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. 2023 Dec 15;27(12):2317-2328.
doi: 10.1021/acs.oprd.3c00300. Epub 2023 Nov 16.

Kilo-Scale GMP Synthesis of Renewable Semisynthetic Vaccine-Grade Squalene

Karl J Fisher  1 , Laura Shirtcliff  2 , Greg Buchanan  1 , Andrew W Thompson  1 , Frank X Woolard  1 , Donald H LaMunyon  2 , Jonathan L Marshall  2 , Maya B Baranouskas  2 , Rodger B Voelker  2 , Jason S Lusk  2 , Charles E Wells  2 , Raodoh Mohamath  3 , Robert Kinsey  3 , William R Lykins  3 , Gabi Ramer-Denisoff  3 , Christopher B Fox  3 , Christopher J Paddon  1 , Derek McPhee  1
Affiliations

Kilo-Scale GMP Synthesis of Renewable Semisynthetic Vaccine-Grade Squalene

Karl J Fisher et al. Org Process Res Dev. .

Abstract

Emulsions of the triterpene squalene ((6E,10E,14E,18E)-2,6,10,15,19,23-hexamethyltetracosa-2,6,10,14,18,22-hexaene, CAS 111-02-4) have been used as adjuvants in influenza vaccines since the 1990s. Traditionally sourced from shark liver oil, the overfishing of sharks and concomitant reduction in the oceanic shark population raises sustainability issues for vaccine adjuvant grade squalene. We report a semisynthetic route to squalene meeting current pharmacopeial specifications for use in vaccines that leverages the ready availability of trans-β-farnesene ((6E)-7,11-dimethyl-3-methylene-1,6,10-dodecatriene, CAS 18794-84-8), manufactured from sustainable sugarcane via a yeast fermentation process. The scalability of the proposed route was verified by a kilo-scale GMP synthesis. We also report data demonstrating the synthesized semi-synthetic squalene's physical stability and biological activity when used in a vaccine adjuvant formulation.

Keywords: Squalene; allylic sulfone; farnesene; reductive desulfonylation; semi-synthesis; thiourea inclusion complex crystallization; vaccine adjuvant.

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Conflict of interest statement

Competing Interests All authors declare no competing non-financial interests, but the following declare competing financial interests: C.J.P., G.B., A.W.T. and D.J.M. own shares and possess stock options in Amyris, Inc. F.X.W. owns shares in Amyris, Inc. C.B.F. is an inventor on patents and/or patent applications involving terpenoid emulsion adjuvant formulations. K.J.F. and F.X.W. are inventors on a patent application involving the synthesis of squalene from farnesene.

Figures

Figure 1:
Figure 1:
Emulsion physicochemical stability. Emulsions were stored at 2–8C for 12 months and monitored by dynamic light scattering and pH measurement. (A) Emulsion droplet diameter, (B) size polydispersity index, (C) pH. The dotted lines represent pre-established specification ranges.
Figure 2:
Figure 2:
Emulsion adjuvant activity in mice immunized with split, inactivated H5N1 vaccine antigen. Vaccine antigen was mixed with emulsion immediately prior to intramuscular immunization of C57BL/6 (n = 8) mice. The control group was immunized with antigen alone. All mice were immunized twice, three weeks apart. Serum and splenocytes were collected three weeks after the 2nd immunization. Data are represented as box-whisker plots with bars representing median values, boxes representing 1st-3rd quartiles, and whiskers representing the maximum and minimum values. Statistical comparisons were performed by one-way ANOVA with Tukey’s correction for multiple comparisons, with p-values <0.1 reported on the plots. (A) Antigen-specific serum total IgG (IgGT) endpoint titer measured by ELISA. (B) Antigen-specific serum IgG1 endpoint titer measured by ELISA. (C) Antigen-specific serum IgG2c endpoint titer measured by ELISA. (D) Antigen-specific IL-5-secreting splenocytes measured by ELISpot. (E) Antigen-specific IFNγ-secreting splenocytes measured by ELISpot. (F) Antigen-specific IL-17a-secreting splenocytes measured by ELISpot.
Figure 3:
Figure 3:
Infrared absorption spectrophotometry comparing semi-synthetic squalene (red) with European Pharmacopeia shark squalene standard (blue)
Scheme 1:
Scheme 1:
Retrosynthesis of squalene
Scheme 2:
Scheme 2:
Planned route to vaccine-grade squalene. Glossary: Et2N = N,N-diethylamine; n-BuLi = n-butyllithium; AcOH = acetic acid; 50% NaOH = 50% aqueous sodium hydroxide; TolSo2Na = toluenesulfinic acid sodium salt; LiEt3BH = lithium triethylborohydride; Pd(dppp)Cl2 = palladium diphenylphosphino dichloride. cGMP = IPEC-PQG Joint Good Manufacturing Practices Guide for Pharmaceutical Excipients (v.5, 2002).
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