Squalene Emulsion Manufacturing Process Scale-Up for Enhanced Global Pandemic Response
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pharmaceuticals Article Squalene Emulsion Manufacturing Process Scale-Up for Enhanced Global Pandemic Response Tony Phan 1, Christian Devine 1, Erik D. Laursen 1, Adrian Simpson 1, Aaron Kahn 1 , Amit P. Khandhar 1, Steven Mesite 2, Brad Besse 2, Ken J. Mabery 3, Elizabeth I. Flanagan 4 and Christopher B. Fox 1,5,* 1 Infectious Disease Research Institute, 1616 Eastlake Avenue E #400, Seattle, WA 98102, USA; [email protected] (T.P.); [email protected] (C.D.); [email protected] (E.D.L.); [email protected] (A.S.); [email protected] (A.K.); [email protected] (A.P.K.) 2 Microfluidics Corp., 90 Glacier Drive #1000, Westwood, MA 02090, USA; [email protected] (S.M.); [email protected] (B.B.) 3 Pall Corp., 20 Walkup Drive, Westborough, MA 01581, USA; [email protected] 4 Sartorius Stedim North America Inc., 565 Johnson Avenue, Bohemia, NY 11716, USA; [email protected] 5 Department of Global Health, University of Washington, Seattle, WA 98102, USA * Correspondence: [email protected]; Tel.: +1-206-858-6027 Received: 8 July 2020; Accepted: 24 July 2020; Published: 28 July 2020 Abstract: Squalene emulsions are among the most widely employed vaccine adjuvant formulations. Among the demonstrated benefits of squalene emulsions is the ability to enable vaccine antigen dose sparing, an important consideration for pandemic response. In order to increase pandemic response capabilities, it is desirable to scale up adjuvant manufacturing processes. We describe innovative process enhancements that enabled the scale-up of bulk stable squalene emulsion (SE) manufacturing capacity from a 3000- to 5,000,000-dose batch size. Manufacture of concentrated bulk along with the accompanying viscosity change in the continuous phase resulted in a 25-fold ≥ process efficiency enhancement. Process streamlining and implementation of single-use biocontainers resulted in reduced space requirements, fewer unit operations, and minimization of cleaning requirements. Emulsion physicochemical characteristics were measured by dynamic light scattering, laser diffraction, and HPLC with charged aerosol detection. The newly developed full-scale process was demonstrated by producing two 5,000,000-dose batches of bulk concentrated SE. A scale-up of adjuvant manufacturing capacity through process innovation enables more efficient production capabilities for pandemic response. Keywords: squalene emulsion; vaccine adjuvant; nanoemulsion; process scale-up; emulsion manufacturing; adjuvant manufacturing; pandemic response 1. Introduction Squalene emulsions (SE) represent an important component of pandemic preparedness. An estimated 200M doses of vaccines containing squalene-based oil-in-water (o/w) emulsions have been administered to humans, making them the most widely used class of adjuvant formulations, except for aluminum salts [1]. Squalene-based emulsions have been shown to help potentiate immune responses against a variety of diseases in preclinical and clinical testing, such as malaria, tuberculosis, leishmaniasis, meningitis, etc. [2], and various squalene-based formulations (e.g., MF59®, AS03™, and AF03™) have been developed by large pharmaceutical companies and included in licensed influenza vaccine products. Pandemic vaccines, in particular, can benefit from the antigen dose sparing Pharmaceuticals 2020, 13, 168; doi:10.3390/ph13080168 www.mdpi.com/journal/pharmaceuticals Pharmaceuticals 2020, 13, 168 2 of 14 effects of squalene emulsions. Indeed, since vaccine production capacity is currently inadequate to meet the demand of a global pandemic, squalene emulsions may play an essential role in expanding vaccine coverage [3]. For example, GSK is making available their AS03 adjuvant for COVID-19 vaccine development partners [4]. While some governments maintain stockpiles of squalene-based emulsions for pandemic preparedness [5], additional adjuvant manufacturing capacity is needed to achieve global pandemic response capabilities [6]. In particular, developing countries with vaccine manufacturing programs could benefit from the increased availability of adjuvants. Previous technology transfer efforts have sought to establish local adjuvant manufacturing capacity [7]. Nevertheless, such efforts have been limited to a relatively small manufacturing scale. The Infectious Disease Research Institute (IDRI) has developed a squalene-phospholipid stable emulsion (SE) that has moved into clinical testing with influenza vaccines, demonstrating dose-sparing capacity [8]. The composition of SE differs from that of other commercially produced squalene emulsions, with SE being the only clinical stage squalene emulsion to employ phospholipid as the primary emulsifier [1]. In the present report, we describe the scale-up of bulk SE adjuvant manufacturing from 3000 to 5,000,000 doses/batch, including the development of innovative process efficiency improvements to allow for a concentrated bulk emulsion manufacturing capacity even in a relatively small Current Good Manufacturing Practice (cGMP) facility. 2. Results and Discussion Scale-up objectives. The pre-determined goal of the project was to establish the capability to manufacture 50 million total doses of squalene emulsion adjuvant within 3 months of a pandemic declaration. IDRI’s cGMP facility is a small 4000 ft2 facility staffed by a team that works a single shift. Assuming some days may be needed for equipment maintenance, we estimated that 50 working days in a 3-month period could be dedicated to adjuvant manufacturing, translating into a 1M dose daily batch size required to meet the pre-determined objective. Due to the facility space constraints, manufacturing multiple batches in parallel lines was not a feasible alternative. To support Phase 1/2 clinical trials, SE had generally been manufactured at the 1-L scale, which is equivalent to ~3000 doses after accounting for manufacturing losses and assuming 250 µL for a human dose (SE is typically manufactured at 4% v/v oil which is designed for bedside mixing with vaccine antigen at 1:1 v:v ratio for a final injection composition of 2% v/v oil, see Table1). Thus, the pre-determined objective required scaling from 3000 doses to 1M doses per day, a ~300-fold increase in scale (i.e., from 1-L batch size to 300-L batch size). Performance criteria to assess success of manufactured emulsions were as follows: particle diameter (Z-ave) of 100 nm as measured by dynamic light scattering, oil and excipient content ≤ within 20% of target values as measured by HPLC with charged aerosol and diode array detection, and a homogeneous milky-white visual appearance. Table 1. Squalene emulsion (SE) composition. Component Concentration in 4% v/v Oil SE (mg/mL) Concentration in 30% v/v Oil SE (mg/mL) squalene 34 257 α-tocopherol 0.2 1.5 DMPC 7.6 57 poloxamer 188 0.36 2.7 glycerol 22.7 170 ammonium phosphate, monobasic 2.7 20.5 ammonium phosphate, dibasic 0.17 1.2 water-for-injection (WFI) QS QS Small scale manufacturing process. The basic manufacturing process and composition for small scale SE (1-L batch) was reported by Fox et al. [9]. Since 2013, some changes in the reported SE Pharmaceuticals 2020, 13, 168 3 of 14 composition have been made, namely replacing egg phosphatidylcholine with synthetic DMPC [10] α andPharmaceuticals the addition 2020, of13,a x smallFOR PEER amount REVIEW of -tocopherol as an antioxidant (Table1)[ 11]. The resulting small3 of 15 scale process is shown in Figure1. Some ine fficient or difficult-to-scale steps can be noted in Figure1, namely water bath sonicationDMPC to disperse components7.6 (diffi cult to scale), separate57 containers required for oil and aqueouspoloxamer phases (space 188 contraints in cGMP0.36 suite for multiple large containers),2.7 and a high number of passes required on the Microfluidizer processor (at an anticipated flow rate of ~3 L/min, glycerol 22.7 170 a 300-L batch size would require 27 h to process 16 passes). Based on these calculations, it was decided ammonium phosphate, monobasic 2.7 20.5 to develop innovations to improve process efficiency and reduce space or equipment requirements ammonium phosphate, dibasic 0.17 1.2 prior to a full scale-up. water-for-injection (WFI) QS QS Figure 1. Small-scale ( 1 L) process flow diagram for production of SE. Figure 1. Small-scale (≤1≤ L) process flow diagram for production of SE. Removal of heated sonication step. To enable process simplification and scale-up feasibility, we evaluatedRemoval theof heated impact sonication of removing step. the To elevated enable temperature process simplification sonication and step. scale Sonication-up feasibility, in a heated we waterevaluated bath the was impact originally of removing presumed the to elevated be necessary temperature to efficiently sonication disperse step. the Sonication emulsifier in (DMPC) a heated in thewater oil bath phase was to promoteoriginally more presumed rapid particleto be necessary size reduction to efficiently in subsequent disperse processing the emulsifier steps. (DMPC) However, in eliminationthe oil phase ofto thepromote heated more sonication rapid particle step appeared size reduction to have in subsequent no detrimental processing effect onsteps. the However, resulting physicalelimination characteristics of the heated of sonication the manufactured step appeared emulsions to have (Table no2 ).detrimental Therefore, effect subsequent on the emulsion resulting processphysical development characteristics omitted of the themanufactured heated sonication emulsions step. (Table 2). Therefore, subsequent emulsion processDispersed