New process analytical technology and downstream processing for the manufacture of in vitro transcribed messenger RNA

Garbett, Lauren (2025). New process analytical technology and downstream processing for the manufacture of in vitro transcribed messenger RNA. University of Birmingham. Ph.D.

Garbett2025PhD.pdf
Text - Accepted Version
Restricted to Repository staff only until 31 July 2029.
Available under License All rights reserved.
Download (6MB) | Request a copy

Abstract

The first Medicines and Healthcare products Regulatory Agency (MHRA) approved COVID- 19 vaccine being an mRNA vaccine demonstrated one of the key advantages of mRNA therapeutics: the speed of manufacture once the target has been identified. However, given that this was the first mRNA therapeutic to be approved, there is no set manufacturing process with many manufacturing options available, particularly in the downstream processing (DSP) stages. The current shift within the pharmaceutical industry from batch to continuous manufacturing requires a high level of process understanding, and the inclusion of relevant process analytical technology (PAT). However, there is currently a lack of in-line PAT methods for mRNA manufacture. This thesis aims to develop DSP methods and in-line PAT suitable for batch and continuous operation for this purpose.

Here, the advantages of using heat in DSP methods are investigated, using the novel Travelling Heating Zone Reactor (THZR) system for affinity chromatography with an oligo(dT) resin. Additionally, the use of circular dichroism (CD) as a PAT is studied, with the goal of utilising a capillary-based CD system, developed at the University of Birmingham, as an in-line PAT during the DSP steps. The potential use of CD in distinguishing between single-stranded RNA (ssRNA) and double-stranded RNA (dsRNA) is also explored.

Initial batch studies are carried out using a 0.2 mL pre-packed column submerged in a water bath to screen various elution temperatures and buffer conditions to take forward to THZR operation. Based on these preliminary studies, 65 °C was selected as the elution temperature, and a high and low salt buffer condition was chosen (10 mM sodium phosphate, 1 mM EDTA, and 500 mM and 150 mM NaCl for the high and the low salt condition respectively, at pH 7.4). The THZR was then used for three applications: batch purification of crude IVT mRNA mixtures, the continuous concentration of pure mRNA, and the quasi-continuous separation and concentration of crude IVT mRNA. For all of these 3 case studies, the mRNA was eluted in a tall sharp peak, mostly within a single pass of the THZ, leading to little to no mRNA remaining bound to the column. Gel electrophoresis and CD analysis demonstrated the elution of purified, intact mRNA.

CD was demonstrated to be able distinguish between purified and crude IVT mRNA mixtures, particularly at the 210 and 270 nm wavelengths. The ratio between these two wavelengths also gave information on whether the mRNA was single stranded or double stranded, with magnitude of the peak at 270 nm being ~1.5 – 2 times greater than that for the 210 nm peak for ssRNA, whereas for dsRNA the two peaks are of similar magnitude. These two wavelengths were then monitored in-line during further water-bath studies, where both wavelengths demonstrated the ability to recognise purified mRNA from the non-binding species, such as the DNA template, enzyme, and free nucleotides, which the commonly used UV absorbance at 260 nm is not able to achieve.

This project provides novel DSP and PAT methods, contributing to the advancement of existing mRNA production methods, whilst aiding in the transition from batch to continuous mRNA manufacture.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):