Analytical Procedures For Quality of mRNA Vaccines

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Analytical Procedures for

Quality of mRNA Vaccines


and Therapeutics
Draft Guidelines: 3rd Edition
Accelerating product development through a common understanding of quality

Vast potential of mRNA technology. The development of A common set of methods is needed. Since mRNA
prophylactic vaccines to combat COVID-19 has shown the technology is relatively new, regulatory guidelines and
potential of mRNA technology, particularly in infectious industry standards to guide non-proprietary aspects of
diseases, and paved the way for a range of novel vaccines mRNA quality during development and manufacturing
and therapies to come. Developing and manufacturing are still evolving. These include areas such as verifying
vaccines based on this modality have proven faster than identity, controlling impurities, and measuring content for
other platforms, making mRNA a promising solution for dosing. Without a common set of methods for determining
addressing future pandemics, as well as other infectious quality, developers and manufacturers must develop their
diseases, such as rabies, Zika, and cytomegalovirus own in-house methods and protocols, taking attention and
infection. Several mRNA-based therapeutic products are resources away from a company’s successful development
also in clinical pipelines for cystic fibrosis and various and commercialization of mRNA technology unique to the
cancers. medical product.

Managing quality is critical. To build public trust and


confidence in innovative products like mRNA vaccines
and therapeutics, they must be, safe, effective, and of
high quality. Like other biologics, manufacturing mRNA-
based products is complex. As changes are made to
raw materials, starting materials, processing steps, and
formulation during the progression from early research and
development to large-scale manufacturing, robust testing is
needed to ensure the quality and safety of the final product.
Methods for determining product quality that are suitable
for preclinical or early clinical development phases may
not be fit for purpose at commercial scale. Product quality
attributes that are not properly identified and addressed can
impact the integrity of the product, leading to poor clinical
outcomes, causing costly delays, and threatening regulatory
approval.

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Analytical Procedures for Quality of
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Draft guidelines

There are four main types of RNA therapeutic General Chapters <1235> Vaccines for Human Use—General
strategies: Considerations and <1239> Vaccines for Human Use—Viral
mRNA, antisense RNA (asRNA), RNA interference (RNAi) Vaccines.
and RNA aptamers. Of the four, only mRNA-based products
In February 2022, USP and our BIO3 Expert Committee
involve delivery of in vitro transcribed mRNA into a target
released the first edition of the draft guideline to solicit
cell, where cellular machinery translates the mRNA into
feedback from global stakeholders on the included methods
a protein. There are three different modalities that utilize
and to encourage submission of any alternative methods
mRNA.
and additional supporting documentation, including
1) Vaccination – where mRNA encoding specific target validation packages related to the methods presented in the
antigen(s) is administered to elicit protective immunity. draft guidelines. USP received over 300 comments, edits,
2) Cell therapy – where mRNA is transfected into the and several donations of methods. In the second edition,
cells ex vivo to alter cell phenotype or function. stakeholder feedback was addressed, and the donated
These cells are then delivered into the patient. methods were incorporated.

3) Replacement therapy – where mRNA is In April 2023, the second edition of the guidelines
administered to the patient to compensate was launched by USP to gather further feedback from
for a defective gene or a protein. stakeholders worldwide. During this phase, USP actively
sought public comments on the revised mRNA draft
The global mRNA therapeutic and vaccine market is guidelines, receiving input from various stakeholders
expected to expand significantly in the next ten years due alongside several donated methods. With the release of
to the proven effectiveness of the platform (i.e., Covid-19 the third version, USP has meticulously addressed public
vaccines), speed to design and produce the product, as well comments and integrated donated methods into the
as greater flexibility compared to traditional approaches.
guidelines. Additionally, USP has conducted evaluations
mRNA-based therapeutics and vaccines are in development
of methods for several critical quality attributes (CQA) and
for a variety of indications, including infectious diseases,
incorporated these methods into the draft guidelines.
metabolic diseases, cancer, and cardiovascular diseases.
Major updates in this version include the following:
Creating shared understanding is critical to advancing
adoption of this technology. To address this need, USP is • Replaced Table 1 with a new version adapted
developing a set of analytical methods for mRNA quality to from proposed USP Chapter <1040> which has
support developers, manufacturers, regulatory agencies, been published in Pharmacopeial Forum.
and national control laboratories worldwide. The goal • Added donated LC-MS method for 5’ capping efficiency
is to create an overall shared understanding of mRNA
• Added donated LC-MS method for 3’ poly(A) tail length
quality attributes, with the aim of accelerating product
development, guiding successful manufacturing scale-up, • Updated immunoblot method
and bolstering regulatory confidence in the utilization of • Added residual NTPs and capping agent
best practices and suitable quality controls when developing
in mRNA method by AEX-HPLC
and manufacturing this new modality.
• USP has evaluated the following methods
Based on needs identified by various stakeholders, USP
– LC-MS method for 3’ poly(A) tail length
and our BIO3 – Complex Biologics and Vaccines Expert
Committee developed this draft guideline as a first step – Immunoblot method for dsRNA impurity
towards developing a procedural guideline for testing – AEX-HPLC method for residual NTPs
of mRNA vaccines. It includes analytical procedures and
and capping agent in mRNA
best practices to support the assessment of common
quality attributes of mRNA vaccines and therapeutics. – CGE method for mRNA integrity
This draft guideline builds on best practices described in – IP-RP-HPLC method for mRNA purity

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Analytical Procedures for Quality of
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To further support the quality and consistency of These proposed compendial methods would be published
analytical methods used for mRNA-based vaccines and in the Pharmacopeial Forum for public comment prior to
therapies, USP has initiated evaluation of several methods publication as Chapters in USP-NF. In other cases, where
in USP laboratories, with the goal of qualifying and/ standardization is more challenging due to the variety
or validating these methods. In some cases, where the of mRNA products in development, methods and other
method is applicable to a broad range of mRNA products, associated information may be published outside of the
methods may be advanced as documentary standards compendial process (e.g., draft guidelines, technical notes,
(for example general chapters with validated methods). or scientific publications).

Additional collaboration is needed


To advance these draft guidelines, we invite industry, academic and government experts with experience or interest
in mRNA vaccines and technology to provide feedback on the methods detailed in these draft guidelines and to
recommend additional information supporting the understanding of mRNA quality. We encourage the submission of
any alternative analytical methods as well as supporting documents (e.g., validation documentation). By collaborating
with USP, participants play an active role in shaping standards and solutions that contribute to building the supply of
safe, effective, quality medicines that people around the world can trust.

To provide feedback on this proposed draft or inquire about other aspects of this work, contact
USPVaccines@usp.org. More information can be found on our website at http://www.usp.org/mrna-quality.

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Analytical Procedures for Quality of
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Draft guidelines

Introduction
Naturally occurring mRNA is produced in eukaryotic cells by the transcription of DNA in the nucleus by RNA polymerase.
The mRNA molecules are transported out of the nucleus to the cytoplasm, where they serve as templates for translation by
ribosomes to produce proteins. In this way, the information stored in the genome is used to express specific proteins. An mRNA
cannot create any protein other than the protein for which it is coded.
Two main forms of mRNA vaccines have been developed: non-replicating mRNA vaccines (conventional) and self-amplifying
mRNA (SAM) vaccines as shown in Figure 1 below. The conventional non-replicating mRNA vaccine construct consists of a 5′
7-methylguanosine cap structure, a 5′ untranslated region (UTR), the open reading frame (ORF) encoding the target protein, a 3′
UTR, and a 3′ poly (A) tail. SAM vaccines are derived from alphavirus genomes, where the mRNA molecule additionally encodes
replicases that can direct intracellular mRNA amplification. In both forms of mRNA vaccines, the UTR regions are important for
regulating protein expression, blocking exonuclease-mediated mRNA degradation, and enhancing translation efficiency. The
UTRs, 5’ cap, and poly(A) tail also help stabilize the mRNA molecule itself inside the cell.

Figure 1: Two Main Forms of mRNA Vaccines


5’ 3’ 3’

CAP UTR ORF UTR —AAAAAA CAP UTR Replicase ORF UTR —AAAAAA

A) Non-replicating mRNA B) Self-amplifying mRNA (SAM)

There are several ways mRNA drug substances can be manufactured including with the use of PCR-generated template or non-
linearized plasmid with the terminator sequence. The mRNA drug substance template can be prepared by amplification by host
cells (e.g., Escherichia coli). In either case, each lot of the plasmid DNA that is used in manufacturing of the mRNA vaccines,
must be tested to confirm its identity, purity, and quality before release. Plasmid DNA is considered a GMP-level starting material
and must be manufactured in accordance with the principles of GMP from the initial cell bank stage onward. Best practices
to manage cell banks and testing recommendations is described in General Chapters <1042> Cell Banking Practices for
Recombinant Biologics.
Table 1 below summarizes the attributes and suggested methods which are described in more detail in the proposed General
Chapter currently in PF <1040> Quality Considerations of Plasmid DNA As a Starting Material for Cell and Gene Therapies. The
goal of this general chapter is to describe considerations for the manufacture and release testing of plasmids for use as starting
materials. The document was published in the Pharmacopeial Forum (PF) 49(6) in November 2023 for 90-day public comment
period. Currently, the public comments received are being reviewed by the expert panel followed by the expert committee.

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Table 1: Plasmid DNA Quality Attributes, USP Chapter <1040>

Quality Attribute Analytical Procedures Example Acceptance Criteriaa


Appearance—color and Visual Inspection from 〈631〉b or Ph. Eur. Clear, colorless solution but may be molecule and/or
clarity 2.2.2 process specific
〈790〉,c〈1790〉d, Ph. Eur. 2.9.20, Ph. Eur.
Visible particulates Free of visible particulates
2.9.19
pH 〈791〉e End use-specific
A260
Concentration End use-specific
Size exclusion HPLC
Restriction map Identical to theoretical or identical to reference map
Identity
Sequencing Identical to reference sequence
Identity of homologous Restriction map
regions including long
PCR The integrity of these regions should be understood as
terminal repeats (LTRs),
part of the overall control strategy.
inverted terminal repeats
Sequencing
(ITRs), and poly(A) tails
Agarose gel electrophoresis
Anion exchange (AEX) HPLC Criteria should be end use-specific. Here are some
possible attributes of interest: Control for appropriate
Plasmid DNA topology Capillary gel electrophoresis (CGE)
percentage total supercoiled with low percentage of
Analytical ultracentrifugation open circular, linear, and other forms
Electron microscopy
Colorimetric [e.g., bicinchoninic acid
(BCA), Lowry]
Residual protein End use-specific; 1–10% (w/w)f
Host cell protein enzyme-linked
immunosorbent assay (ELISA)
Quantitative polymerase chain reaction
Residual host DNA End use-specific; 1–10% (w/w)f
(PCR)
Agarose gel electrophoresis (AGE) with
an appropriate stain
Fluorescent labeling of RNA measured
Residual host RNA by fluorimetry End use-specific; 1–10% (w/w)f

Reverse phase HPLC

RT-PCR
ELISA
End use-specific; consider both patient and
Residual antibiotic(s) Mass spectrometry
environmental safety
Reverse phase HPLC
Endotoxins <85>g End use-specific; ≤10–100 EU/mg
Bioburdenh <61>i End use-specific; in general, ≤1 CFU/10 mL

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Analytical Procedures for Quality of
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Quality Attribute Analytical Procedures Example Acceptance Criteriaa


No growth [NOTE—Scale of manufacturing may make
Sterility h
<71> j
sampling per 〈71〉 difficult. See
5.1.12 Bioburden and Sterility for more details.]
Should not be present in microbial generated
plasmid DNA. Testing is optional de- pending on
Mycoplasmak <63>l
application. May be a requirement to rule out human
contamination.
a Example acceptance criteria are provided when available, unless it is so specific and specific to the process (end use-specific).
b Color and Acromicity <631>
c Visible Particulates in Injections 〈790〉.
d Visual Inspection of Injections 〈1790〉.
e pH <791>.
f May need to consider patient safety guidelines when setting these limits.
g Bacterial Endotoxins Test <85>.
h Testing of both bioburden and sterility are not needed. One or the other should be chosen based on application and agreement with health authorities. If sterility is the testing approach that
is chosen, sampling per <71> might not be feasible due to manufacturing scale, etc. See 5.1.12 Bioburden and sterility for more details.
i Microbial Enumeration Tests <61>.
j Sterility Tests <71>.
k Mycoplasma does not infect prokaryotic cells, so by default it should not be present in plasmids prepared from microbial sources. However, this test may be required for entry of the DNA
starting material into eukaryotic manufacturing facilities and by regulatory authorities.
l Mycoplasma tests <63>.

Manufacturing of mRNA drug substance can be broken down to 2 essential steps: upstream enzymatic processes and
downstream chromatography and ultrafiltration-based purification. The upstream process requires high quality template DNA
for optimal in vitro transcription (IVT) yields. The DNA plasmid is enzymatically linearized and purified before it is used in large
scale manufacture of the mRNA intermediate, in a cell-free system via in vitro transcription from the linearized plasmid DNA
template. Due to the degeneracy of the genetic code, the mRNA sequence can be optimized with respect to codon usage for
the more efficient translation and stability. In addition, modified nucleosides can be used to enhance the function of the mRNA.
The 5´-cap can be introduced co-transcriptionally by adding capping reagents to the IVT mix. Also, the 3´-poly (A) tail can be
added enzymatically or can be encoded in the DNA template. This all depends on the specific manufacturing process. The
mRNA drug substance is then purified and formulated to make the drug product. The mRNA-based therapeutics and vaccines
require a delivery system such as polymers, polymer-based nanoparticles, lipids, or lipid nanoparticles for entry into recipient
cells. While there are ranges of types of vehicles available to deliver mRNA, LNPs currently are the most advanced system
that has been demonstrated to be safe and effective. LNPs protect mRNA from degradation and enable cell entry through
endocytosis. Once in the endosome, mRNA molecules are released from the endosome into the cytoplasm providing templates
to produce multiple copies of the expressed protein. In an mRNA-based vaccine, the expressed protein serves as an antigen to
stimulate an immunological response, which is the desired outcome of vaccination.
When mRNA is used as a therapy, modifications can be introduced into the mRNA molecule to enhance functionality including
translation efficiency. This is achieved through modifications which decrease nucleoside modification, inhibit nuclease
resistance, and decrease immunogenicity of the mRNA itself. When mRNA is used as vaccines, the exogenous mRNA can
activate innate immune cells via Toll-like receptors (TLR) 3, 7, and 8, other receptors like RIG-I, or effector proteins. TLR ligation
leads to the production of cytokines which results in the generation of adaptive T and B cell responses. Signaling of TLR7
augments production of proinflammatory cytokines, increases antigen presentation, and improves memory B cell survival.
The quality of mRNA drug substance and drug product is determined by their design, development, and specifications applied
to them during the development and manufacturing process (Figure 2). Quality control checks on starting materials such as
nucleotides/ nucleosides, enzymes (for synthesis, linearization, and capping), solvents and buffers, polymers, nanoparticles,
lipids/ lipid nanoparticles must be incorporated into the testing program. In cases where animal and/or human derived
materials are used, they should be tested for adventitious agents. For a well characterized product, analytical methods for
characterization, lot release and stability testing of drug substance and drug product must be developed and performed to
monitor critical quality attributes and other product attributes. Stability studies must be performed to define and confirm

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product shelf life, understand potential degradation pathways, and support comparability assessments. When scaling up or
otherwise modifying the manufacturing process, comparability studies may include both routine (e.g., release) and non-routine
(e.g. characterization) testing (ICH guideline Q5E). This guideline provides methods for assessment of quality attributes for
characterization and release testing for bulk purified mRNA drug substance, as listed in Table 2. The mRNA drug substance
requires formulation (e.g., LNPs) for delivery; suggested methods to support lot release and stability testing of LNP-mRNA
encapsulated drug product characterization and release testing are listed in Table 3 below. These tables define the quality
profiles for drug substance and drug product with method options. These tests are examples and developers will need to
evaluate and decide in conjunction with health authorities what is appropriate for a given product; alternative methods,
technologies, equipment, and reagents may be selected on a case-by-case basis. Among these, stability-indicating parameters
may include mRNA quantity, integrity, degree of encapsulation, potency, particle size, polydispersity and/or impurities
associated with the mRNA and lipid components. Note however that stability-indicating methods must be assessed and defined
on a case-by-case basis for each product and relevant stage, based on experience.

Figure 2: mRNA Production Process and Testing

Upstream Downstream

LNP
Template In vitro Template Flow Filtration Flow Filtration
Sterile Drug Encapsultation Drug
DNA mRNA DNA Quencing Dilution Conc & Buffer Chromatography Conc & Buffer
Filtration Substance & Final Product
Linearization Transcription Digestion Exchange 1 Exchange 2
Formulation

Release assays
Each lot of plasmid must be analyzed Lot-to-lot comparability Characterization
− appearance,
prior to release − confirm identity, to demonstrate & Release assays
concentration,
purity & quality successful scale up − appearance,
purity, quality,
and tech transfer of the concentration,
identity, potency,
production process purity, safety
safety

Stability testing − long term stability


studies, real time stability study as well as
accelerated temperature conditions must
be conducted

Table 2: Characterization and release testing for mRNA Drug Substance

Quality Attribute Method

High throughput sequencing (HTS)

Identity mRNA sequence identity confirmation Sanger sequencing

Reverse Transcriptase – PCR (RT-PCR)

Quantitative reverse-transcription PCR (RT -qPCR)

Content RNA concentration Reverse-transcription digital PCR (RT–dPCR)

Ultraviolet Spectroscopy (UV)

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Table 2: Characterization and release testing for mRNA Drug Substance (Continued)

Quality Attribute Method

Capillary electrophoresis D

Integrity mRNA intactness Capillary gel electrophoresis (CGE) D

Agarose gel electrophoresis


Ion pair reversed-phase high-performance liquid
mRNA purity
chromatography (IP-RP-HPLC)
Reverse-phase liquid chromatography mass
spectroscopy (RP–LC-MS/MS) D
5’ capping efficiency Ion pair reversed-phase high-performance liquid
chromatography (IP-RP-HPLC)
Liquid chromatography mass spectroscopy (LC-MS/MS) D

Liquid chromatography mass spectroscopy (LC-MS/MS) D


3’ poly(A) tail length Ion pair reversed-phase high-performance liquid
chromatography (IP-RP-HPLC)
Immunoblot
Product related impurities - dsRNA
Purity Enzyme-linked immunosorbent assay (ELISA)
Product related impurities - aggregate Size exclusion-high-performance liquid chromatography
quantitation (SEC-HPLC) D
Product related impurities - percentage
Reversed-phase HPLC (RP-HPLC) D
of fragment mRNA
Process related impurities - residual DNA
quantitative PCR (qPCR)
template
Process related impurities - quantitation Reverse-phase liquid chromatography mass
of free/non-incorporated nucleosides spectroscopy (RP–LC-MS/MS) D
Process related impurities - residual NTP Anion exchange high-performance liquid
and capping agent chromatography (AEX-HPLC) D
Process related impurities - residual T7
Enzyme-linked immunosorbent assay (ELISA)
RNA polymerase content
Potency Expression of target protein Cell-based assay

Endotoxin USP <85>


Safety
Bioburden USP <61>, <62>, <1115>

Appearance USP <790>

Other Residual solvents USP <467>

pH USP <791>

D Donated methods

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Table 3: Characterization and release testing for mRNA Drug Product

Quality Attribute Method

Sanger sequencing

RNA identification Reverse Transcriptase – PCR (RT-PCR)


Identity
Identity of lipids Reversed-phase high-performance liquid
chromatography with charged aerosol detector
(RP-HPLC-CAD)

RNA concentration/RNA encapsulation efficiency Fluorescence-based assay

Content Reversed-phase high-performance liquid


Lipid content chromatography with charged aerosol detector
(RP-HPLC-CAD)
LNP size and polydispersity Dynamic light scattering (DLS)
Integrity
RNA size and integrity Capillary gel electrophoresis (CGE) D
Product related impurities - aggregate Size exclusion-high-performance liquid chromatography
quantitation (SEC-HPLC) D
Purity
Product related impurities - percentage of Ion pair reversed-phase high-performance liquid
fragment mRNA chromatography (IP-RP-HPLC) D
Potency Expression of target protein Cell-based assay

Endotoxin USP <85>


Safety
Sterility USP <71>

Appearance USP <790>

Residual solvents USP <467>

Osmolality USP <785>

Other Subvisible particles USP <787>

Extractable volume USP <1>, <698>

Container closure integrity USP <1207>

pH USP <791>
D Donated methods

[Note: all temperatures are written in “ ° “, denoting Celsius.]

Identity

Identity of Encoded RNA Sequence by HTS


Multiple commercial instruments are available for mRNA sequencing. A common form of this technique involves library
preparation, cluster generation, sequencing, and bioinformatic data analysis, including quality control determinations. Library
preparation involves mRNA enrichment and isolation through the hybridization of the mRNA poly(A) tail to a poly(T) oligomer
attached to a solid support, typically a magnetic bead. The isolated mRNA is fragmented in the presence of divalent cations

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and at high temperature, or through other appropriate mechanical cleavage methods. The mRNA fragments are then used as
the templates to make double-stranded (ds) complementary DNA (cDNA) using reverse transcriptase and random primers. DNA
adapters and indexes are then ligated onto the ends of the ds cDNA that are in preparation for amplification. The constructed
library of cDNA fragments is then subjected to amplification using specific primer sets that are complementary to those used
during library construction along with fluorescent labeled deoxynucleoside triphosphates (dNTPs) and dideoxynucleotide
triphosphates (ddNTPs). The ddNTPs act as reversable terminators that prohibit any further attachment of nucleotides at the 3′
end in a given sequencing cycle. Once completed, most sequencing instruments use optical detection to determine nucleotide
incorporation during DNA synthesis, while others may use electrical detection. Appropriate software and bioanalytical tools are
then used to determine the sequence of the starting mRNA molecule.
Purification and fragmentation of mRNA: One of the key processes in HTS is the enrichment of mRNA for the subsequent
library construct. Fragmentation and library preparation should be done directly on the RNA.
SDS lysis buffer: 1% SDS, 10 mM of EDTA
RNA fragmentation buffer (10X): 1M Tris, pH 8.0 and 100 mM of MgCl2
Stop solution: 200 mM of ethylenediaminetetraacetic acid (EDTA), pH 8.0
For the RNA purification step, for each reaction, add the following mixture in each well of the 96 well plate. Mix 14.5 µL of SDS
lysis buffer, 48 µL of 6M GuHCl and 7.25 µL of proteinase K (20mg/mL). Add 1 – 10 µg of mRNA sample (A260/280 ratio of RNA
should be around 2:1). Mix well and incubate at room temperature for 10 min and then heat at 65° for 10 min prior to the addition
of 145 µL of RNA clean-up beads.1 Wash beads three times in 70% ethanol using a magnetic bead stand and then elute RNA
into the 30 µL resuspension buffer. Assess the quality of RNA by using Agilent Fragment Bioanalyzer system or CGE method
(integrity methods provided below).
Alternatively, mRNA-sequencing (mRNA-Seq) protocol can be applied using the poly(A)-selection strategy for purifying mRNA
by filtering RNA with 3′ polyadenylated (poly(A)) tails to include only mRNA. First the total RNA is briefly denatured, followed by
hybridization of polyadenylated 3’ ends to oligo(dT) beads. All other non-polyadenylated transcripts such as rRNA, tRNA, and
degraded RNA are washed away in the final step.
For mRNA fragmentation, mix 1–18 µL of purified mRNA, 2 µL of RNA fragmentation buffer (can be prepared fresh or purchased)
and nuclease-free water to final volume of 20 µL in a sterile PCR tube.2 Incubate in a preheated thermal cycler for 5 min at
94°. Transfer the tube to ice and add 2 µL of Stop solution. Clean fragmented RNA using ethanol precipitation. Mix 22 µL of
fragmented RNA, 2 µL of 3M sodium acetate at pH 5.2, 1-2 µL of 10 mg/mL linear acrylamide and 60 µL of >99.8% ethanol in
a sterile 1.5 mL microcentrifuge tube. Mix well and incubate at −80° for 30 min. Centrifuge the tube in a microcentrifuge at
appropriate rotation, time, and temperature (at e.g. maximum speed (10,000 x g) for 25 min at 4°). Carefully remove supernatant
and wash the pellet with 300 µL of 70% ethanol. Repeat the wash step and remove 70% ethanol. Air dry the pellet for up to 10
min at room temperature to remove residual ethanol and resuspend in 14.5 µL of nuclease-free water.
Synthesis of first strand cDNA: RNA fragments are reverse transcribed to cDNA because the DNA is more stable and allows for
amplification using DNA polymerases. mRNA can be transcribed from the coding strand (has the same sequence as mRNA) or
template strand (used for transcription). This process will use the cleaved RNA fragments into the first strand of cDNA using
random primers and reverse transcriptase.
First strand buffer (5X): Mix 250 mM of tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) at pH 8.3, 375 of mM KCl,
and 15 mM of MgCl2
Second strand buffer (2X): Mix 0.2 M of HEPES at pH 6.9, 20 mM of MgCl2, 5 mM dithiothreitol and 0.14 M KCl
10 mM dNTP mix: Mix 10 mM of each nucleotide (dATP, dCTP, dGTP and dTTP) in 0.6 mM of Tris-HCl.
In a 200-µL PCR tube, add 1 µL of gene specific primers and 11.1 µL (≤ 1 µg of total RNA or 50 – 100 ng of poly(A)+ RNA) of mRNA.3
Incubate the sample in a PCR thermal cycler at 65° for 5 min and then place on ice immediately. Set the thermal cycler to 25°.
For each reaction, mix the following reagents in the order listed in a separate PCR tube. Add 4 µL of First strand buffer prepared

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fresh or from a kit,4 2 µL of 100 mM DTT, 0.4 µL of 25 mM dNTP mix (prepared fresh or from a kit), 0.5 µL RNase Inhibitor to a
final volume of 6.9 µL per reaction. Add 6.9 µL of mixture to the PCR tube and mix well. Heat the sample in the preheated PCR
thermal cycler at 25° for 2 min. Add 1–2 µL (~200 U) of reverse transcriptase enzyme (1 µL for less than 5 kb cDNA and 2 µL for
longer) to the sample and incubate the sample in a thermal cycler with programed of 25° for 10 min, 42° for 50 min, 70° for 15
min then hold at 4°. Then place the tube on ice.
Synthesize second strand cDNA: This process purifies the products of the ligation reaction to select a size for enrichment with
a gel, beads, or other appropriate methods. The following gel method described is only an example.
To the first stand of cDNA mix, add 62.8 µL of ultra-pure water. To this mixture, add 10 µL of Second strand buffer, and 1.2 µL of
25 mM dNTP mix.5 Mix well and incubate on ice for 5 min. Add 1.0 µL of RNaseH, and 5.0 µL of DNA Polymerase I. Mix well and
incubate at 16° in a thermal cycler for 2.5h. Purify the sample using a PCR purification kit, following the instructions provided by
the manufacturer, and elute in 50 µL of elution buffer supplied in the kit. Final product will be in the form of double-stranded
DNA. Here, samples can be stored at –15° to –25° or on ice before moving on to performing end repair protocol.
End repair: This process cleaves 3′ overhangs into blunt ends which can occur due to attachment of non-templated
nucleotides.
Preheat a heat block at 20°. In a 1.5 mL RNase-free tube, add 50 µL of eluted DNA, 27.4 µL of RNase–free water, 10 µL of 10X end
repair buffer,6 1.6 µL of 25 mM dNTP mix, 5 µL T4 DNA Polymerase , 1 µL of Klenow DNA Polymerase, and 5 µL T4 PNK to a total
volume of 100 µL. Incubate the sample in a heat block at 20° for 30 min. Purify the sample using PCR purification kit, following
the instructions provided by the manufacturer, and elute in 50 µL of elution buffer supplied in the kit. Final product will be in the
form of double-stranded DNA. Here, samples can be stored at –15° to –25° or on ice before moving onto the next step.
Adenylate 3′ ends: This process adds an “A” base to the 3’ end of the blunt phosphorylated DNA fragments.
Preheat a heat block at 37°. In a 1.5 mL RNase–free tube add 32 µL of eluted DNA, 5 µL of A-tailing buffer,7 10 µL of 1 mM dATP,
and 3 µL of Klenow exo (3’ to 5’ exo minus) to a total volume of 50 µL.8 Incubate the sample in a 37° heat block for 30 min. Purify
the sample using PCR purification kit following the instructions provided by the manufacturer,9 and elute in 23 µL of elution
buffer. Final product will be in the form of double-stranded DNA. Here, samples can be stored at –15° to –25° or on ice before
moving on to performing end repair protocol.
Ligate adapters: This procedure ligates multiple indexing adapters to the ends of the ds cDNA, preparing them for hybridization
onto a flow cell.
In a 1.5 mL RNase–free tube, add 23 µL of eluted DNA, 25 µL of 2X Rapid T4 DNA Ligase Buffer, 1 µL of PE Adapter Oligo Mix,10
and 1 µL of T4 DNA Ligase to a total volume of 50 µL. Incubate the sample at room temperature for 15 min, then purify the
sample using a PCR purification kit following the instructions provided by the manufacturer and elute in 10 µL of elution buffer.
Ensure complete removal of ethanol. Here, samples can be stored at –15° to –25° or on ice before moving on to performing end
repair protocol.
Purification of cDNA templates: This process purifies the products of the ligation reaction on a gel to select a size for
enrichment.
Prepare a solution with 2% agarose gel in distilled water and 1X TAE buffer (final concentration) to a final volume of 50 mL. Load
the samples onto the gel. On the first and the third well load 2 µL of DNA ladder (the use of ladder that covers a range of bp from
100 bp to 2000 bp), and on second well load 10 µL DNA elute from the ligation step mixed with 2 µL of 6X DNA Loading Dye.11
Run the gel at 120 V for 60 min. Remove the gel slice (≤400 mg) by using a clean gel excision tip before following instructions in
the Gel Extraction Kit, to purify the sample and elute in 30 µL of elution buffer. Here, samples can be stored at –15° to –25° or on
ice before moving on to performing end repair protocol.
Enrichment of purified cDNA templates: This procedure uses PCR to selectively enrich those DNA fragments that have adapter
molecules on both ends, and to amplify the amount of DNA in the library.
In a 200-µL PCR tube, per reaction add 10 µL of 5X phusion buffer,12 1.0 µL of PCR primer PE 1.0,13 1.0 µL of PCR Primer PE 2.0,14

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0.5 µL of 25 mM dNTP mix, 0.5 µL of Phusion DNA Polymerase,15 and 7.0 µL of nuclease–free water to a total volume of 20 µL per
reaction. Add 30 µL of the purified ligation mixture to the PCR tube before amplification. PCR amplification can be done by 30s
at 98°, then 15 cycles of 10s at 98°, 30s at 65°, 30s at 72°, 5 min at 72° and hold at 4°. Purify the sample using a PCR purification
kit,16 following the instructions provided by the manufacturer, and elute in 30 µL of elution buffer. Here, samples can be stored
at –15° to –25° or on ice.
Validation of library: Quantify your libraries. There are variety of ways to validate the library including using qPCR, ddPCR,
Bioanalyzer, or Micro Chip. Next, check the size and purity of the sample. This can also be analyzed by a native PAGE.
Analysis of the sequencing data: Vendor supplied software is used to analyze the run data files and determine the sequence
of the starting mRNA molecule. Alternatively, tools such as Sailfish, RSEM, and BitSeq can also help determine the sequence.
There are three steps to HTS analysis. First is the FASTQ “raw” data file generation using the vendor supplied software. Second,
using the trimming and alignment tool for BAM/SAM files which have reads that are aligned to genome, and finally, identification
of mutations/variants. Appropriate system suitability controls should be included within the assay to ensure a valid test; assay
validity criteria must be established in a case-by-case basis, but could be based on parameters such as number of reads
required, Q30, controls, % of reads before and after trimming, % of unmapped reads, etc.

Identity by Sanger Sequencing


Sanger sequencing is a standard sequencing technique that yields information about the identity and order of the four
nucleotide bases in a segment of DNA. It is a technique that uses dye-labeled chemical analogs that are missing the hydroxyl
group required for extension of the DNA chain called ddNTPs of the nucleotide bases. The method is performed by generating
double stranded cDNA from mRNA and then sequencing by sanger sequencing. The detailed description provided is an
example of a potentially suitable analytical procedure.
[NOTE- The protocol below is written for sequencing with Applied Biosystems BigDye Direct chemistry and BigDye XTerminator
clean-up. It provides one-tube simplicity for Sanger sequencing and post-sequencing processing. For other sequencing
chemistries, please see the documentation supplied with the kit.]
TE buffer solution: 10 mM of Tris-Cl, 1 mM of EDTA, pH 8.0
cDNA Synthesis (prior to Sanger Sequencing): Combine 10 µL of cDNA Synthesis master mix (containing a gene specific
primer or random hexamers and oligo-dT primers ),17 1 – 15 µL of sample (100 ng – 2.5 µg of mRNA recommended), and water to
final volume of 50 µL. Vortex the mixture briefly and centrifuge for 5–10 s at 1,000 x g. Put samples in the thermal cycler and run
the program detailed in Table 4. Samples can be held at 4° for up to 8 h or freeze at –20° for longer storage. However, individual
reverse transcriptase may differ in terms of optimal working temperatures. Set the temperature conditions as per the specific RT
instructions.

Table 4. Thermal Cycler Conditions (Prior to Sanger Sequencing)

Steps
Polymerase
Annealing Polymerase Extension Hold
Inactivation
Temperature (0) 25 50 80 4
Time (min) 10 15 10 Indefinitely

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PCR amplification: Primers should be in pairs consisting of forward and reverse primers that focus on specific regions of the
target gene.
[NOTE- BDD sequencing requires M13 sequence tag additions to the primers. Add M13 forward (5’-TGTAAAACGACGGCCAGT-3’)
and reverse (5’-CAGGAAACAGCTATGACC-3’) sequences to the 5’-end of primers specific for your gene(s)].
Resuspend dried and desalted primers to final concentration of 100 µM with 1xTE buffer solution. Next, make a working stock
of PCR amplification primers. Add 492 µL of TE buffer solution to each labeled microcentrifuge tubes for each primer pair. Add
4 µL each of both the forward and reverse primers to the appropriate microcentrifuge tubes. Each one should be 0.8 µM in this
amplification primer mix.
In each well of a 96-well PCR plate, combine 1.5 µL of amplification primer mix in triplicate, 5 µL of PCR BigDye Direct PCR
Master Mix,18 1 µL of cDNA sample (20-40 ng of cDNA), and water to final volume of 10 µL. Setup duplicate reactions, since one
will be used for forward and another for reverse direction sequencing. Make sure to include a separate positive control sample
and a separate negative control sample (no-template). Seal the plate, vortex the mixture briefly and centrifuge for 5–10s at 1,000
x g. Put samples in the thermal cycler and run the program detailed in Table 5.

Table 5. Thermal Cycler Conditions (Amplification)

Steps
Polymerase Cycle (40 cycles)
Hold
Activation Denaturation Annealing Extension
Temperature (0) 95 96 62 68 4
Time (min) 10 min 3s 25 s 30 s Indefinitely

Cycle sequencing: Remove the seal from the plate and add 2 µL BigDye Sequencing Master Mix, 1 µL of forward or reverse
primer to each of the wells.19
[NOTE—Add forward primer to one of the duplicate PCR reactions, and the reverse primer to the other reaction.]
Seal the plate, vortex the mixture briefly, and centrifuge for 5–10 s at 1000 x g. Put samples in the thermal cycler and run the
program as detailed in Table 6.

Table 6. Thermal Cycler Conditions (Cycle Sequencing)

Steps
Post Cycling (25 cycles)
PCR Post PCR Polymerase
Hold
Clean Inactivation Activation Denaturation Annealing Extension
up
Temperature (0) 37 80 96 96 50 60 4
Time (min) 15 min 2 min 1 min 10 s 5s 75 s Indefinitely

Sequencing clean-up: There are methods available for removal of unincorporated nucleotides and salts. The following protocol
uses BigDye Xterminator20 Sequencing clean-up. This kit works directly on the reactions in the wells of the Cycle Sequencing
plate, without the transfer of the reaction to new tubes or other manipulations.

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Centrifuge the reaction plate for 1 min at 1,000 x g. Each well requires 45 µL of BigDye Xterminator SAM solution and 10 µL
Xterminator solution. Combine enough of these solutions sufficient for each reaction plus a 20% extra volume.
Add 55 µL of this mixture to each of the cycle sequencing wells. Make sure to vortex the XTerminator mixture after every 8-12
additions to re-homogenize the mixture.
Collection of data: Load the plate into the capillary electrophoresis instrument. Select or create an appropriate run module
according to capillary length, number of capillaries, and polymer type on the instrument. Make sure BDX running options are
chosen. The electrophoresis will separate the labeled chain-terminated fragments by length with single-nucleotide resolution.
Once the run is finished, the instrument will generate a file that can be converted into a sequence.
Data analysis: There are multiple ways to analyze data. One way is to use sequence analysis software to generate a report
documenting the resulting sequence and QC metrics of the run. Verify the sequence is correct using alignment algorithms
such as BLAST. If needed, software is available that will call low frequency somatic variants at a detection level as low as 10%
frequency (Applied Biosystems Minor Variant Finder).

Identity by Non-Quantitative RT-PCR


Non-quantitative reverse transcription PCR (RT-PCR) and digital PCR can be used to identify mRNA. Alternatively, a general
overview of the qRT-PCR is provided below and is performed in two steps: reverse transcription (first strand of cDNA synthesis),
and PCR amplification. The purpose is solely the identification of the mRNA and not quantification of the mRNA drug substance.
Note that in general, RT-PCR provides less information by comparison to the sequencing approaches as a maximum of 100 nt
can be verified by PCR, in contrast to the full sequence provided by HTS and Sanger methods. However, RT-PCR can be selected
as a suitable method for mRNA identity testing under certain circumstances. Thus, the justification of selection of an identity
method for DP and DS testing should take all relevant aspects from the product itself, manufacturing process and analytical
methods into consideration.
10 mM dNTP mix: Mix 10 mM of each nucleotide (dATP, dCTP, dGTP and dTTP) in 0.6 mM Tris-HCl.
First strand buffer (5X): Mix 250 mM of Tris-HCl at pH 8.3, 375 mM of KCl, and 15 mM of MgCl2.
PCR buffer (10X): 200 mM of Tris HCl at pH 8.4 and 500 mM of KCl.
First strand cDNA synthesis: Prepare the following mixed solution.

Table 7. First-Strand cDNA Solution

Component Volume
Gene specific primer, random hexamers, oligo-dT primer
1 µL
(2 pmol)
mRNA (1-500 ng) X µL
10 mM dNTP mixture 1 µL
RNase-free water Final volume to 12 µL

Heat the mixture at 65° for 5 min and then quickly cool on ice for 2 min. Centrifuge for 5–10 s at 1,000 x g. Next, prepare a
reverse transcription reaction system by combining the following solutions.

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Table 8. Reverse Transcription Reaction Solution

Component Volume
cDNA mixture from above 12 µL
First strand buffer (5X) 4 µL
RNase-free water Final volume to 19 µL

Gently vortex the mixture for few minutes. If random primers are used, incubate at 25° for 2 min, then add 1 µL (200 U) of
Reverse Transcriptase to the reaction tube and mix gently with pipette. Incubate at 42–50° for 50 min.
[NOTE—There are different types/suppliers of reverse transcriptase with different condition requirements therefore use as per
manufacturer’s instructions]
Inactivate and stop the reverse transcription reaction by heating at 70° for 15 min. Sample can be used immediately for
subsequent PCR reactions or can be stored at –20° for short-term storage and –80° for long-term storage.
RT-PCR: Using Table 9 prepare a 50 µL reaction solution.

Table 9. RT-PCR Reaction Solutions

Component Volume
PCR buffer (10X) 5 µL
50 mM MgCI2 1.5 µL
10 mM dNTP mixture 1 µL
Forward primer (10 µM) 1 µL
Reverse primer (10 µM) 1 µL
Taq DNA polymerase (5 Units/µL) 1 µL
cDNA from first strand reaction 2 µL
ddH20 Final volume to 50 µL

Gently mix the reaction and place it in the thermal cycler using the following program. This program is an example based on a
specific enzyme/process and operators should follow conditions based on the reagents and amplicon they are working with.
Follow procedures as per manufacturer’s guidance.

Table 10. Thermal Cycler Conditions

Temperature (o) Time Cycle


94 2 min 1
94 30 s
Tm - 5 30 s 15-40
72 1 min
72 5 min 1
4 Hold 1

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Content

Content by qPCR
Quantitative PCR (qPCR) can be performed to quantify mRNA using either the fluorescent DNA probes or DNA probe. Following
method uses TaqMan as fluorescent dye and a 1/20 volume of the cDNA preparation as template and SuperScript III Platinum
kit.21
[NOTE- There are currently four different fluorescent DNA probes for RT-qPCR; SYBR Green, TaqMan, Molecular Beacons and
Scorpions. Follow manufacturer’s instructions for each. Adjustment may be required for the use of other kits or other real time
PCR instruments.]
mRNA content by qPCR: Thaw all components including primer/probe mix. Additionally, primer/probe mix can also be
purchased. Mix thoroughly by vortexing each tube for 30s at maximum speed to ensure homogeneity. Centrifuge briefly to
collect contents at the bottom. Prepare the following reaction mixture on ice.
Master Mix: Prepare the following mixed solution.

Table 11. Master Mix

Component Volume
Reaction mix 2X 12.5 µL
MgSO4 0.4 µL
Forward primer (10 µM) 1 µL
Reverse primer (10 µM) 1 µL
Probe (10 µM) 0.5 µL
mRNA (1-500 ng) X µL
Superscript IIIRT/Platinum Taq Mix 1 µL
RNase-free water Final volume to 25 µL

Heat the mixture at 65° for 5 min, and then quickly cool on ice for 2 min. Centrifuge for 5–10s at 1,000 x g.
Assay controls: Each RT-qPCR should include controls in addition to the unknown sample.
Negative control: A known negative template control that is sterile with nuclease-free water. This is used as a control for PCR
reagent function and cross-contamination.
Positive control: Used as control for PCR reagent function including primer and probe integrity.
Mix thoroughly by vortexing each tube for 10s at maximum speed. Centrifuge briefly and allow the reaction tubes to equilibrate
to room temperature for no more than 10 min. Add 20 μL of master mix into each well of a optical plate chilled on ice. Make sure
to include at least 2 wells of negative control and 2 wells of positive control.
Run the plate on thermocycler using the following cycling conditions.

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Table 12. Thermal Cycler Conditions

Cycling Step Temperature (o) Time Cycles


Enzyme activation 95 10 min None
Denaturation 95 15 sec 40
Annealing/extension 60 30-60 sec 40

Data analysis: Follow instructions for data acquisition and analysis based on the system used.

Content by digital PCR


Digital PCR can be used to confirm the identity of mRNA without a standard curve. mRNA drug substance is reverse transcribed
to cDNA and amplified, followed by running the samples on a digital or droplet digital PCR System.22
10 mM dNTP mix: Mix 10 mM of each nucleotide (dATP, dCTP, dGTP and dTTP) in 0.6 mM of Tris-HCl.
First Strand buffer (5X): Mix 250 mM of Tris-HCl at pH 8.3, 375 mM of KCl, and 15 mM of MgCl2.
Primer/probe mix (20X): Mix 10 μL of 100 μM of forward primer, 10 μL of 100 μM of reverse primer, 5 μL of 100 μM labeled
probe and 75 μL of PCR-grade water.
First strand cDNA synthesis: Prepare the following mixed solution.
[NOTE—To increase the efficiency of cDNA synthesis, the reverse transcription reaction should include a target gene-specific
primer that is the same primer used as reverse primer for each target in the ddPCR reaction.]

Table 13. First-Strand cDNA Solution

Component Volume
Gene specific primer (2 pmol) 1 µL
mRNA (1-500 ng) X µL
10 mM dNTP mixture 1 µL
RNase-free water Final volume to 12 µL

Heat the mixture at 65° for 5 min, and then quickly cool on ice for 2 min. Centrifuge for 5–10 s at 1,000 x g. Next, prepare a
reverse transcription reaction system by preparing the following mixed solution.

Table 14. Reverse Transcription Reaction Solution

Component Volume
cDNA mixture from above 12 µL
First strand buffer (5X) 4 µL
RNase-free water Final volume to 19 µL

NOTE—There are several types/suppliers of reverse transcriptase with different condition requirements therefore use as per
manufacturer’s instructions]

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Gently vortex the mixture for few seconds. If gene specific primers are used, incubate at 25° for 2 min then add 1 μL (200 U) of
reverse transcriptase to the reaction tube and mix gently with pipette. Incubate at 42–50° for 50 min.
[NOTE—If reverse primer of PCR is used as a reverse transcription primer, it is recommended to perform the reaction at 45–50°,
otherwise, general recommendation is to perform the reaction at 42°.]
Inactivate and stop the reverse transcription reaction by heating at 70° for 15 min. Sample can be used immediately for
subsequent PCR reactions or can be stored at –20° for short-term storage and –80° for long-term storage.
Expression by dPCR: Thaw all components including primer/probe mix. Additionally, primer/probe mix can also be purchased.23
Mix thoroughly by vortexing each tube for 30s at maximum speed to ensure homogeneity. Centrifuge briefly to collect contents
at the bottom of the tube. Prepare the following reaction mixture on ice.

Table 15. dPCR Reaction Mixture

Component Volume per Reaction (µ


µL) Final Concentration
Supermix 5 1x
Reverse transcriptase 2 20 U/ µL
300 mM DTT 1 15 mM
Target primers/probe Variable 900 nM/ 250 nM
RNA/DNase-free water Variable NA
Total RNA Variable 100 fg − 100 ng per reaction
Total volume 20 NA

Mix thoroughly by vortexing each tube for 10s at maximum speed. Centrifuge briefly and allow the reaction tubes to equilibrate
to room temperature for no more than 10 min.
Droplet generation: Load 20 µL of each reaction mixture from above into a sample well of a DG8 cartridge.24 Add 70 µL of
Droplet Generator Oil to the bottom row of the cartridge designed for “oil”. Fit rubber DG80 Gasket onto the Cartridge and
place it on the Droplet Generator. This process should take about 1 min. Droplets are held in the top row. Using a multi-channel
pipettor, transfer 45 µL droplets into 96-well PCR plate and cover the plate with foil sheet immediately. Seal the plate using the
PCR Plate Sealer at 180° for 5s.
Run the plate on thermocycler using the following cycling conditions.

Table 16. Thermal Cycler Conditions

Cycling Step Temperature (o) Time Cycles


Reverse transcription 42-63 60 min 1
Enzyme activation 95 10 sec 1
Denaturation 95 30 sec 40
Annealing/extension 52 1 min 40
Enzyme deactivation 98 10 min 1
Hold 4 Infinite 1

[ NOTE— To determine acceptable temperature ranges for reverse transcription, perform a thermal gradient from 42° to 51.5°
while fixing the annealing/extension step at 52°. Using the optimized reverse transcription temperature, perform a thermal
gradient from 50° to 63° to identify acceptable annealing/extension temperature ranges.]

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Data analysis: Follow instructions for data acquisition and analysis based on the system used.

Content by Ultraviolet Spectroscopy


(See Ultraviolet-Visible Spectroscopy <857>)
This method is used to calculate RNA concentration in the bulk solution. The absorbance of a diluted RNA sample is measured
at 260 nm and 280 nm, and the concentration is calculated using the Beer-Lambert Law equation.
[NOTE: The A260/A280 ratio is dependent on both pH and ionic strength.]
[NOTE: The use of an alternative methods (such as a fluorescence-based assay) for RNA content is also possible at the DS stage.]

Sample solution: Dilute RNA matrix of the mRNA sample to prepare dilutions within the linear range of the method and to
obtain a solution with a maximum absorbance at 260 nm.
Perform a background correction by making readings from a blank at 320 nm, 260 nm, and 280 nm. The absorbance at
respective wavelengths are acquired with calibrated spectrometer referencing with appropriate blanks.
[NOTE— Acquire a full UV spectrum to detect offsets caused by blank/sample mismatch or additional error sources (particle
scattering caused by dust or undissolved particulates, contaminations). For a simple correction of light scattering contribution,
OD values at 260 nm and 280 nm can be subtracted by OD value at 320 nm of the blank solution (buffer solution only). For a
best correction, second derivative method on most UV spectrum can be used.]

Analysis: The molar extinction coefficient is determined by measuring the absorbance values of an RNA standard (in buffered
solution) dilution series in the sample matrix. The slope of the obtained standard curve can be used to determine the molar
extinction coefficient of RNA in the specific matrix.
Determine the absorbance of the Sample solution using Beer-Lambert Law equation. For highly diluted RNA samples, cuvettes
with optical path lengths other than 1 cm might be preferable.
Beer-Lambert Law equation
A=εbC

A = absorbance
ε = molar absorption coefficient
b = path length, 1 cm
C = concentration

[NOTE— The mass extinction coefficient of single-stranded RNA is: 0.025(μg/ml)−1 cm−1 (absorbance max at 260 nm)].

Integrity

RNA Integrity
A high-resolution analytical method that can measure the integrity of RNA molecules by length is crucial for quality assurance,
understanding potency, and for optimization of manufacturing processes. The Fragment Analyzer system is a parallel capillary
electrophoresis instrument that can analyze 12, 48 or 96 samples at once of the linearized plasmid DNA purity and IVT RNAs.
Capillary gel electrophoresis involves filling a capillary with a separation gel matrix with a fluorescent dye. A microliter size
injection is made on the capillary using voltage injection and the RNA fragments bind the fluorescent dye as they migrate
through the capillary by size using electrophoretic separation. Size comparison is performed against a reference ladder sample
that has RNA fragments of defined size. Data acquisition and analysis can be performed using the instrument’s software to
determines the size and concentration of the RNA fragments present in the sample.

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RNA Integrity by Fragment Analyzer


RNA ladder: Use a suitable RNA ladder.25
RNA diluent marker solution: Use a suitable RNA diluent marker solution.26
Intercalating dye solution: Use a suitable intercalating dye solution.27
Separation gel: Use a suitable separation gel.28
Blank solution: Use a suitable blank solution.29
Capillary conditioning solution (5X): Use a suitable 5X capillary conditioning solution.30
Capillary conditions solution (1X): Mix 1-part Capillary conditions solution with 4 parts WFI
Inlet Buffer (5X): Use a suitable Inlet Buffer
Inlet Buffer: Prepare the Inlet Buffer (5X) by mixing 1-part Inlet buffer with 4 parts WFI
RNA separation gel: Prepare by mixing Intercalating Dye Solution (1/10,000 dilution) with the RNA Separation gel to create a
sufficient volume depending on the number of samples to be analyzed.
RNA ladder solution: Thaw ladder on ice. Heat-denature the ladder at 70° for 2 min, immediately cool to 4° and keep on ice.
Use 2 μL of the 96 ng/μL ladder for every run.
mRNA sample preparation: Heat-denature the RNA samples at 70° for 2 min and immediately cool to 4° and keep on ice
before use. The mRNA input sample must be within a suitably validated concentration range (e.g. 1 ng/μL to 100 ng/μL). If the
concentration of the sample is above this range, dilute with RNase-free water. Prepare each sample in duplicate.
Sample plate preparation: Using a fresh RNase-free 96-well sample plate, pipette 22 μL of the RNA diluent marker (15 nt) to
each well in a row that is to contain sample or RNA ladder solution. Pipette 2 μL of each denatured RNA sample into the assigned
well on the plate containing 22 μL of RNA diluent marker solution (15 nt). The RNA ladder solution must be run in parallel with the
samples for each experiment to ensure accurate quantification and sizing. Pipette 2 μL of denatured RNA ladder solution into
the 22 μL of RNA diluent marker solution (15 nt) in the designated ladder well. Mix the contents of each well by pipetting up and
down 10x or by using a plate shaker set to 3000 rpm for 2 minutes. Fill any unused wells within the row of the sample plate with
24 μL of Blank solution. After mixing each well, centrifuge the plate to remove any air bubbles. Check the wells of the sample
plate to ensure there are no air bubbles trapped in the bottom of the wells. The presence of trapped air bubbles can lead to
injection failures.
Instrument set up: Pipet 1 mL of fresh Inlet buffer into a deep well plate and place plate into drawer ‘B’ on the instrument.
Pipet 200 μL of 0.25x TE Rinse buffer into each well of a 96 well plate and place in drawer ‘M’. Load prepared gel and prepared
conditioning solution into the instrument.
Separation procedure and analysis: Load prepared sample plate into the instruments and enter sample names. Select Add to
Queue and select the corresponding method based on the appropriate capillary length of the installed capillary array. Select
OK to add the method to the queue. Click the ‘GO’ button to start the separation. For IVT RNA separations, it is recommended
to increase the separation time by 5 minutes to ensure that the entire sample separates out. This can be done when the method
is loaded and clicking on Edit Method, then increasing the time to 45 minutes. After the separation is completed, the samples
can be automatically analyzed, and reports generated with the instrument data analysis software.

RNA Integrity by Capillary Gel Electrophoresis


A high-resolution analytical method that can measure the integrity of RNA molecules by length is crucial for quality assurance,
and for optimization of manufacturing processes. The following two methods for capillary gel electrophoresis (CGE) with Light
Induced Fluorescence (LIF) detection is used to evaluate the total RNA integrity. Option A is suitable for separation of RNA
fragments varying from 200 to 6,500 bases and option B from 50 to 9,000 bases.

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Separation Buffer

Option A: Protocol: 1X TBE buffer: 89 mM Tris, 89 mM boric acid, 2 mM EDTA, pH 8.3.


Option B: RNA 9000 Purity & Integrity Kit31: Buffer not required.
Separation Gel
Option A: 1% Polyvinylpyrrolidone (PVP) at 1.3 MDa in 1X TBE buffer with 4 M Urea and 50,000x dilution or 0.002% SYBRTM
Green II Stain31. Suitable for separation of RNA fragments varying from 200 to 6,500 bases.
Option B: RNA 9000 Purity & Integrity Kit31: Validated and ready-to-use separation gel. SYBRTM Green II Stain included and
diluted at optimized concentration for mixing with the separation gel for single stranded RNA integrity and purity analysis.
Suitable for separation of RNA fragments varying from 50 to 9,000 bases.
RNA ladder and marker
Option A: Dilute RNA ladder in nuclease-free water to 25 μg/mL.32 If necessary, spike the ladder with a 1.2 Kb RNA marker to
assess the separation method. Denature the solution for 5 minutes at 65° and place the sample mixture on ice or in an ice water-
bath to cool the sample down for a minimum of 5 minutes.
Option B: RNA 9000 Purity & Integrity Kit: Add 2 μL of the ssRNA ladder to 48 μL of nuclease-free water or Sample Loading
Solution. Heat the sample at 70ᵒ for 10 minutes, and immediately after the heating step, put the mixture on ice or in an ice
water-bath to cool the sample down for a minimum of 2 minutes.
[NOTE: RNA ladders should be used as guides for size estimation and/or indicators for analytical variability. The user should
consider RNA chemical modifications, the purine: pyrimidine ratio, and GC-content that may affect the expected migration time
of analytes in comparison to the RNA ladder or markers.]
Sample preparation
To release the RNA from the mRNA-LNP particle for capillary electrophoresis (CE) analysis without a purification step, mix 10 μL
of the mRNA-LNP sample(s) with 20 μL of a 0.3% Triton X-100 solution and incubate at room temperature for 20 minutes. Next,
heat the sample(s) at 70ᵒ for 5 minutes, and immediately after the heating step cool down the sample(s) by placing on ice or
in an ice-water bath for a minimum of 5 minutes. Lastly, add 60 μL of CE-grade water to the sample(s) and analyze by CE. This
procedure can also be used for the analysis of free or in vitro transcribed RNA. An RNA detection range from 50 ng/mL to 50 μg/
mL has been reported using the PA 800 Plus Pharmaceutical Analysis System or BioPhase 8800 System.
[NOTE - Occasionally, higher molecular weight (HMW) RNA products may be detected by capillary electrophoresis. The nature
of these HMW products is of great interest in the field. Reports have shown that breaking up the mRNA-LNP or dissolving the
released RNA material from the mRNA-LNP in the presence of water-based formamide solution (>80%) can dissociate these
HMW products. The user should use caution interpreting these HMW RNA products.]

Cartridge
PA 800 Plus Pharmaceutical Analysis System: EZ cartridge pre-assembled with bare fused-silica capillary (50 μm I.D., 30 cm
total length, 20 cm effective length).
BioPhase 8800 System: Pre-assembled BioPhase bare fused-silica capillary cartridge (8 capillaries, 30 cm total length).
Capillary gel electrophoresis
Option A: Suitable for RNA fragments ranging from 200 to 6,500 bases. Perform capillary electrophoresis by reverse polarity
200 V/cm electrical field (6 kV) at 25° within 18 minutes of running time. Sample introduction into the inlet of the capillary
can be achieved electrokinetically at 5kV for 3 seconds. The sample tray temperature can be set at 10° with the LIF detector
configured to 488 nm laser with an emission filter of 520 nm. A calibration curve using the RNA ladder described above can be
generated to estimate the size of an unknown sample peaks. The user can add samples as needed to obtain the desired number
of replicate measurements for statistical analyses.

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Option B: Suitable for RNA fragments ranging from 50 to 9,000 bases. The recommended capillary electrophoresis settings for
the PA 800 Plus Pharmaceutical Analysis System or the BioPhase 8800 System include a 6 kV reverse polarity with a capillary
temperature of 30ᵒ with a ramp time of 2.0 minutes and a duration of 22 minutes. Automated sample introduction into the inlet
of the capillary can be achieved first by a water-plug step using 0.5 psi for 5 seconds followed by pressure injection at 1.0 psi for
5 seconds. The sample tray can be set at 10ᵒ and the LIF detector configured to 488 nm laser with an emission filter of 520 nm.
This separation method can analyze up to 8 samples or replicates in a single run using the BioPhase 8800 System. For detailed
instrument settings refer to RNA 9000 Purity & Integrity Kit application guide.
Data analysis
32 Karat software is compatible with the PA 800 Plus Pharmaceutical Analysis System. Samples run with the BioPhase 8800
System can be analyzed with BioPhase software. 32 Karat data files are compatible with the BioPhase software. Automated data
processing can determine main product percent purity composition based on corrected peak area %.

RNA Integrity by Agarose Gel Electrophoresis


MOPS buffer (10X): Dissolve 41.86 g of 200 mM MOPS (free acid), 6.80 g of 50 mM sodium acetate, 3.72 g of 10 mM EDTA-2H20
and 3.80 g of 10 mM EGTA (free acid) in 850 ml of RNase-free water. Adjust pH to 7.0 ± 0.2 with 10 M NaOH and adjust volume to
1000 mL with RNase-free water. Filter the solution through 0.2 µm pore size filter.
Running buffer (1X): Dilute 10X MOPS buffer with deionized water, 1:50.
Loading dye: Add 10X MOPS buffer, 0.5 M EDTA (pH 8.0) and bromophenol blue to deionized water to the final concentration of
2.1X electrophoresis buffer, 1mM EDTA and 0.04% bromophenol blue. Add ethidium bromide for a final concentration of 10 µg/
mL. Filter through a 0.2 µm syringe filter.
Loading buffer (2X): Prepare enough of the loading buffer by combining 14 volumes of loading dye with 1 volume of 37%
formaldehyde.
[NOTE— Loading dye mixed with formaldehyde is not stable upon storage and must be used within a few hours.]
mRNA sample preparation: Add the freshly prepared 2X loading buffer to each RNA sample (1:1 v/v). Close tubes tightly, mix the
contents, and spin briefly in a microcentrifuge. Denature the sample by heating at 70° for 5 min, then cool to room temperature.
RNA markers (0.5–9 kb long): Dilute 2 µL of the marker with 3 µL of nuclease-free water and mix with 15 µL of loading dye.
Analysis: Heat 1 g of agarose (for a 1% gel) in 72mL of deionized water until dissolved. Cool agarose to 60°. Place in fume
hood. Add 10 mL of 10X MOPS buffer and 5.5 mL of prewarmed 37% formaldehyde. Pour the gel in the tank and add enough
Running buffer to cover the gel by a few millimeters. Tightly cover the gel casting assembly with plastic wrap during agarose
solidification to prevent formaldehyde losses from the gel. Remove the comb.
Load the gel and electrophoresis at 5 V/cm until the bromophenol blue has migrated as far as two-thirds the length of the gel.
Visualize the gel on a UV transilluminator. The bands can also be quantified by densitometry using known RNA standards.
Acceptance criteria: Visual observation of the marker should show distinct bands and a single band for the intact RNA sample,
like those of the in-house control.
DNAzyme-mediated mRNA capping efficiency assay by LC-MS
A DNAzyme is a synthetic oligonucleotide capable of catalytically cleaving RNA to which it hybridizes. Applying DNAzyme to
cleave the specified 5’ end of the in-vitro transcribed mRNA allows for subsequent analysis of capped and uncapped 5’-RNA
fragments by LC-MS.

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Analytical Procedures for Quality of
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[NOTE – DNAzyme must be optimized for each RNA sequence, otherwise it will not bind to the sample.]
Annealing Buffer: 15 mM NaCl, 5 mM Tris-HCl, pH 7.5, 0.1 mM EDTA
Annealing Reaction: 100 pmol-454 pmol of mRNA and DNAzyme (2-fold excess as a minimum) are mixed in annealing buffer,
heated for 3 min at 95oC and slowly cooled to 37oC.
Cleavage reaction: One-tenth of the volume of 10x cleavage buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.5, 200 mM MgCl2) is
added and the mixture is incubated for 3 hours at 37oC.
Phosphatase treatment: The post DNAzyme reaction mixture is treated with a mixture of T4 polynucleotide kinase and CIP by
adding one-tenths of the volume of T4 PNK T4 buffer, 10 units of each enzyme per 1 pmol of mRNA and incubating mixture for 1
hour at 37oC.
Quenching the reaction: Add appropriate volume of 0.5 M EDTA to phosphatase-treated reaction mixture.
Sample workup: The reaction mixture is concentrated and desalted using 3K MWCO ultracentrifugation filters. The resulting
concentrated sample is diluted with Mobile phase A to target 2 pmol/μL RNA with at least a 1:1 ratio of filter solution and Mobile
phase A and used for LCMS analysis.
Alternatively, RNA is isolated from the reaction mixture using phenol-chloroform extraction and NaOAc-Ethanol precipitation.
The resulting RNA pellet is re-suspended in Mobile phase A and is used for LCMS analysis.
Chromatographic system
(See Chromatography <621>, System Suitability.)
[NOTE - Alternative settings can be applied, if justified and validated for intended use.]

Mode: LC-MS
Solution A: 1% Hexafluoroisopropanol (HFIP), 0.1% Diisopropylethylamine (DIPEA), 2 µM EDTA in water
Solution B: 0.075% HFIP, 0.0375% DIEA, 2 µM EDTA in 65/35 v/v ACN/Water
Column: ACQUITY UPLC BEH C18 Column, 130Å, 1.7 µm, 2.1 mm X 100 mm reverse phase
Flow rate: 400 μL/min
Detector: UV 260nm and ESI-MS
Column Temperature: 70ᵒ
Autosampler compartment temperature: 10ᵒ
Injection volume: 50-100 μL (100 pmoles)

Table 17. LC Gradient Table

Time (min) Solution A (%) Solution B (%)


0.0 98 2
3.0 98 2
3.1 94 6
22.1 85 15
33.1 80 20
33.2 0 100
35.2 0 100
35.3 98 2
42.0 98 2

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ESI parameters: Gas temperature 263°, gas flow 13 L/min, nebulizer pressure 40 psi, sheath gas temperature 425°, sheath
gas flow 12 L/min, capillary voltage 3500 V, nozzle voltage 0 V
Analysis
Measure the peak areas of the respective peaks.

Purity
mRNA 5′- CAP by RP–LC-MS/MS
This method allows accurate detection and quantitative assessment of ribose-methylated and ribose-unmethylated cap
structures, which can be separated and quantified using LC-MS/MS. To this end, it is necessary to digest the mRNA using
Nuclease P1 to produce cap dinucleotides.
Solution A: 30 mM of ammonium acetate buffer. The pH is adjusted to pH 7.5 by addition of ammonium hydroxide
Solution B: Acetonitrile
[NOTE- Use only LC-MS grade chemicals.]
Mobile phase: See the gradient below.

Table 18. RP-HPLC Gradient Table

Time (min) Solution A (%) Solution B (%)


0 100 0
1 100 0
7 70 30
9 50 50
10.5 50 50
12.5 100 0
20 100 0

Sample preparation: Treat up to 20 µg mRNA with 0.6 U nuclease P1 in 20 mM ammonium acetate (pH 5.3) and 0.1 mM
zinc chloride. Incubate the reaction at 50°C for 1 h. Nuclease P1 hydrolyze RNA to nucleoside monophosphates. Since
phosphodiester bonds of cap structures are spared out, it also results cap dinucleotides (e.g., m7GpppN). Optionally, the
resulting nucleoside monophosphates can be hydrolyzed to nucleoside level using Alkaline Phosphatase.
Chromatographic system
[NOTE: Alternative method settings can be applied, if justified and validated for intended use.]
Column: Poroshell 120EC-C18 3x150 mm 2.7 µm
Column temperature: 25°
Flow rate: 0.5 mL/min

Mass parameters
Mass spectrometer: Triple quadrupole (QQQ) equipped with an electrospray ion source (ESI).
Mode: Positive ion mode, MRM (multiple reaction monitoring).
ESI parameters: Gas temperature 300°C, gas flow 7 L/min, nebulizer pressure 40 psi, sheath gas temperature 350°C, sheath
gas flow 12 L/min, capillary voltage 3500 V, nozzle voltage 0 V.
QQQ parameters: Depending on which cap dinucleotides must be detected. It is recommended to optimize the instrument
parameters (fragmentor voltage, collision energy, and cell accelerator voltages) for each mass spectrometer to achieve
optimal sensitivity.

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Analytical Procedures for Quality of
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Table 19. Examples for QQQ Parameters

Cell
Compound Precursor Ion Product Ion Fragmentor Collision
Accelerator
Name (m/z) (m/z) (V) Energy (V)
Voltage (V)
m7GpppAm 801 136 135 68 7
m GpppA
7
787 136 135 60 7
m7GpppGm 817 166 135 68 7
m7GpppG 803 248 135 20 7

Analysis
Measure the peak areas of the dinucleotide containing the ribose methylation and of the dinucleotide not containing the
ribose methylation.
Calculate the percentage of ribose-methylated cap-dinucleotides:
Result = [ A rm / (Arm + Au)] * 100
Arm = area of the ribose-methylated cap dinucleotide peak
Au = area of the ribose-unmethylated cap dinucleotide peak

mRNA 5’-CAP by IP−RP−HPLC


A cap is required at the 5’ end of the mRNA molecule to protect the molecule from degradation and to facilitate successful
protein translation. Capping efficiency is thus a critical quality attribute for mRNA-based vaccines. Capped and uncapped
mRNA fragments can be separated and quantitated using ion pair reverse-phase high performance liquid chromatography
(IP RP-HPLC). It may be necessary to perform site-specific cleavage of the mRNA molecule using ribonuclease H to produce
smaller specific mRNA fragments in the sample that can be adequately resolved using IP RP-HPLC.
Solution A: 100 mM of triethylammonium acetate buffer, pH 7.0
Solution B: Solution A with 25% (v/v) acetonitrile
Mobile phase: See the gradient table below.

Table 20. IP-RP-HPLC Gradient Table

Time (min) Solution A (%) Solution B (%)


0 90.0 10.0
36 85.5 14.5

RNase cleavage buffer: Prepare a solution of 20 mM of HEPES-KOH, 50 mM of KCl and 10 mM of MgCl2, pH 9.0.

Sample solution: To increase the resolution, select a site-specific RNA cleavage probe with 2′-O-methyl modifications, except at
the 3′ end which has 4 to 6 deoxyribonucleic acids (DNA) at the cleavage site. The RNA cleavage probe concentration should
be 120% of the mRNA concentration to ensure complete hybridization of the mRNA. The RNA cleavage probe is product specific
and should be chosen to produce a 5′-cap fragment of sufficient size (~25 nt) for the IP–RP–HPLC analysis. The RNA cleavage
probe-RNA complex mixture should be between 0.5 and 2.0 mM in RNase cleavage buffer. Anneal the RNA cleavage probe to
mRNA by heating to 90° and then cooling slowly to room temperature. Add RNase H to a final concentration of 20 units per 100
μL reaction volume. Incubate the reaction at 37° for 3 h.

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Analytical Procedures for Quality of
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Chromatographic system
(See Chromatography <621>, System Suitability.)
[NOTE: Alternative method settings can be applied, if justified and validated for intended use.]

Mode: LC
Detector: UV 260 nm
Column: Acquity Premier Oligonucleotide C18 column, 130 Å,1.7μm,2.1 x 100 mm
Column temperature: 50°
Flow rate: 0.5 mL/min
Injection volume: 15 µL

Analysis
Sample: Sample solution
Measure the areas of the 5’ capped mRNA peaks and of the uncapped mRNA peaks.
Calculate the percentage of uncapped mRNA:

Result = [A�/(A� + Ac)] × 100

A� = area of the uncapped mRNA peak


Ac = area of the 5’ capped mRNA peak

Percent Poly(A) Tail Length by IP-RP-HPLC


A poly(A) tail is required at the 3’ end of the mRNA molecule to protect the molecule from degradation and to facilitate
successful protein translation. The presence of a poly(A) tail is a critical quality attribute for a mRNA vaccine. mRNA molecules
with and without a poly(A) tail (tailless) can be separated and quantitated using ion pair reverse-phase high performance liquid
chromatography (IP-RP-HPLC). It may be necessary to perform site-specific cleavage of the mRNA molecule using RNAse T1 to
produce smaller specific mRNA fragments in the sample that can be adequately resolved using IP-RP-HPLC.
[NOTE— Poly(A) tail is dependent upon the manufacturing process and the design of the mRNA itself]

Buffer A: 100 mM of triethylammonium acetate buffer, pH 7.0


Buffer B: Solution A containing 25% acetonitrile.
Mobile phase: See the gradient table below.

Table 21. RP-HPLC Gradient

Time (min) Buffer A (%) Buffer B (%)


0 57 43
0.5 50 50
3.5 46 54
4 46 54
4.5 57 43
5 57 43

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Analytical Procedures for Quality of
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Chromatographic system
(See Chromatography <621>, System Suitability.)
[NOTE: Alternative settings can be applied, if justified and validated for intended use.]

Mode: LC
Detector: UV 260 nm
Column: RNASep, 7.8 x 50 mm; packing non-porous, PS/DVB resin matrix
Column temperature: 61°
Flow rate: 0.9 mL/min
Injection volume: 10 µL

Analysis
Samples: Sample solution
Measure the areas of the peaks. There could be multiple peaks due to different lengths of poly(A) tail and different tailless
fragments.
Calculate the percentage of poly(A) mRNA:
Result = [A b/ (A b + A e)] × 100

A b = area of the poly(A) mRNA peak


A e = area of the tailless mRNA peak
Inaccurate results can occur due to over estimation of poly(A) tail percentages. If the enzyme fails to digest properly, it can
result in underestimation for tailless fragments or overestimation for poly(A) mRNA. Second, peak areas are proportional to
the lengths of digestion products: longer poly(A) tail have stronger signal than shorter tailless mRNA. This could lead to over-
estimation percentage of poly(A) mRNA.

Poly(A)-tail Length and Polydispersity Analysis in mRNA by LC-MS


Liquid chromatography mass spectroscopy (LC-MS) method can be used to analyze poly(A) tail length ≤ 130 nucleotides (nt).
The method involves initial digestion of the mRNA with a combination of nucleotide-specific endonucleases that leave the
long poly(A) fragments intact while reducing the rest of the mRNA to mononucleotides and small oligonucleotide fragments.
The resulting digest is analyzed using IP-RP-HPLC-ESI-MS method. The data is processed using deconvolution software to
determine composition, lengths, and polydispersity of the poly(A) species.

mRNA digestion procedure: Add 50-100 pmole mRNA in digestion buffer and incubated at 90ᵒ for 10 min. Add appropriate
amounts of RNAse A and RNAse T1 to the mRNA solution and incubate the reaction at 37ᵒ C for 1 -3 h. Analyze the reaction
mixture by LCMS.

Chromatographic system
(See Chromatography <621>, System Suitability.)
[NOTE: Alternative settings can be applied, if justified and validated for intended use.]

Mode: LC-MS
Solution A: 1% Hexafluoroisopropanol (HFIP) and 0.2% TEA in water
Solution B: 50/50 Acetonitrile/methanol
Column: ACQUITY UPLC BEH C18 Column, 130Å, 1.7 µm, 2.1 mm X 100 mm reverse phase
Flow rate: 250 μL/min
Detector: UV 260 nm and ESI-MS
Column Temperature: 70ᵒ
Autosampler compartment temperature: 10ᵒ
Injection volume: 100 μL

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Table 22. RP-HPLC Gradient Table

Time (min) Solution A (%) Solution B (%)


0 99 1
1.0 99 1
16.0 86 14
16.2 0 100
17.2 0 100
17.5 99 1
23.0 99 1

ESI parameters: Gas temperature 250°, gas flow 13 L/min, nebulizer pressure 40 psi, sheath gas temperature 275°, sheath gas
flow 12 L/min, capillary voltage 3500 V, nozzle voltage 0 V

Analysis: Analyze the poly(A) peak by MS to determine composition, lengths, and polydispersity of the poly(A) species

Measure the Poly A tail distribution of mRNA using the following equation:

Weighted Average of the Poly A tail length = SUM(Rounded A count * Intensity)/ SUM(Intensity)

[NOTE - To determine the Rounded A count - open the Deconvolution peak and copy and paste the values in excel. Highlight
the masses with rel% between 20 - 100, then divide all the highlighted masses by 329 and round to the nearest integer to
estimate the number of A’s.]

mRNA Purity by IP-RP-HPLC


Ion-pair reverse-phase high performance liquid chromatography (IP-RP-HPLC) can be utilized for the determination of the integrity
of the transcribed mRNA.

Chromatographic system
(See Chromatography <621>, System Suitability.)
[NOTE - Alternative settings can be applied, if justified and validated for intended use.]

Mode: LC
Solution A: 100 mM Triethylammonium acetate (TEAA), 1 mM EDTA in water, pH 7.3
Solution B: 100 mM TEAA, 1 mM EDTA, 25% Acetonitrile in water, pH 7.3
Column: RNASep, 7.8 x 50 mm; packing non-porous, PS/DVB resin matrix
Flow rate: see gradient table below.
Detector: UV 260 nm, collect 3D data from 200-400 nm
Column Temperature: 65ᵒ
Autosampler compartment temperature: 10ᵒ
Maximum column backpressure: 4000 psi
Injection volume: 5 μL
Sample preparation: Dilute mRNA samples to 0.2 mg/mL of water.
Blank preparation: Prepare blank vials with 100-200 μL of water into HPLC vials. Run blank samples bracketing each set of
samples.

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Table 23. RP-HPLC Gradient Table

Time (min) Solution A (%) Solution B (%)


0 0.3 30
1 0.3 30
1.1 0.3 40
2 0.3 40
16 0.3 60
16.5 1 30
19 1 30
19.6 0.3 30
20 0.3 30

Analysis
Measure the percent areas of the respective peaks.

dsRNA by Immunoblot
If dsRNA is present in the mRNA vaccine, it has the potential of being immunogenic. For that reason, dsRNA content should be
determined and controlled.
[NOTE— dsRNA is dependent upon the manufacturing process and the design of the mRNA itself and could fall under
characterization and not for drug substance release assay.]
TBS-T buffer: Prepare a solution of 25 mM Tris-HCl, 150mM NaCl, 0.05% of tween-20, pH7.4.
Blocking buffer: Prepare a solution 5 % nonfat dried milk or 5% ECL blocking reagent in TBS-T buffer.
Incubation buffer: Prepare a solution 1 % nonfat dried milk or 1% ECL blocking reagent in TBS-T buffer.
dsRNA Antibody Solution: Dilute the reconstituted antibody 1:5000 in Incubation Buffer.33
[NOTE- Dilute the reconstituted antibody as per your antibodies manufacturer’s instructions. This recommendation is specific to
this antibody only.]
Detection Antibody Solution: Dilute the reconstituted HRP-conjugated donkey anti-mouse IgG 1:5000 in Incubation Buffer.34
Detection Reagent: Chemiluminescent (ECL) substrate for low femtogram protein level detection
Procedure and Analysis: Blot 200ng of the mRNA test sample and a dsRNA reference sample at the limit of detection onto
a positively charged Nylon membrane (0.45µM) and dry for 30 min. Incubate membrane with Blocking Buffer for 1 h. Rinse
membrane with TBS-T buffer twice. Incubate membrane with dsRNA Antibody Solution at room temperature for 1 h. Rinse
membrane 4 times and wash 6 times, 5 min per wash, with TBS-T buffer. Incubate membrane with Detection Antibody Solution
at room temperature for 1 h. Rinse membrane 4 times and wash 6 times, 5 min per wash, with TBS-T buffer. Detect the
membrane with Detection Reagent. Capture images with an appropriate digital imaging system.
[NOTE- Buffer/matrix interference must be factored into the final format since signal intensity could be higher depending on the
buffer used.]

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dsRNA by ELISA
ELISA can be used to measure a specific antigen in a biological sample. It can be used to quantitate the amount of dsRNA. This
example uses K1 IgG2a monoclonal antibody.

[NOTE— dsRNA is dependent upon the manufacturing process and the design of the mRNA itself and could fall under
characterization and not for drug substance release assay.]

Sample diluent buffer: Prepare the solution by combining equal volume of 0.1 M NaCl in 1X TE solution, and RNA storage
solution.

To determine the concentration of dsRNA, generate a standard curve using antigens of a known concentration then calculate
the concentration of dsRNA using the optical density (OD).

ELISA can be performed by capturing anti-dsRNA monoclonal antibody, by diluting K1 IgG2a to a concentration of 300ng/mL
in blocking buffer. Add 100 μL of this mixture to a protein A coated 96-well microplate. Incubate the plate overnight at 5ᵒ. Wash
plate with 1X PBS containing 0.05% Tween-20. Dilute samples in sample diluent buffer. Add 200 μL of this mixture to the plate
and incubate for 2 hrs at RT with shaking at 500 rpm. Wash plate with 1X PBS containing 0.05% Tween-20. After washing add 50
μL of neat K2 IgM hybridoma supernatant to the plate and incubate for 1 hr at RT with shaking at 500 rpm. Wash plate with 1X
PBS containing 0.05% Tween-20. Dilute HRP-conjugated goat anti-mouse IgM, chain specific polyclonal antibody to 1:16,000-
fold in blocking buffer. Add 100 μL of this to the plate and incubate for 1 hr at RT with shaking at 500 rpm. Wash plate with 1X
PBS containing 0.05% Tween-20. Add 100 μL 1-Step Ultra TMB-ELISA substrate Solution to the plate following the instruction
provided with the substrate. Incubate as instructed.

Analysis
Read the absorbance values at 450 nm on a plate reader. The concentration of dsRNA in the test sample can be determined
from the standard curve prepared from a 142-bp dsRNA standard and the response of the standard concentrations fit to a
4-parameter logistic equation.

mRNA Aggregation by SEC-HPLC


Size Exclusion Chromatography (SEC-HPLC) can be utilized for its quick and reliable method in many applications such as
purification or aggregate quantification. This SEC method allows a quick and reliable to quantify the percentage of aggregates
of mRNA sample. Further characterization of each SEC peak can be performed using RP-HPLC as an orthogonal method (see
below).

Chromatographic system
(See Chromatography <621>, System Suitability.)
Mode: LC
Mobile phase: 150 mM Phosphate buffer, pH 7.0
Column: SRT SEC-1000, 5 µ, 1000Å, 7.8x300 mm
Flow rate: 1.0 mL/min
Detector: UV260 nm
Column Temperature: 250
Sample: mRNA sample (single-stranded, 1000 nucleotides, 300-600 kDa) (0.5 mg/mL)
Injection volume: 5 μL

Analysis
Measure the peak areas of the respective peaks (main peak, high molecular weight, and low molecular weight).

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mRNA Percent of Fragments by RP-HPLC


Ion-pair reverse-phase high performance liquid chromatography (IP RP HPLC) can be utilized for the determination of the
integrity of IVT-transcribed mRNA.

Chromatographic system
(See Chromatography <621>, System Suitability.)
[NOTE: Alternative settings can be applied, if justified and validated for intended use.]
Mode: LC
Solution A: 100 mM triethylammonium acetate (TEAA), pH 7.0
Solution B: 100 mM TEAA/ 25% Acetonitrile, pH 7.0
Column: Proteomix RP-1000, 5 µm, 1000Å, 2.1x100 mm
Flow rate: 0.3 mL/min
Detector: UV 260 nm
Column Temperature: 50ᵒ
Sample: mRNA sample (single-stranded, 1000 nucleotides, 300-600 kDa) (0.5 mg/mL)
Injection volume: 10 μL

Table 24. RP-HPLC Gradient Table

Time (min) Solution A (%) Solution B (%)


0 90 10
1 90 10
5 40 60
20 30 70

Analysis
Measure the peak areas of the respective peaks

Residual DNA Template (qPCR)


(See Residual DNA Testing <509>)
The following method is suitable for measurement of residual host cell DNA in mRNA vaccines drug substance. Extraction may
not be required for drug substances; therefore, a quantitative polymerase chain reaction (qPCR)-based method can be directly
used for the measurement of residual host cell DNA template. For discussion of the principles and best practices for this type
of testing, see Nucleic Acid-Based Techniques—Approaches for Detecting Trace Nucleic Acids (Residual DNA Testing) 〈1130〉,
which may be a helpful resource.
Sample Preparation: There are several procedures for nucleic acid extraction. Use method suitable for nucleic acid extraction
that is appropriate for mRNA to be examined. One such procedure is described in detail below and validated for starting DNA
concentrations ranging from 0.01 to 50 pg/µL.
Resuspension solution: Dissolve Tris-HCl and EDTA to obtain a solution of 10 mM and 1.0 mM, respectively. Add hydrochloric
acid or sodium hydroxide to adjust to a pH of 8.0.
DNA standard stock solution: Dilute reference material to a concentration of 1 µg/mL in Resuspension solution.
Sample solutions: Samples for testing may require dilution or reconstitution to 1) overcome matrix interference affecting the
DNA recovery, 2) yield an appropriate starting volume, or 3) bring the analyte concentration within the quantitative range of the
qPCR method. Sample solutions may be diluted in water or in Resuspension solution if necessary. For drug substance samples,

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Sample solutions should contain sufficient starting material to allow determination of the residual DNA content, if present at the
specification limit.
Positive control solution: Prepare by spiking DNA standard stock solution to Sample solutions at a concentration appropriate for
the assay (specification, or otherwise justified).
Negative control solution: Water or Resuspension solution is used in place of Sample solutions in the extraction procedures
and will be extracted with any samples (if extraction is necessary). The Negative control solution is tested using the qPCR-based
method to determine the DNA content contributed by the background and to demonstrate that there is no potential cross-
contamination during the assay. This is also known as the no template control.
qPCR Analysis
2X Master mix: A suitable buffer containing magnesium chloride, deoxyadenosine triphosphate, deoxyguanosine triphosphate,
deoxycytidine triphosphate, deoxyuridine triphosphate, deoxythymidine triphosphate, and highly purified DNA polymerase. Mix
well immediately before use.
DNA stock primers and probes: Determine the fragment of the DNA template that needs to be amplified and design the
forward and reverse primers.
Prepare individual 10 µM solutions of the primer pairs and probe specific to mRNA vaccines, using DNAse-free water.
DNA probe solution: Dilute DNA stock probe to 2.5 µM with DNAse-free water.
Standard solutions: Dilute the DNA standard stock solution to obtain 5 or more suitable standards within the concentration
range of 0.001–100 pg/µL.
Analysis of samples: Sample solutions, Positive control solution, Negative control solution, and Standard solutions
[NOTE—If samples are extracted, then extracted Sample solutions and extracted Control solutions will be used.]
Transfer 25 µL of the 2X Master mix to each well of a 96-well qPCR plate. Add 5 µL each of the DNA stock forward primer, the
DNA stock reverse primer, and the DNA probe solution of the appropriate species to each well. Add 10 µL of either (extracted)
Sample solutions, Standard solutions, (extracted) Negative control solution, or (extracted) Positive control solution to their
respective wells.
[NOTE—The qPCR reaction volume may be scaled as appropriate to accommodate different instruments.]
Mix, seal the plate tightly, and centrifuge for 1 min at 1000 × g. Place the plate in a suitable qPCR thermal cycler. Incubate for 2
min at 50°, then for 10 min at 95°, followed by 40 cycles, with each cycle consisting of 95° for 15s and 60° for 1 min.
[NOTE—Some instruments and reagents require a preincubation step. Carefully follow specific instrument/reagent
recommendations.]
Monitor the signal of the labeled probe using a suitable fluorescence detector. Determine the threshold value using the
instrument-specific recommendations. Record the cycle thresholds (Ct) for each sample.
Calculations:
Plot the log quantity of DNA of the Standard solutions versus the Ct.
Calculate the slope and the intercept.
Using these values and the following equation, calculate the quantity of DNA in each well:
Result=10 (Ct −b/m)
Ct = cycle threshold of the Sample solutions
b = intercept of the line for the Standard solutions
m = slope of the line for the Standard solutions
Calculate the quantity of DNA in each of the Sample solutions. Correct for any dilution or concentration of the sample.

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Quantitation of Free/Non-Incorporated Nucleosides in mRNA by RP-LC-MS/MS


This method allows for detection and quantitation of most modified nucleosides in synthetic mRNA. This includes not only
modifications that were intentionally introduced via their triphosphates during in vitro transcription, but also impurities
introduced via commercial NTP charges of synthetic or biological origin, as well as impurities such as oxidized nucleosides. The
method also detects m7G or ribose-methylated modifications released from cap structures. Using LC-MS/MS, it is possible to
analyze mRNA at nucleoside level to detect and quantify dozens of different modified nucleosides in a single run. The method
can easily be adapted for quality control of NTP charges used for in vitro transcription.
Solution A: 5 mM of ammonium acetate buffer. The pH is adjusted to pH 5.3 by addition of acetic acid
Solution B: Acetonitrile It is obligatory to use only LC-MS grade chemicals.
Mobile phase: See the gradient below.

Table 25. RP-HPLC Gradient Table

Time (min) Solution A (%) Solution B (%)


0 100 0
10 92 8
20 60 40
23 100 0
30 100 0

Sample preparation
Up to 10 µg RNA are digested to nucleoside level using 0.6 U nuclease P1, 0.2 U snake venom phosphodiesterase, 0.2 U bovine
intestine phosphatase and 10 U benzonase in 5 mM Tris (pH 8) and 1 mM magnesium chloride for 2 h at 37°. Optionally, it is
possible to add deaminase inhibitors like pentostatin (A-deaminase inhibitor, 200 ng) and tetrahydrouridine (C-deaminase
inhibitor, 500 ng) to avoid nucleoside degradation.
[NOTE: Alternative settings can be applied, if justified and validated for intended use.]
Chromatographic system
Column: Synergi Fusion 4 µm particle size, 80 A pore size, 250 x 2.0 mm; Phenomenex
Column temperature: 35°
Flow rate: 0.35 mL/min
Detector: UV 254 nm
Mass parameters
Mass spectrometer: Triple quadrupole (QQQ) equipped with an electrospray ion source (ESI)
Mode: Positive ion mode, dMRM (dynamic multiple reaction monitoring)
ESI parameters: Gas temperature 300°, gas flow 7 L/min, nebulizer pressure 60 psi, sheath gas temperature 400°, sheath gas
flow 12 L/min, capillary voltage 3000 V, nozzle voltage 0 V
QQQ parameters: Depending on which modified nucleoside must be detected. It is recommended to optimize the instrument
parameters (fragmentor, collision energy, cell accelerator voltage) for each mass spectrometer to achieve optimal sensitivity.

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Table 26. Examples for QQQ Parameters

Cell
Compound Precursor Product Ion Retention Delta Ret Fragmentor Collision
Accelerator
Name Ion (m/z) (m/z) Time (min) Time (min) (V) Energy (V)
Voltage (V)
m7G 298 166 6.5 4 80 13 4
Am 282 136 14.8 3 105 17 4
m5C 258 126 7.6 3 75 13 4
8oxoG 300 168 9.5 3 100 13 4

Analysis – Relative quantification


Measure the peak areas of the respective modified nucleoside by MS/MS.
The amount of modified nucleoside is normalized to the amount of adenosine, to account for differences in the injected RNA
amount. For this purpose, extract the peak areas of adenosine from the UV chromatogram recorded at 254 nm.
Quantification is performed in a relative manner by adding isotope-labeled standards to each sample.

A(MS mod) = area of the MS peak of the respective modification


n(ISTD) = amount of internal standard in each sample
A(MS ISTD) = area of the MS peak of the internal standard
A(UV adenosine) = area of the UV peak of adenosine

Alternatively, another main nucleoside (C, U or G) can be chosen for normalization. For example, in the case of enzymatic
polyadenylation which leads to unknown or different amounts of adenosine.
Analysis – Absolute quantification
Measure the peak areas of the respective modified nucleoside by MS/MS.
The amount of modified nucleoside is normalized to the amount of adenosine, to account for differences in the injected RNA
amount. For this purpose, extract the peak areas of adenosine from the UV chromatogram recorded at 254 nm.
Absolute quantification is performed by using external calibration solutions. Prepare calibration solutions with concentrations
of 0.1, 0.5, 1, 5, 10, 50, 100 and 500 nM for MS/MS detected modifications each containing equal amounts of internal standard.
Inject 10 µL of each dilution to achieve a calibration in a range from 1 – 5000 fmol. For adenosine, prepare calibration solutions
with concentrations of 0.1, 1, 10 and 100 µmol. Inject 5 µL of each dilution to achieve a calibration in a range from 0.5 – 500
pmol. The response factor corresponds to the slope of the linear fit of the calibration curves (peak areas are plotted against the
respective amount).

x(mod per main nucleoside) = absolute quantification, amount of modification per main nucleoside)
A(MS mod) = area of the MS peak of the respective modification

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Analytical Procedures for Quality of
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A(MS ISTD) = area of the MS peak of the internal standard


n(ISTD) = amount of internal standard in each sample
rf(MS mod/ MS ISTD) = response factor of the ratio of the respective modification and the internal standard
A(UV adenosine) = area of the UV peak of the respective modification
rf(UV adenosine) = response factor of the respective modification

For mRNA of defined known sequence:


The amount of modified nucleoside can be normalized to the amount of RNA molecules.

x(mod per RNA) = absolute quantification, amount of modification per RNA molecule
N(adenosine) = number of the respective modification in the RNA sequence
Alternatively, another main nucleoside (C, U or G) can be chosen for normalization. For example, in the case of
enzymatic polyadenylation which leads to unknown amounts of adenosine

Residual T7 RNA Polymerase by ELISA


ELISA can be used to assess residual protein impurities specific for the detection of T7 RNA polymerase.
To determine the concentration of T7 RNA polymerase, generate a standard curve using antigens of a known concentration then
calculate the concentration of T7 RNA polymerase using the optical density (OD).
Coating plate: Dilute rabbit anti-T7 RNA polymerase polyclonal antibody to 3 μg/mL in 1X PBS. Coat high binding 96-well
microplate with 100 μL of this mixture and incubate overnight at 5ᵒ. Wash plate with 1X PBS containing 0.05% Tween-20.
Block with Blocker Casein in PBS for 1.5 h at RT with shaking at 500 rpm. Wash plate with 1X PBS containing 0.05% Tween-20.
Dilute T7 RNA polymerase and test samples in 1X PBS with 0.05% Tween and 0.1% BSA. Add 100 μL of this mixture to the 96-
well microplate and incubate for 2 h at RT with shaking at 500 rpm. Wash plate with 1X PBS containing 0.05% Tween-20. Dilute
horseradish peroxidase-conjugated rabbit anti-T7 RNA polymerase antibody to 3.3 μg/mL in Blocker Casein in PBS. Add 50
μL of this solution to the plate and incubate for 1 hr at RT with shaking at 500 rpm. Wash plate with 1X PBS containing 0.05%
Tween-20. Add 100 μL 1-Step Ultra TMB-ELISA substrate Solution to the plate following the instruction provided with the
substrate. Incubate as instructed.
Analysis
Read the absorbance values at 450 nm on a plate reader. The concentration of T7 RNA polymerase in the test sample can
be determined from the standard curve prepared from a purified T7 polymerase standard and the response of the standard
concentrations fit to a 4-parameter logistic equation.

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Analytical Procedures for Quality of
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Residual NTPs and capping reagent in mRNA by AEX-HPLC


Anion exchange high performance liquid chromatography (AEX-HPLC) can be utilized to verify and quantify the presence of
residual NTPs and capping reagent in mRNA.
Preparation of Samples and Controls
NTP stock solution: Prepare intermediate solution by diluting mixed NTPs to 1 mM in DNase/RNase free water into a
microcentrifuge tube.
Capping stock solution: Prepare intermediate solution by diluting capping agent to 10 mM in DNase/RNase free water into a
microcentrifuge tube.
NTP control: Add 8 μL of 1 mM mixed intermediate dilution, 50 μL of 100 mM NaCl and 142 μL of water. Vortex and spin down in
a microcentrifuge.
Capping reagent control: Add 3.2 μL of 10 mM mixed intermediate dilution, 50 μL of 100 mM NaCl and 146.8 μL of water. Vortex
and spin down in a microcentrifuge.
mRNA sample: Add 150 μL of mRNA and 50 μL of 100 mM NaCl to a microcentrifuge tube. Vortex and spin down in a
microcentrifuge.
Blank preparation: Prepare blank vials by mixing 50 μL of 100 mM of NaCl with 150 μL of water.
Filtration: Transfer blank, controls, and mRNA samples into a separate 10kDa ultrafiltration unit. Centrifuge the vials at 13,000 x
g for at least 20 minutes. Transfer the filtrate into HPLC vials.
Chromatographic system
(See Chromatography <621>, System Suitability.)
Mode: LC
Solution A: 25 mM TRIS base
Solution B: 25 mM TRIS base, 1 M lithium chloride
Column: DNAPac PA200 RS, 4 μm, 4.6 x150 mm
Flow rate: 1.0 mL/min
Detector: UV 260 nm
Column Temperature: 25ᵒ
Autosampler compartment temperature: 10ᵒ
Injection volume: 5 μL

Table 27. RP-HPLC Gradient Table

Time (min) Solution A (%) Solution B (%)


0 90 10
0.5 90 10
8.0 74 26
8.5 0 100
9.5 0 100
10.0 90 10
15.0 90 10

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Analytical Procedures for Quality of
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Analysis

Measure the peak areas of the respective peaks.


Calculate the mM of NTPs in the mRNA sample using the following equation.

Biological Activity by Cell-Based Assay


Potency testing intends to determine cellular functionality of the DP (uptake, release, translation of RNA). DP potency can be
demonstrated by verifying the cellular translation of the DP-containing mRNA into the encoded protein. There are two ways
to detect cellular translation, I) antibody dependent detection of the target protein (FACS, ELISA, WB etc.) or II) antibody
independent detection (aptamers, MS etc.). The method described here uses ELISA as the protein detection method, but other
methods may also be used.
Functional binding assay of the expressed protein from transfected cells can be used to demonstrate potency of the mRNA-LNP.
[NOTE- This example of cell-based expression uses HepG2 cells. Other cell lines or procedures can be used.]
Sample preparation
Culture HepG2 cells in Minimum Essential Media (EMEM) containing 10% fetal bovine serum. For transfections with mRNA
samples, seed cells at 1.0 X 106 cells in a 12 well tissue culture plate and incubate at 37˚ and 5% CO2 in a humidified incubator for
16-24h prior to the transfection. Transfect cells with 4 μg of RNA using the Lipofectamine MessengerMax reagent and Opti-MEM
according to manufacturer’s instructions. Incubate the transfected cells at 37˚ and 5% CO2 in a humidified incubator for 24 to
72 hrs. After 24-72 hrs., clarify the supernatant containing the expressed protein by centrifugation at maximum rpm for 1-2 min.
Clarified supernatants can be stored at -80˚ if not used immediately.
For transfections with mRNA-LNP samples, seed cells as described above. Transfect cells with a total of 1.25 mg mRNA-LNP in
Opti-MEM. Transfection of the LNP can be facilitated by the inclusion of ApoE3. Dilute ApoE3 to 1 mg/mL in the transfection
media. Add the transfection media to the LNP and incubate at 37˚ and 5% CO2 in a humidified incubator for 3 to 4 hrs. Add
complete growth media to each well and incubate the plates at 37˚ and 5% CO2 in a humidified incubator for 24 to 48 hrs.,
clarify the supernatant containing the expressed protein by centrifugation at maximum rpm for 1-2 min. Clarified supernatants
can be stored at -80˚ if not used immediately.
Next, perform ELISA on the expressed protein from transfected cells to demonstrate potency.
Analysis
Read the absorbance values at appropriate wavelength on a plate reader. The concentration of the mRNA sample can be
determined from the standard curve prepared and the response of the standard concentrations fit to a 4-parameter logistic
equation.

mRNA Drug Product Testing


The mRNA vaccines drug product can be properly analyzed by a list of compendial and non-compendial methods as described
in Table 2 above. This section summarizes each test method utilized for drug product release. In addition, some of the analysis
may require extraction of the RNA from mRNA-LNP or LNP from mRNA-LNP for further analysis using the methods described in
the drug substance section.

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Analytical Procedures for Quality of
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Extraction Methods
RNA Extraction From mRNA-LNP
The purpose of RNA extraction is to obtain high quality purified sample for applications described below for some of the drug
product analysis. There are several common reagents (e.g., TRIzol, chloroform) and kits (using chemical or beads) for RNA
extractions. Follow the instructions provided in the kit. Two examples have been provided below.
Extract mRNA from mRNA-LNP formulation by isopropanol precipitation
[NOTE- Use only glass vials and syringes.]
Extract mRNA from the mRNA-LNP formulation by isopropanol (IPA) precipitation. Perform a 10-fold dilution by adding 100 μL
of mRNA-LNP to 900 μL of 60 mM ammonium acetate in 100% isopropanol. Vortex and mix thoroughly. Centrifuge the sample
at 14,000 x g for 15 minutes at 4ᵒ. Discard supernatant then wash the pellet with 1 mL of 100% isopropanol. Vortex the sample
and centrifuge again at 14,000 x g for 15 minutes at 4ᵒ. Wash the mRNA pellet with 70% ethanol before drying the samples in
speedvac for 20 min at RT. Dry samples can be stored or resuspended in 100 μL of RNase-free water at room temperature for
quantification by UV absorbance.
Extract mRNA from mRNA-LNP formulation using chemical mixture
[NOTE- Use only glass vials and syringes.]
Incubate 100 μL of mRNA-LNP under agitation for 10 min at 50ᵒ in 10 μL of 1% Triton X-100. Next, extract the mRNA by adding
900 μL of a mixture of Phenol\Chloroform\Isoamyl alcohol 25:24:1. Next precipitate from the extracted solution by adding 0.1
volume of 3M sodium acetate, pH 5.2 and 2.5 volume of 100% ethanol. Incubate for 12 h at -20ᵒ. Centrifuge the sample at 12,000
x g for 10 minutes at 4ᵒ. Wash the mRNA pellet with 70% ethanol before drying the samples in speedvac for 20 min at RT. Dry
samples can be stored or resuspended in 100 μL of RNase-free water at room temperature for quantification by UV absorbance.

Lipid Extraction from mRNA-LNP


The purpose of lipid extraction is to separate lipids from other constituents, such as nucleic acids and to preserve these lipids
for further analysis. Polar solvents can be used to separate lipids whereas nonpolar solvents are used to dissolve lipids. There
are several common kits and reagents available for lipid extractions. Follow the instructions provided in the kit. Following is a
methanol:chloroform extraction method.
Extract of LNP from mRNA-LNP formulation using methanol:chloroform
[NOTE- Use only glass vials and syringes.]
Add 100 μL of mRNA-LNP sample into 200 μL of 100% cold methanol in 2 mL glass vial. Vortex and mix thoroughly for protein
precipitation. Add 500 μL of chloroform with glass syringe and vortex. Keep sample on ice for 10 min. Add 200 μL of nucleic
acid free water for phase separation. Again, vortex and keep on ice for 10 min. Insert vial into the 15- or 50-mL falcon tubes
then centrifuge at 600 rpm for 5 minutes. Carefully remove bottom chloroform layer of about 300 μL using syringe and transfer
into a new amber color glass vial with glass syringe. Dry the samples in a speedvac for 20 min at RT or dry under nitrogen gas
stream. Store dried sample in -20ᵒ until analysis. Sample can be reconstituted IPA:methanol (1:1) for analysis.

Identity and Content Determination of Lipids by RP-UPLC-CAD

Reverse phase ultra-performance liquid chromatography with charged aerosol detection (RP-UPLC-CAD) can be used to
determine identity of each individual lipid component and/or the associated degradation products.
[NOTE-Use glass pipets to transfer lipids/LNPs in organic solvents and use glass inserts, vials, and bottle to store lipids and LNP
samples.]

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Analytical Procedures for Quality of
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LNP extraction: Follow Lipid extraction method from above to extract all the components of lipids from mRNA-LNP.
Alternatively, mRNA-LNP can be diluted in 100% ethanol. Analyze the supernatant.
Lipid controls: Dilute each one of the purified lipid controls (cationic lipids, auxiliary lipids, cholesterol, and polyethylene glycol
(PEG) separately. These will be used to identify and quantify lipid components.
Chromatographic system
(See Chromatography <621>, System Suitability.)
[NOTE—Alternative method settings can be applied, if justified and validated for intended use.]
Mode: LC
Column: ACE Excel 2 Super C18 column, 2.1 X 150 mm
Solution A: 0.1% trifluoroacetic acid (TFA) in water
Solution B: 60/40/0.1% isopropyl alcohol/tetrahydrofuran/TFA
Column temperature: 60 °
Autosampler temperature: 15ᵒ
Flow rate: 0.5 mL/min
Injection volume: 5 µL
CAD settings
CAD evaporative temperature: 35°
Power function: 1.0
Gas resolution mode: Analytical
Data rate: 2 Hz
Filter: 3.6
Mobile phase: See the gradient table below.

Table 28. RP-UPLC-CAD Gradient Table

Time (min) Solution A (%) Solution B (%)


0 95 5
1.5 95 5
5.5 52 48
9.5 52 48
10.5 44 56
22.5 44 56
30.5 4 96
32.5 4 96
35 95 5
40 95 5

Analysis
Sample: Sample solution
Measure the areas of the lipid peaks and calculate the percentage of each.

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Analytical Procedures for Quality of
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RNA Encapsulation Efficiency by Ribogreen Assay

Encapsulation efficiency can be measured by a RiboGreen assay. RiboGreen is a fluorescent nucleic acid stain used for RNA
quantitation.
[NOTE—All solutions should be prepared in sterile nuclease-free glassware, using nuclease-free pipettes.]
RNA standard curve:
Prepare a 2 μg/mL solution of RNA in TE (10 mM Tris-HCl (pH 8.0) 1.0 mM EDTA) using nuclease-free tube. Dilute the 2 μg/mL
RNA solution into disposable cuvettes (10 levels) between 1000 – 10 ng/mL. Add 1.0 mL of 1:200 diluted (in TE buffer) RiboGreen
reagent.
Prepare a separate standard curve as described above with the addition of 0.15% of Triton X-100 into the 2 μg/mL RNA solution.
Controls:
Dilute 16S and 23S ribosomal RNA standard 50-fold in TE buffer to make 2 μg/mL working solution.
To determine the concentration of free mRNA, dilute mRNA-LNP sample to 2 μg/mL by adding 1X TE buffer to a final volume of
2000 μL. Add 1000 μL of RiboGreen solution. To release the encapsulated mRNA, add 0.15% of Triton X-100 into the mixture.
Transfer the content to 4 mL disposable cuvette and measure the fluorescence response. at 1ex = 480 nm and 1em = 520 nm.
Transfer the sample to the cuvette port of the microplate reader.
Analysis
Measure the fluorescence response at 1ex = 480 nm and 1em = 520 m. Determine the concentration of samples from calibration
curve that were generated in the presence and absence of detergent and fit to a linear regression model with 1/x weighing.

Particle Size by DLS

Particle size affects biodistribution and cellular uptake and is a critical metric for LNPs. Dynamic light scattering (DLS) can
be used for characterization studies and during quality control release testing to determine the average particle size and
polydispersity index (PDI) of mRNA-LNP samples.
Sample preparation
Dilute LNP samples in a suitable diluent (if necessary) at the concentration used at a drug product level or the working
concentration during production. The actual dilution of the sample may need to be adjusted depending on the DLS system to
enable accurate sizing reads. Perform measurements with the recommended backscatter angle and dispersant refractive index
by the instrument manufacturer. Set viscosity parameters to corresponding DP samples and dispersant, respectively.
Analysis
Run DLS measurements for 100s and measure the mean hydrodynamic diameter of each sample.

RNA Size and Integrity by Capillary Gel Electrophoresis

The PA800 plus Pharmaceutical Analysis System from SCIEX System or the multi-capillary BioPhase 8800 System with Light
Induced Fluorescence (LIF) detection can be used to evaluate the total RNA integrity. The above method for DS can also be
applied to DP post mRNA extraction (see above) or by adding Triton X-100 at 2% (w/v) to the RNA sample mixture to release the
encapsulated mRNA.

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Analytical Procedures for Quality of
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mRNA Aggregation Quantitation by SEC-HPLC

Size Exclusion Chromatography (SEC-HPLC) can be utilized for its quick and reliable method in many applications such as
purification or aggregate quantification.
Chromatographic system
(See Chromatography <621>, System Suitability.)
[NOTE: Alternative settings can be applied, if justified and validated for intended use.]
Mode: LC
Mobile phase: 100 Mm Tris acetate/ 2.5 mM EDTA pH 8
Column: Zenix SEC-300, 3 µm, 300Å, 4.6x150 mm
Flow rate: 0.25 mL/min
Detector: UV260 nm
Column Temperature: 25ᵒ
Sample: mRNA extracted from formulated mRNA-LNP
Injection volume: 5 μL
Analysis
Measure the peak areas of the respective peaks (main peak, high molecular weight, and low molecular weight).

Percentage of mRNA Fragment by IP-RP-HPLC

Ion pair reverse phase high performance liquid chromatography (IP-RP-HPLC) can be utilized to identify impurities formed
through mRNA:lipid reactions.
Chromatographic system
(See Chromatography <621>, System Suitability.)
Mode: LC
Solution A: 50 mM dibutylammonium acetate, 100 mM triethylammonium acetate
Solution B: 50 mM dibutylammonium acetate, 100 mM triethylammonium acetate, 50% acetonitrile
Column: RNASep, 7.8 x 50 mm; packing non-porous, PS/DVB resin matrix
Flow rate: 0.25 mL/min
Detector: UV 260nm
Column Temperature: 25ᵒ
Sample: mRNA extracted from formulated mRNA-LNP
Injection mass: 2μg of mRNA

Mobile phase: See the gradient table below.

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Table 29. IP-RP-HPLC Gradient Table

Time (min) Solution A (%) Solution B (%)


0 75 25
1.5 75 25
4.5 50 50
19 44 56
19.5 0 100
22.5 0 100
30.5 75 25
35.0 75 25
35 95 5
40 95 5

Analysis
Sample: Sample solution
Measure the areas of each peak and calculate the relative percentage of the late eluting peak of the total chromatographic peak
area.

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Analytical Procedures for Quality of
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Endnotes
1. RNAclean XP beads can be obtained from Beckman, Product Code 19. BigDye Direct Cycle Sequencing Kit can be obtained from
A66514 or equivalent ThermoFisher, Product Code 4458688 or equivalent.
2. RNA Fragmentation Reagents can be obtained from Thermo Fisher, 20. BigDye XTerminator Purification Kit can be obtained from
Product Code AM8740 or equivalent ThermoFisher, Product Code 4376486 or equivalent.
3. Random Primers can be obtained from Illumina, Product Code 21. SuperScript III Platnium One-Step qRT-PCR kit obtained from
1004784 or equivalent ThermoFisher, Product Code 11732088 or equivanlent.
4. SuperScript II can be obtained from Invitrogen, Product Code 22. QX200 or QX100 Droplet Digital PCR System from BioRad Product
18064-014 or equivalent Code 186 or equivalent.
5. 25 mM dNTP mix can be obtained from Thermo, Product Code 23. One-Step RT-ddPCR Advanced Kit for Probes from Bio-Rad, Product
R1122 or equivalent Code 1864021.
6. Fast DNA End Repair Kit can be obtained from ThermoFisher, 24. DG8 Cartridges for QX200/QX100 Droplet Generator from BioRad
Product Code K0771 or equivalent Product Code 1864008 or equivalent.
7. A-Tailing Buffer can be obtained from Illumina, Product Code 25. Suitable RNA Ladder can be obtained from Agilent, Product Code
1002105 or equivalent DNF-386-U015 or equivalent.
8. Klenow Exo can be obtained from Illumina, Product Code 11318090 26. Suitable RNA Diluent Marker Solution can be obtained from Agilent,
or equivalent Product Code DNF-370-0004.
9. MinElute PCR Purification Kit can be obtained from QIAGEN, 27. Suitable Intercalating Dye Solution can be obtained from Agilent,
Product Code 28004 or equivalent Product Code DNF-600-U030.
10. PE Adapter Oligo Mix can be obtained from Illumina, Product Code 28. Suitable Separation Gel can be obtained from Agilent, Product
1001782 or equivalent Code DNF-265-0240.
11. 6X DNA Gel Loading Dye can be obtained from ThermoFisher, 29. Suitable Blank Solution can be obtained from Agilent, Product
Product Code R0611 or equivalent Code DNF-301-0008.
12. 5X Phusion Buffer (Finnzymes Oy) can be obtained from Illumina, 30. Suitable 5X Capillary Conditioning Solution can be obtained from
Product Code 1000585 Agilent, Product Code DNF-475-0050.
13. PCR Primer PE 1.0 can be obtained from Illumina, Product Code 31. RNA 9000 Purity & Integrity Kit can be obtained from Sciex,
1001783 Product Code C48231 or equivalent
14. PCR Primer PE 2.0 can be obtained from Illumina, Product Code 32. RNA ladders RNA 6000 ladder with 6 transcripts from Thermo
1001784 Fisher Product Code AM7152 or RNA marker from Promega with 9
transcripts product code G3191.
15. Phusion DNA Polymerase (Finnzymes Oy) can be obtained from
Illumina, Product Code 1000584 33. Suitable dsRNA antibody can be obtained from SCICONS English &
Scientific Consulting, Product Code 10010500 or equivalent.
16. QIAquick PCR Purification Kit can be obtained from QIAGEN,
Product Code 28104 or equivalent 34. Suitable Detection Antibody can be obtained from Jackson
ImmunoResearch, Product Code 715-035-151 or equivalent.
17. Suitable cDNA synthesis master mix can be obtained from
ThermoFisher, Product Code 117565500 or equivalent. 35. Suitable Detection Reagent can be obtained from Cytiva, Product
18. BigDye Direct Cycle Sequencing Kit can be obtained from Code RPN2109 or equivalent.
ThermoFisher, Product Code 4458688 or equivalent.

The inclusion of a reference to any substance, product, method, test, assay, or equipment with respect to which intellectual
property rights may exist shall not be deemed, and is not intended as, a grant of, or authority to exercise, any right or privilege
protected by such patent, trademark, copyright, and/or trade secret. All such rights and privileges are vested in their respective
owners. See also the Intellectual Property Policy Section in USP’s Commitment to Confidentiality: https://www.usp.org/sites/
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