Quality by design for mRNA platform purification based on continuous oligo-dT chromatography

Abstract

Oligo-deoxythymidine (oligo-dT) ligand-based affinity chromatography is a robust method for purifying mRNA drug substances within the manufacturing process of mRNA-based products, including vaccines and therapeutics. However, the conventional batch mode of operation for oligo-dT chromatography has certain drawbacks that reduce the productivity of this process. Here, we report a new continuous oligo-dT chromatography process for the purification of in vitro transcribed mRNA, which reduces losses, improves the efficiency of oligo-dT resin use, and intensifies the chromatography process. Furthermore, the quality by design (QbD) framework was used to establish a design space for the newly developed method. The optimization of process parameters (PPs), including salt type, salt concentration, load flow rate and mRNA load concentration both in batch and the continuous mode, achieved a greater than 90% yield (mRNA recovery) along with greater than 95% mRNA integrity and greater than 99% purity. The productivity of continuous chromatography was estimated to be 5.75-fold higher, and the operating cost was estimated 15% lower, when compared with batch chromatography. Moreover, the QbD framework was further used to map the relationship between critical quality attributes and key performance indicators as a function of critical process parameters and critical material attributes.

Keywords: Continuous manufacturing; MT: Delivery Strategies; mRNA therapeutics; mRNA vaccines; multi-column continuous chromatography process; oligo-dT affinity chromatography; quality by design.

Graphical abstract

Figure 1

Overview of the proposed mRNA-based vaccines and therapeutics manufacturing process and the oligo-dT affinity purification method (A) Illustration of the continuous mRNA-based vaccines and therapeutics manufacturing platform process with examples of CMAs, CPPs and CQAs for the continuous chromatography process. (B) Schematic of the oligo-dT affinity chromatography process.

Figure 2

Oligo-dT chromatography results from one-factor-at-a-time ÄKTA PCC batch runs for assessing the impact of loading salt type CPP, loading salt concentration CPP, eGFP mRNA load concentration CPP and the flow rate CPP on the mRNA yield KPI (measured by AEX HPLC), mRNA integrity CQA (measured by CGE) and mRNA purity CQA (measured by AEX HPLC) The mean CQA and KPI values are shown by dots, and error bars represent standard deviation for three replicates (n = 3). (A) Varied mRNA load concentration (mg/mL), under constant load flow rate of 1 mL/min, constant 0.6 M NaCl concentration in loading buffer, and a constant total loading volume of 10 mL. (B) Varied flow rate (mL/min), under constant mRNA load concentration of 0.4 mg/mL, constant 0.6 M NaCl concentration in loading buffer, and a constant total loading volume of 10 mL. (C) Varied NaCl salt concentration (M), under constant mRNA load concentration of 0.6 mg/mL in constant 10 mL load volume and constant flow rate of 1 mL/min. (D) Varied Gu-HCL salt concentration, under constant mRNA load concentration of 0.6 mg/mL in constant 10 mL load volume and constant flow rate of 1 mL/min.

Figure 3

Results from multi-factorial optimisation of mRNA yield (A) Three-dimensional surface plot for the Oligo-dT chromatography multi-factorial results from ÄKTA PCC batch runs for assessing the impact of Gu-HCl load salt concentration CPP, mRNA load concentration CPP, and load flow rate CPP on mRNA yield KPI (measured by AEX HPLC). (B) Two-dimensional projection plots of (A) onto the side, rear, and bottom planes. Red lines, Gu-HCl concentration at 0.3 M; blue lines, Gu-HCl concentration at 0.6 M; black lines, Gu-HCl concentration at 0.9 M.

Figure 4

Chromatogram of the continuous oligo-dT results performed at load salt concentration of 600 mM Gu-HCl, mRNA load concentration of 0.23 mg/mL and load flow rate of 4 mL/min In total, 3 cycles of 12 load-elutes were performed using 1 mL monolith oligo-dT columns, with a total run time of 70 min excluding the start-up equilibration and shut-down phases. In this run, 41.44 mg of mRNA was purified.

Figure 5

Oligo-dT chromatography chromatogram from ÄKTA PCC continuous runs for assessing the impact of mRNA load concentration CPP and the load flow rate CPP on the mRNA yield KPI (measured by AEX HPLC), mRNA integrity CQA (measured by CGE) and mRNA purity CQA (measured by AEX HPLC) The load volume was 80 mL (20 mL for each column load-elute) per test. The mean CQA and KPI values are shown by dots, and error bars represent standard deviation for three replicates (n = 3) of tests 1–9. (A) Chromatograms of the 9 oligo-dT continuous chromatography tests. LC, load concentration (mg/mL); LFR, load flow rate (mL/min). (B) Yield (mg), mRNA integrity (%), and mRNA purity (%) of the nine tests. (C) Yield (%), process time (min), and productivity (mg/min/mL) of the purification runs.