DoE-derived continuous and robust process for manufacturing of pharmaceutical-grade wide-range LNPs for RNA-vaccine/drug delivery

Abstract

Lipid nanoparticle (LNP) technology has become extremely demanding for delivering RNA-products and other drugs. However, there is no platform to manufacture pharmaceutical-grade LNPs with desired particle size from a wide range in continuous mode. We have developed a unique platform to obtain any specific size-range of LNPs from 60 to 180 nm satisfying pharmaceutical regulatory requirements for polydispersity index, sterility, dose uniformity and bio-functionality. We applied design of experiment (DoE) methodology and identified the critical process parameters to establish the process for global application. Cross-point validation within the response map of DoE confirmed that the platform is robust to produce specific size (± 10 nm) of LNPs within the design-range. The technology is successfully transformed to production scale and validated. Products from R&D, pilot and production batches for a candidate SARS-CoV-2 mRNA-vaccine generated equivalent biological responses. The data collectively established the robustness and bio-uniformity of doses for global RNA-vaccine/drug formulation.

Figure 1

Schematics of the developed process and comparison with existing method. (a) LNP is formed using a simple T-mixer serviced by flow-controlled syringe pumps. The process flow is indicated by arrow; dotted arrow indicates zoom view of the LNP size. (b) The DoE model and result of the DoE experiments revealed that in a sonication field the size of the LNPs gradually increased with the growing time and pH. Desired size of LNP can be achieved by selecting specific ‘operation space’ (dotted oval). (c) Single step buffer exchange is associated with the gradual size enlargement of LNP (top) but a 2-stage buffer exchange restricts LNP size (bottom). (d) Process development decision tree. (e) Different level of sonication power produced different level of effect on size enlargement of LNP. (f) A schematic diagram of relative position of the flow-path of fluid in sonication field of a water bath sonicator. (g) Process flow for small volume (1 ml and 10 ml) batches. (h) Process flow for large batch in continuous mode.

Figure 2

DoE of mRNA-LNPs formation, cross-point validation and buffer change. (a) Gradual changes in LNP size in respect of varying time and pH; specific size ranges of LNPs are depicted in different colors. Numerical values in white circle indicate representative conditions for the model validity test. (b) Relevant data for PDI values are shown. (c) A representative histogram set from triplicate experiments of model validity test is shown after LNP formation. (d,f) The effect of buffer change on LNP size during stabilization and formulation, and (e,g) relevant PDI values, respectively. n = 3 for all experiments.

Figure 3

Characterization of the method for scale-up process and evaluation of the bioequivalency of doses obtained from scale-up process. (a) The sizes of LNPs do not change during different steps of dose manufacturing and handling; solid line, LNP size and dotted line, relevant PDI values. (b) The changes in LNP size for each steps are shown for individual batch for all 3 batch sizes and found non-significant. Relevant PDI values are shown at the bottom in dark shade. (c) The yield for different sizes of batches. (d,e) Copy numbers of mRNA and mRNA encapsulation efficiency in LNP for different size batches, respectively. Data were analyzed using one-way ANOVA and found non-significant; n = 4 for all experiments of (a–e). (f) Antibody titer in response of vaccination in rabbits from 3 different size batches (n = 6); data were compared by Mann–Whitney test, ****p value < 0.0001, ***p value < 0.001, **p value < 0.01, *p value < 0.05. (g) Neutralization of GFP-expressing SARS-CoV-2 pseudovirus in ACE2-expressing HEK cells by vaccinated sera (1 ×, 10 × and 100 × represents vaccines manufactured from different batch sizes). The data shown here is from 640 × dilution of sera. Compare with a commercially available anti-spike antibody all vaccinated sera showed reduced number of GFP-positive cells (n = 4). (h) The neutralization curve shows the immunized rabbit sera outclassed the commercial SARS-CoV-2 antibody with no significant differences for 3 different size batches (n = 4); the IC₅₀ values were found at 480 ± 30 × dilutions. (i) HIV1-based SARS-CoV-2 pseudovirus neutralization by vaccinated sera was evaluated by RT-PCR (n = 4); the IC₅₀ values were found at 280 ± 40 × dilutions and superior than the commercial antibody.