Inside of mRNA therapeutics manufacturing facility

Thermo Fisher Scientific is one of the few primary manufacturers of nucleotides in the industry. We offer nucleotides for therapeutics development and further manufacturing, including NTPs, modified nucleotides, and CAP analogs [7-methylguanosine or m 7 G (5’)ppp(5’)G]. These nucleotides are synthesized from high-quality raw materials in state-of-the-art manufacturing facilities in multiple locations. The nucleotides are available in a variety of formats, formulations, and volumes for convenience and flexibility.

Thermo Scientific TheraPure nucleotides were developed to meet the needs of mRNA-based therapeutic development strategies. TheraPure reagents provide high-quality manufactured, commercial-scale, critical raw materials with a high degree of qualification, traceability, and regulatory documentation. Our routine QC on all TheraPure reagents includes:

  • Controlled microbial enumeration testing
  • Controlled bacterial endotoxin level testing
  • TSE/BSE component-free
  • Performance testing based on the intended use

A

TheraPure ATP showing greater than 99% purity (from HPLC)

B

TheraPure CTP showing greater than 99% purity (from HPLC)

C

TheraPure GTP showing greater than 99% purity (from HPLC)

D

TheraPure GTP showing greater than 99% purity (from HPLC)

Figure 1. Relative HPLC profiles of NTPs illustrating purity. HPLC analysis shows greater than 99% triphosphate purity for (A) ATP, (B) CTP, (C) GTP, and (D) UTP. All impurities are characterized and are non-critical for IVT applications.

TheraPure nucleotides (GTP, CTP, ATP, and UTP)

The TheraPure NTPs are extensively tested and verified for use in a wide variety of molecular biology applications, including in vitro transcription (IVT), and are suitable for use as starting materials for production of nucleic acid-based (mRNA) therapeutics or other Active Pharmaceutical Ingredients (API). Our TheraPure NTPs are manufactured in accordance to ISO 9001, ISO 13485 quality management standards and principles of ICH Q7, EU GMP Part II, and in line with many of the principles defined in U.S. 21 CFR 820 ‘Quality System Regulation’ for medical devices.

The specifications for TheraPure NTPs include:

  • TSE/BSE free, beta-lactam-free, mycoplasma, melamine and nitrosamine risk-free product, reducing risks for customers and their workflows
  • Greater than 99% purity confirmed by HPLC
  • Functionally tested in in vitro transcription
  • Stable for 3 years at –20°C

Applications

  • In vitro transcription
  • aRNA synthesis
  • siRNA synthesis
  • RNA amplification
  • Ligation
  • Phosphorylation

Advantages of TheraPure NTPs compared to research use only NTPs

TheraPure NTPs provide a proven choice for mRNA therapeutic development so you can smoothly transition to commercialization with confidence. In addition to providing beta-lactam-free, and melamine and nitrosamine risk-free formulations, our TheraPure NTPs have intended use statements compatible with use in further manufacturing of therapeutics, safety testing, and regulatory documentation—measures to provide quality starting material to help minimize risk, ease the burden on your quality systems, and support your regulatory submission.

Table 1. QC requirements between TheraPure NTPs and research use only NTPs.

 TheraPure NTPsCatalog NTPs
ParameterGMP gradeResearch and clinical development gradeResearch use only (RUO) NTPs
Color and clarity✓✓Not available
Concentration✓✓✓
pH✓✓✓
Identity✓✓✓
Purity✓✓✓
Base purity and impurities✓✓✓
Endo- and exonucleases✓✓✓
Ribonucleases✓✓✓
λmax✓✓✓
Testing in transcription✓✓✓
Microbial enumeration✓✓Not available
Bacterial endotoxin assay✓✓Not available

Table 2.  Comparison of QC specification between TheraPure NTPs and catalog NTPs.

 TheraPure NTPsCatalog NTPs
ParameterGMP gradeResearch and clinical development gradeResearch use only (RUO) NTPs
Color and clarityPh. Eur. 2.2.1 + 2.2.2, method IIPh. Eur. 2.2.1 + 2.2.2, method IIN/A
Concentration100 ± 3 mM
pH7.3 – 7.5
IdentityRRTRRT2RRT2
Purity and impurities≥ 99% triphosphate
Base purity≥ 99.5%
Endo- and exonucleasesNot detectable
RibonucleasesNot detectable
λmaxATP259 ± 2 nm
CTP271 ± 2 nm
GTP253 ± 2 nm
UTP262 ± 2 nm
Testing in transcriptionConforms
Microbial enumeration< 1 cfu/mL< 5 cfu/mLN/A
Bacterial endotoxin assay< 1 EU/mL< 1 EU/mLN/A

Table 3. TheraPure NTPs portfolio with available salt concentrations.

ParameterTheraPure NTPs
GMP gradeResearch and clinical development grade
ATP, Sodium salt 100 mM✓✓
ATP, Sodium salt 200 mMContact us for availability✓
ATP, Tris salt 100 mMContact us for availability✓
ATP, Tris salt 200 mMContact us for availability✓
CTP, Sodium salt 100 mM✓✓
CTP, Sodium salt 200 mMContact us for availability✓
CTP, Tris salt 100 mMContact us for availability✓
CTP, Tris salt 200 mMContact us for availability✓
GTP, Sodium salt 100 mM✓✓
GTP, Sodium salt 200 mMContact us for availability✓
GTP, Tris salt 100 mMContact us for availability✓
GTP, Tris salt 200 mMContact us for availability✓
UTP, Sodium salt 100 mMContact us for availability✓
UTP, Sodium salt 200 mMContact us for availability✓
UTP, Tris salt 100 mMContact us for availability✓
UTP, Tris salt 200 mMContact us for availability✓

TheraPure modified nucleotides

From platform development through commercialization, TheraPure modified nucleotides offer:

  • High purity
  • Low bacterial endotoxin
  • TSE/BSE free
  • Scalability for manufacturing

Table 4. Specification of TheraPure modified nucleotides.

ParameterPseudo UTP 100 mM, Na+1-Me pseudo UTP 100 mM, Na+1-Me pseudo UTP 200 mM, Tris+5-Methoxy UTP 100 mM, Na+5-Methoxy UTP 200 mM, Tris+1-Ethyl pseudo UTP 200 mM, Tris+N4-Ac-CTP 100 mM, Na+
HPLC>99%
Spectral UV analysisConforms to reference
pH7.7 ± 0.27.7 ± 0.27.7 ± 0.27.0 ± 0.27.0 ± 0.27.7 ± 0.27.0 ± 0.5
Concentration100 ± 3 mM100 ± 3 mM100 ± 3 mM100 ± 3 mM100 ± 3 mM200 ± 6 mM100 ± 3 mM
λmax271 ± 2 nm271 ± 2 nm271 ± 2 nm280 ± 2 nm280 ± 2 nm271 ± 2 nm243 ± 2 nm
EndodeoxyribonucleaseNone detected
ExodeoxyribonucleaseNone detected
RNase activityNone detected
Protease activityNone detected
Endotoxin≤1.0 EU/mL

Cap analogs and ARCA cap

Cap analog [m7G(5')ppp(5')G] is used for the synthesis of 5' capped RNA molecules in in vitro transcription reactions. These molecules are translated more efficiently in in vitro translation systems than uncapped mRNAs. Cap analog is also used as a highly specific inhibitor of the initiation step of protein synthesis.

Anti-Reverse Cap Analog (ARCA) is a modified cap analog to ensure incorporation in the forward orientation during in vitro transcription.  As a result, unlike transcripts synthesized with conventional cap analog, 100% of the transcripts synthesized with ARCA at the 5' end are translatable leading to a strong stimulatory effect on translation.

Advantages of ARCA cap:

  • Synthesize more protein from invitro-transcribed RNA
  • Express RNA transcripts more efficiently both in vitro and in vivo
  • Stabilize transcripts in vivo with included reagents for poly(A) tailing
More protein synthesized from ARCA capped compared to standard RNA

Figure 3. Transcripts containing ARCA are more highly translated in transfected cells. Comparison of protein expression from standard and ARCA-capped luciferase RNAs, plus and minus poly(A) tail, at different time points after transfection. (A) Either standard cap analog-capped or ARCA-capped luciferase in vitro transcribed RNA (1 µg) was transfected into HeLa cells. Cells were harvested and lysed at 8 hr, 10 hr, 12 hr, 24 hr, and 48 hr post-transfection. Luciferase activity was measured and plotted against transfection time. (B) Poly(A)-tailed luciferase RNA (1 µg) prepared in either a standard mMESSAGE mMACHINE or a mMESSAGE mMACHINE Ultra reaction was transfected as above. Luciferase activity was measured and plotted against time following transfection.

Related areas

mRNA purification solution   |   Gene therapy research   |   OEM and commercial supply   |   In vitro transcription

Customization capabilities

Experienced supplier of IVT reagents for your mRNA therapeutics development

We know that not all nucleotide requirements are alike. We are committed to providing you with TheraPure reagents customized to your needs—quality nucleotides you know and trust, personalized to your specifications.

  • Add or remove components
  • Change formulaton
  • Custom QC tests
  • Custom packaging options including size/volume, containers, labels, etc.

If you are interested in custom TheraPure nucleotides or services, contact us

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