间线几乎没有延误空间。

一体化执行如何解决订书肽生产挑战？

该项目在其他CDMO无法破解多重挑战后，被转移至药明康德，公司旗下WuXi TIDES平台通过一体化方法帮助解决这些相互关联的挑战。从项目启动之初，WuXi TIDES团队就认识到，这并不仅仅是一项收率提升工作，而是一个更广泛的CMC执行挑战，需要多个工作模块协同推进、同步解决问题。

方法一：确保UAA供应，降低来源风险

对于该订书肽项目而言，能否获得可靠来源的非天然氨基酸，可能直接影响工艺开发能否按计划推进。在本案例中，合成该肽分子需要两种非商业化的UAA，带来了潜在原料供应风险和时间线不确定性。为降低这一风险，团队在短短6周内自主设计并开发了UAA合成路线，并最终交付了25公斤纯度高于99%的UAA。这为后续肽合成和纯化工艺开发提供了保障，同时帮助避免外部供应不确定性造成的延误。

方法二：重新设计RCM条件，提高转化率和粗品纯度

团队重新优化了RCM工艺，以提高转化率并降低下游杂质负担。在此前的工艺中，为弥补低转化率问题，催化剂用量被反复提高。然而，仅增加催化剂用量可能无法解决RCM效率低下的根本问题。相反，高催化剂用量反而会增加杂质负担，并提高下游纯化的复杂度，包括较高的残留钌（ruthenium）水平、纯化困难以及更大的杂质控制压力。相比之下，团队通过优化溶剂体系、底物浓度和催化剂添加策略等关键反应参数，在分子构象约束较强的情况下提高了RCM转化率，降低了催化剂用量。

方法三：降低钌残留并改善prep-HPLC纯化

残留钌控制和纯化稳健性同样是使订书肽工艺适用于临床规模生产的关键。此前的粗品API含有较高水平的残留钌，外观颜色较深，这缩短了prep-HPLC色谱柱填料的寿命，并增加了纯化复杂性。为解决这一问题，团队在prep-HPLC前实施了柱前金属清除策略。该方法使成品API中的残留钌水平大幅降低，同时该策略还帮助延长色谱柱填料的使用寿命，节省了生产成本，并将纯化周期缩短了30%。

结果：八个月内将不稳定工艺推进至千克级GMP API交付

通过这些协同干预，团队将一个低收率、不稳定的实验室工艺转化为更稳健、可放大的GMP生产工艺。最终收率提高超过2倍，在八个月内交付公斤级GMP API。对于一个涉及连续RCM反应且金属残留控制具有挑战性的复杂三重订书肽而言，这意味着工艺性能和临床供应准备度的显著提升。

通过并行CMC执行加速CTM交付

除API工艺优化外，项目速度还取决于并行CMC执行。在GMP API生产推进的同时，分析方法开发与验证也同步开展。团队得以及时确认金属残留指标、杂质谱分布和稳定性指导数据，降低了后期返工风险并支持及时放行。

在API放大生产推进的同时，制剂团队也在约五周内筛选了30多个处方原型（formulation prototypes）。该环节的关键挑战在于开发一种适合临床给药的超高浓度注射制剂，同时保持溶解性、可注射性和物理稳定性。通过系统性的处方筛选与优化，团队最终实现了浓度达到百毫克每毫升级以上的稳定注射用肽溶液。

一体化执行推动2期CTM交付在11个月内完成

由于UAA供应、API工艺优化、分析方法开发、处方筛选和CTM生产以协同方式推进，各工作模块之间没有出现空档期。CTM生产、包装和放行在三个月内完成，整个项目从转移至WuXi TIDES到2期CTM放行共耗时11个月。

该案例表明，临床规模订书肽生产不仅涉及复杂化学问题，更考验一体化CMC执行能力。通过统筹UAA供应、RCM工艺优化、钌残留控制及检测、prep-HPLC纯化、高浓度处方开发和CTM生产等关键环节，一体化平台能够帮助将难以放大的工艺转化为符合GMP要求的生产工艺。在这一临床2期项目中，药明康德支持了复杂订书肽项目临床试验物料的及时交付，也体现出其更广泛的目标：解决复杂开发与生产挑战，赋能客户推动有前景的疗法更快惠及有需要的患者。

Stapled Peptide Manufacturing: How WuXi AppTec’s CRDMO Platform Helps Advance Complex Peptides to Clinical Supply

Key Takeaways

  • Stapled peptides offer important therapeutic potential, but medium- to large- scale manufacturing can be challenging because of complex sequences, sourcing of unnatural amino acids, multiple ring systems, catalyst residue control, purification, and formulation requirements.

  • In this case study, WuXi AppTec helped to address poor process robustness, high catalyst loading, unacceptable ruthenium residue, prep-HPLC purification challenges, and high-concentration formulation requirements through an integrated approach.

  • The project delivered kilogram-scale GMP API within 8 months, reduced residual ruthenium in the final API by approximately 75-fold compared with the crude material, achieved a peptide solution above 100 mg/mL. Project onboarding to Phase 2 CTM release was completed in 11 months.

Peptide therapeutics have become an increasingly important modality in modern drug discovery and development. From insulin and GLP-1 receptor agonists to therapies for oncology, as well as metabolic, infectious, and rare diseases, peptide-based medicines have demonstrated meaningful clinical value across a broad range of indications.Today, over 100 peptide drugs are available globally, and many more are advancing through preclinical and clinical pipelines.

Stapled Peptides in Drug Development: Better Stability, Stronger Target Engagement, and CMC Challenges

Peptide therapeutics bridge small molecules and biologics, combining high target specificity, design flexibility, and strong biological activity. Yet many native peptides have limited drug-like properties, which can restrict their therapeutic use.

Chemical modification is a key strategy to improve peptide drug-like properties.Macrocyclization, including peptide stapling, can constrain peptide conformation and enhance proteolytic stability.By stabilizing α-helical structures often involved in protein–protein interactions, stapled peptides may expand the therapeutic potential of peptide-based medicines.

Ring-closing olefin metathesis (RCM) is one of the most established methods for creating stapled peptides. Pioneering work by Helen Blackwell and Nobel Laureate Robert H. Grubbs demonstrated that RCM could generate covalently cross-linked peptide helices, helping establish hydrocarbon stapling as an important strategy in stapled peptide design.

However, the structural features that make stapled peptides scientifically compelling can also make them challenging to manufacture. Scaling from laboratory synthesis to kilogram-scale clinical trial material requires more than reaction feasibility. Clinical-scale stapled peptide manufacturing can face Chemistry, Manufacturing, and Controls (CMC) challenges, including unnatural amino acid (UAA) sourcing, RCM conversion, and metal residue control.For clinical-stage biotech companies, these issues are not merely isolated chemistry hurdles but development risks that can affect timelines, cost, quality, and clinical supply.

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Case Study: A Phase 2 Stapled Peptide Facing Low Yield, High Catalyst Loading, and CTM Timeline Pressure

These challenges came into focus for one biotech company developing a Phase 2 triple stapled peptide. The existing manufacturing process suffered from low purity, low and unstable yield, high catalyst loading, and difficult catalyst residue control. At the same time, the program required an ultra-high concentration injectable formulation and timely Clinical Trial Material (CTM) delivery, with limited room for delay.

How Can Integrated Execution Solve Stapled Peptide Manufacturing Challenges?

After the project experienced extensive challenges at another CDMO, the molecule was transferred to WuXi AppTec, where the WuXi TIDES team helped to address these interconnected challenges.From the beginning, the team recognized that this was not simply a yield-improvement project. It was a broader CMC execution challenge that required coordinated problem-solving across multiple workstreams.

Approach 1: Securing UAA Supply to Reduce Sourcing Risk

For stapled peptide programs, reliable access to non-commercial unnatural amino acids can be critical to keeping process development on schedule. In this case, the peptide required two non-commercial UAAs, creating potential sourcing risk and timeline uncertainty.To reduce this risk, the team developed an in-house synthetic route in 6 weeks and delivered 25 kilograms of UAA with greater than 99% purity.This secured a consistent supply of high-quality building blocks for peptide synthesis and process development, while helping avoid external supply delays.

Approach 2: Re-Engineering RCM Conditions to Improve Conversion and Crude Purity

In parallel with other CMC workstreams, the team re-engineered the RCM process to improve conversion and reduce downstream impurity burden. In the previous process, catalyst loading had been repeatedly increased to compensate for low conversion. However, increasing catalyst loading alone may not resolve the root causes of poor RCM efficiency. Instead, high catalyst loading increases both impurity burden and downstream purification complexity, including elevated residual ruthenium, difficult purification, and greater impurity-control pressure. By contrast,the team optimized key reaction parameters, including the solvent system, substrate concentration, and catalyst addition strategy. These changes improved RCM conversion despite increasing conformational constraints, reduced catalyst burden, and increased crude purity by 33%.

Approach 3: Reducing Ruthenium Residue and Improving Prep-HPLC Purification

Residual ruthenium control and purification robustness were critical to making the stapled peptide process suitable for clinical-scale manufacturing. The previous crude material contained a high level of residual ruthenium and appeared dark, which shortened prep-HPLC column lifetime and increased purification complexity. To improve metal impurity control before purification, the team implemented a pre-column metal-scavenging strategy before prep-HPLC.This strategy reduced residual ruthenium by approximately 75-fold from the crude material to the final API. It also helped protect column integrity and reduced the purification cycle time by 30%.

Results: From an Unstable Process to Kilogram-Scale GMP API in 8 Months

Through coordinated CMC interventions, the team transformed a low-yielding, unstable process into a more robust and scalable manufacturing route.Final yield increased by more than twofold, and several kilograms of GMP API were delivered within 8 months.For a complex triple-stapled peptide involving sequential RCM reactions and challenging metal-residue control, these improvements strengthened both process performance and clinical supply readiness.

Accelerating CTM Delivery Through Parallel CMC Execution

Beyond API process optimization, speed also depended on parallel CMC execution. While GMP API production was underway, analytical method development and validation advanced in parallel. Metal quantification, impurity profiling, and stability-indicating methods were aligned early to reduce the risk of late-stage rework and support timely release.

In parallel with API scale-up, the drug product team screened more than 30 formulation prototypes within approximately 5 weeks. The key challenge was to develop an ultra-high concentration injectable formulation suitable for clinical dosing while maintaining solubility, injectability, and physical stability.Through systematic formulation screening and optimization, the team ultimately achieved a stable injectable peptide solution at a concentration exceeding 100 mg/mL.

Integrated Execution Enabled Phase 2 CTM Release in 11 Months

Because UAA supply, API process optimization, analytical development, formulation screening, and CTM manufacturing were advanced in a coordinated manner, the project moved forward without idle time between functions.CTM manufacturing, packaging, and release were completed within 3 months, and the overall project—from project onboarding to Phase 2 CTM release—was completed in 11 months.

This case shows that clinical-scale stapled peptide manufacturing is not only a chemistry challenge, but also an integrated CMC execution challenge. But by coordinating UAA supply, RCM process optimization, ruthenium residue control, prep-HPLC purification, high-concentration formulation, and CTM release, an integrated platform can help transform a non-scalable process into a GMP-ready manufacturing pathway. In this Phase 2 program, WuXi AppTec’s WuXi TIDES team helped support timely clinical trial material delivery for a complex stapled peptide, reflecting a broader goal: solving difficult development and manufacturing challenges so that promising therapies can advance faster toward the patients who need them.

参考资料：

[1] Li Y, Wu M, Fu Y, Xue J, Yuan F, Qu T, Rissanou AN, Wang Y, Li X, Hu H. Therapeutic stapled peptides: Efficacy and molecular targets. Pharmacol Res. 2024 May;203:107137. doi: 10.1016/j.phrs.2024.107137. Epub 2024 Mar 23. PMID: 38522761.

[2] Lau YH, de Andrade P, Wu Y, Spring DR. Peptide stapling techniques based on different macrocyclisation chemistries. Chem Soc Rev. 2015 Jan 7;44(1):91-102. doi: 10.1039/c4cs00246f. Epub 2014 Sep 8. PMID: 25199043.

[3] Stapled peptide. Retrieved May 13, 2026 from https://en.wikipedia.org/wiki/Stapled_peptide#cite_note-pmid24601557-8

[4] Moiola M, Memeo MG, Quadrelli P. Stapled Peptides-A Useful Improvement for Peptide-Based Drugs. Molecules. 2019 Oct 10;24(20):3654. doi: 10.3390/molecules24203654. PMID: 31658723; PMCID: PMC6832507.

[5] Robert H. Grubbs. Retrieved May 13, 2026 from https://www.nobelprize.org/prizes/chemistry/2005/grubbs/facts/

[6] Al Musaimi O. FDA's stamp of approval: Unveiling peptide breakthroughs in cardiovascular diseases, ACE, HIV, CNS, and beyond. J Pept Sci. 2024 Nov;30(11):e3627. doi: 10.1002/psc.3627. Epub 2024 Jun 17. PMID: 38885943.

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