Successfully Transitioning mRNA Production to cGMP Requires Early Process Engagement for Developing Efficient, Cost-effective Manufacturing

Messenger RNAs (mRNAs) are most known for being the active ingredient in COVID-19 vaccines (1). However, mRNA’s clinical potential is also being actively leveraged for therapeutic applications, such as protein replacement, oncology, autoimmune diseases, cell and gene therapy, among others, resulting in 335 ongoing clinical studies (2) involving mRNA.

Bringing mRNA biotherapeutics successfully to the clinic and later to commercialization requires development of appropriate upstream and downstream processes suitable for cost-effective manufacturing of mRNA with the required critical quality attributes (CQAs).

Manufacturing of mRNA at cGMP standards is challenging due to the limited number of available technologies suitable for production (3) and requires careful consideration and alignment of several tasks to build a functional end-to-end solution. Figure 1 shows the complexity of different activities that must be interlinked to prepare a robust and successful cGMP mRNA manufacturing campaign.

Typically, several steps need to be taken to successfully bring an mRNA molecule towards a cGMP manufacturing campaign (see Figure 2). The detailed work-flow starts with template design followed by in vitro transcription (IVT) of the mRNA and downstream purification steps. The next stage involves process development and scale up and these decisions will have a major impact and knock-on effects when manufacturing at cGMP scale. This is where engaging with a development partner such as Lonza that has built considerable expertise in the production of mRNA, as both a drug substance (DS) and a drug product (DP) (4,5) can help de-risk the project and increase the chances of achieving a successful cGMP campaign.

What you’ll discover in this article

In this article, we will discuss why early process optimization is key to developing an efficient, cost-effective cGMP manufacturing process to deliver high-quality mRNA. We will provide insights through case studies on how a Design of Experiments (DoE) approach combined with experience can optimize the upstream, by increasing yields while reducing by-products and impurities resulting in a simplified downstream. We will also detail with a case study - how to make the downstream process adaptable and flexible. Finally, we will illustrate how partnering with Lonza in the early process stages may help to develop a scalable, robust, and economically viable cGMP campaign.

mRNA Process Steps - Are All Steps Scalable and cGMP Compatible?

IVT - mRNA Capping

Before transferring an mRNA production process from R&D to a larger scale, it is important to define the quality target product profile, and then ensure that such product characteristics are met in each unit operation. This strategy will help to develop a platform process where units of operation can be combined or exchanged while being scalable and cGMP compatible.

One of the most important decisions to make when transferring an mRNA production process to cGMP manufacturing is to define the capping strategy. Currently, two main choices are available:

1. co-transcriptional, or
2. post-transcriptional (enzymatic) capping

Both approaches provide efficient capping of the mRNA 5’ end but the selected option will have an effect on yield, stability of the transcript during IVT reaction, as well as on the design of the downstream process - Figure 3 provides an overview of the impact of the capping strategies in the overall manufacturing of mRNA. Therefore, it is important to define from the outset the desired balance between yield, process speed and quality of the final DS and then select the raw materials and process steps that best address such strategic decisions.

Upstream Considerations for cGMP Production

A robust upstream process consists of a scalable IVT reaction in which key parameters are efficiently controlled because of exhaustive characterization of the chemical reaction space. This approach will allow complete definition of ranges and limits of the process. Optimization of reaction parameters, including enzyme kinetics, will reduce formation of by-products and impurities and can reduce downstream complexity. An in-depth knowledge of the IVT process will help maximize the balance between yield, quality and manufacturing costs. This will in turn mitigate and reduce risks by fully understanding Critical Process Parameters (CPPs).

Case Studies

Study 1: Optimizing IVT conditions.

The main goal of this study was to develop a platform mRNA IVT process using plasmid DNA (pDNA) as a template. To determine optimal IVT conditions, we performed a Design of Experiment (DoE) screening complemented with a few conditions derived from internal know-how resulting in a screening panel of over 50 different IVT combinations. The data obtained were analyzed and compared to standard, starting IVT conditions. Our results (Figure 4) show that some conditions resulted in a two-threefold increase in mRNA yields. In subsequent studies we defined a smaller chemical space (only 20 conditions) sufficient to identify the most suitable IVT conditions for the tested mRNA, thus significantly reducing the workload and still allowing us to achieve our goal of developing a scalable, robust process that could be used with a range of DNA templates in terms of both mRNA size and DNA source (bacterial produced pDNA or chemical synthetized DNA).

Study 2: Impact of template sequence and size on IVT performance

To determine the effect of template sequence and size on mRNA yield, we used our optimized IVT conditions (determined in Study 1) to produce a large mRNA molecule (large) and a standard mRNA molecule small). We compared mRNA yields from both processes and our results (Figure 5) showed that the larger construct which was sequence optimized counterintuitively resulted in higher yields than the smaller construct. This indicates that optimizing the sequence can improve yields irrespective of the size of construct used. Furthermore, it implies that in addition to sequence optimization, a screening to find optimal IVT conditions should be performed even for small mRNA templates. As in this study, there is potential to improve the yield of the smaller mRNA by at least 30 %, which would have a significant impact on yield, as well as minimizing costs of goods for a cGMP process.

Study 3: Optimizing IVT conditions using dbDNA

Doggybone DNA (dbDNA) can be used as an alternative to pDNA as a DNA template for IVT. In this study, the goal was to optimize our platform mRNA IVT process for use with dbDNA.

We used two different starting concentrations of dbDNA (40 ng/µL and 50 ng/µL) with 22 of our optimized IVT conditions (determined in Study 1). We assessed the mRNA yields from the screens and our results (Figure 6) demonstrated that we obtained a higher concentration of mRNA (6.0 mg/mL +/- 0.3) using the lower concentration of starting template dsDNA (40 ng/µL) compared to the higher concentration (50 ng/µL) dbDNA template which generated mRNA at 5.5 mg/mL +/- 0.2. These results indicate that we can adapt our IVT screening method and use it with different DNA templates – pDNA as well as dbDNA. The screening platform is independent of the DNA nature and/or source and can be used to assess the optimal starting DNA concentration for IVT reactions.

Assessing mRNA IVT Critical Raw Materials

mRNA production by in vitro transcription requires a range of critical raw materials. Raw materials supplier selection for: nucleotides, capping reagents, and enzymes, impacts IVT process yield, impurities profile, as well as downstream processing scales and costs. We routinely evaluate different suppliers of raw materials and assess their products according to criteria such as quality, price, volume required, security of supply chain, and cGMP compliance.

Case study 4: Performance of T7 Polymerases in IVT

In this study, we performed yield comparability assessments with T7 polymerases from three different suppliers. While IVT conditions were optimized for supplier 1 our results (Figure 7) demonstrated that the T7 polymerase from supplier 3 produces almost two-fold higher yield than the T7 polymerase from supplier 1, showing that a single IVT raw material can drive major changes in process yields. However, optimizing yields needs to be balanced with the cost of the raw material, mRNA quality and, as the process is transferred to cGMP, quality compliance aspects also need to be reviewed.

Downstream Considerations for cGMP Production

In the downstream processes intrinsic physicochemical characteristics, defining each specific mRNA construct, should drive the selection of the most appropriate strategy. Downstream processing should be optimized for reduced duration with a minimum number of steps, in order to guarantee mRNA quality and to minimize loss of material - thereby lowering the costs for raw materials. Scalability and cleanability of equipment (if required) from process development to cGMP manufacturing should be simplified and process yields can be increased by choosing process development equipment that can generate critical process data from an early development stage.

As mentioned above, generation of higher yields decreases the cost of raw materials, as well as reduces process development time, resulting in shorter project timelines. However, the amount and quality of data gathered during process development must be sufficient to define Critical Process Parameters (CPPs) and support design space definition, thus increasing process robustness and ensuring predictive process modeling. As with the upstream process, achieving these optimization goals will mitigate and reduce risks during scale up and transfer to cGMP.

Case Study 5: Diafiltration Cross Flow Filtration Scouting

The aim of the study was to determine the optimum cross-flow filtration conditions able to maximize mRNA product recovery while minimizing process time and cost. We assessed four different cross flow feed conditions (see Table 1). We loaded set concentrations of mRNA (small and large constructs) previously pre-purified from a chromatography step. We then compared all four crossflow conditions and our results (Figure 8) show that as the cross flow feed rate (L/min/m2) increased, the flux rate (LMH) also increased. More importantly, we measured high mRNA recovery (>80%) at all feed rates, indicating that our cross-flow method can operate successfully and independently of feed rate and mRNA size and could be used as a platform method to purify a range of mRNA constructs.

Conclusions

When making the transition to cGMP mRNA manufacturing the number of tasks that need to be accomplished to build an end-to-end workflow is often underestimated even by biopharma companies that are used to operating in state-of-the-art cGMP facilities. To build a bridge between innovation and manufacturing, the development team or CDMO should be asked to answer questions regarding early development work such as how robust is the IVT screening process? And to what extent is the process optimized for variances in mRNA constructs? In the downstream, the main questions to ask should be about how to design the downstream process. Which and how many units of operation? Some specific questions such as how many process parameters will be evaluated for ultrafiltration/diafiltration (UF/DF)? What chromatographic steps need to be added? Are the units selected capable of providing mRNA at the desired quality? Early decisions will also impact the process equipment dimensioning and determine if the cGMP facility is fit for purpose. Optimizing IVT conditions, assessing different raw materials and considering the effect of downstream process parameters can have a major impact on process time, mRNA yield and purity which could make the difference between success or failure of a cGMP manufacturing campaign.

Increase your chances of success with an experienced team

Therefore, partnering with an experienced team able to provide early engagement in process development to establish a platform, in which every step is characterized and optimized to accommodate for variance, is critical to improve overall campaign performance. Engaging with a knowledgeable development partner such as Lonza who can guide you along that process will increase your chances of success and accelerate your route towards cGMP readiness with your mRNA therapeutics.

Want to know more about transitioning to cGMP mRNA manufacturing?

Find out more about our mRNA manufacturing services and discover how you can experience seamless tech transfers of your processes into our manufacturing network. Alternatively, you can contact our friendly experts here.

References

1. Stanton, D. Moderna contracts Lonza to help scale up COVID-19 mRNA vaccine candidate. Bioprocess Insider. 2020 https://bioprocessintl.com/bioprocess-insider/facilities-capacity/moderna-contracts-lonza-to-help-scale-up-covid-19-mrna-vaccine-candidate/
2. Clinicaltrials.gov Database Search for Clinical Trials with mRNA therapeutics | Recruiting, Active, not recruiting https://www.clinicaltrials.gov/ct2/results?term=mRNA+therapeutics&Search=Apply&recrs=a&recrs=d&age_v=&gndr=&type=&rslt= Accessed 16.06.2023.
3. Clemens, J, et al. Current and Emerging Technologies to Optimize mRNA Manufacturing https://www.lonza.com/-/media/Lonza/knowledge/mRNA/22_biologics_mRNA_White_Paper_Update_LoRes_v4.pdf
4. Mullin, R. Lonza to build mRNA and small-molecule plants in Visp. C& EN. 2021 ; 99, (16) https://cen.acs.org/business/outsourcing/Lonza-build-mRNA-small-molecule/99/i16
5. Press release 2021: Lonza and Moderna Announce Further Collaboration for Drug Substance Manufacturing of COVID-19 Vaccine Moderna in the Netherlands https://www.lonza.com/news/2021-06-02-07-02