FROM RESEARCH TO COMMERCIAL QUANTITIES:A SEAMLESS SUPPLY OF OLIGONUCLEOTIDESOligonucleotides are poised for high growth over the next five years, with themarket doubling in value during this timeframe. This growth is driven by thehigh application potential of oligonucleotides to be used in the treatmentsof a variety of medical conditions, the growing number of FDA approved
oligonucleotide drugs, an increased focus on customized or personalized
medicine, and a continued emphasis on the development of therapeutics forrare disease.
As a result, contract development and manufacturing organizations (CDMOs),such as Ajinomoto Bio-Pharma Services, are seeing increased demand foroligonucleotide synthesis. However, with this developing market comessome considerable challenges for drug manufacturers in the ability to costeffectively, quickly and easily scale from research amounts to commercialneeds.
In this white paper, we will discuss how Ajinomoto Bio-Pharma Services
meets this seamless supply challenge by providing development, scale-upand synthesis capabilities of research to commercial quantities of customized
oligonucleotides.

OLIGONUCLEOTIDES

Once the oligonucleotide has been synthesized,
an appropriate deprotection method is selectedbased on the optimal conditions of the specificoligonucleotide. Purification by HPLC or MPLC isthen completed to ensure the correct purity andthe oligonucleotide goes through desalination by
ultrafiltration to remove any excess salts. From
there, the oligonucleotide can be lyophilized orfilled as a sterile solution.

Custom oligonucleotide manufacturing involves
making changes to nucleobases, the sugar
backbone, or the phosphodiester bonds tocreate a specific DNA or RNA molecule, creating
for example, BNA (bridged nucleic acids) andLNA (locked nucleic acids). These chemical
modifications have led to the development of newdrugs for various diseases and therapies that
previously were difficult to treat using conventional
Before the oligonucleotide is released to themedical treatments.
customer, analytical testing is completed toAdditional nucleic acid technologies include the ensure product quality. This testing can include
custom synthesis of antisense oligonucleotides visual inspection, molecular weight confirmation,(ASO), small interfering RNA (siRNA) and sequence analysis, UV measurements, and tests
microRNA (miRNAs), which are used to modulate for purity, sodium and endotoxin.

gene and protein expression, aptamer RNAs,
which modulate protein functions, as well as CpG Solid phase oligonucleotides can be synthesizedoligodeoxynucleotides (CpG ODN), which are used as either in vivo or GMP grade. In vivo grade issuitable for pre-clinical studies, while GMP is
as vaccine adjuvants.
required for clinical and commercial products.
Given the broad variety of platforms and Ajinomoto Bio-Pharma Services has the capabilitymodalities, oligonucleotides hold a strong to synthesize oligonucleotides in in vivo grade andpromise and a growing portfolio for researchers with GMP compliance to meet a wide range ofand biopharma drug companies in developing product needs.
effective, targeted, next-generation therapeutic
drugs and vaccines.

SOLID PHASE SYNTHESIS
Customoligonucleotide
manufacturing
is accomplished through traditional solid
phase synthesis (chain elongation) using
fully automated oligonucleotide synthesizers,
which can manufacture to specific quantity
batches. The customized configurations onthese machines allow for synthesis flexibility
and yield improvement, at any scale from µg
to 20 kg quantities.
FROM RESEARCH TO COMMERICAL QUANTITIES: A SEAMLESS SUPPLY OF OLIGONUCLEOTIDESLARGE SCALE SYNTHESIS

toHistorically, scale up to large scale production ofoligonucleotides has been a challenge due to theincrease of manufacturing costs. Using smallerscale synthesizers to develop numerous batchesof oligonucleotides leads to cost-inefficiencies
from reagents and materials, making it difficult tobring a commercial scale drug therapy to market.
Recently, large scale synthesizers for kilogram
quantities of oligonucleotides have been madeavailable in the market, which has enabled larger,
more cost-efficient production batches. In 2019,
Ajinomoto Bio-Pharma Services opened a large
scale oligonucleotide API manufacturing facility,
in order the better serve the increasing demand of
the oligonucleotides market.
This 2,000 m2 manufacturing facility provides aClass 100,000 clean area for cGMP manufacturingof oligonucleotide APIs, a multipurpose room

customsynthesize

heavily

modifiedoligonucleotides, and an OligoProcess synthesizer,
for large scale solid phase oligonucleotide
manufacturing. This customized synthesizer
provides the capability to produce up to 20 kg
quantities, making it suitable for late stage clinical
trials and commercial production. The facility also
houses R&D labs for the development of novel
oligonucleotide manufacturing technologies.
As a manufacturing alternative, solution (or liquid)phase synthesis provides advantages to large
scale solid phase synthesis through cost reduction,scalability and purity. However, depending on thetype of oligonucleotide (natural DNA/RNA, LNA,
BNA, 2’-F, 2’-OMe, etc.), the physiochemical
properties of some oligonucleotides can makeusing solution phase synthesis incompatible. To
overcome this obstacle, PEG and other anchormolecules can be used to negate the lipophilic andinsolubility of the intermediary oligonucleotide.

FROM RESEARCH TO COMMERICAL QUANTITIES: A SEAMLESS SUPPLY OF OLIGONUCLEOTIDESAJIPHASE® SYNTHESISOne such approach is AJIPHASE®, a hybrid of solidand solution phase syntheses that uses an anchorto make the molecules very soluble in non-polarsolvents, providing a homogenous mixture. Afterthe reaction occurs, the anchor is filtered out
along with the excess reagents and byproducts,
providing the recovered intermediate, all in one
reactor.
This advanced technology allows Ajinomoto BioPharma Services to manufacture commercial
quantities of various oligonucleotides underGMP. When compared to traditional solid phase
synthesis, the AJIPHASE® technology uses lesssolvents and reagents, while providing high purity
and equivalent quality, high yield batch sizes.

This becomes especially important in long chain
RNA, as yield goes down as the chain length
increases. To manufacturing long chain RNA,
enzymatic condensation is used to combine longchimera DNA/RNA by fragment enzymatic ligation
methods. This gives high purity and accurate
molecular weight, but at a high cost to manufacture
in quantities larger than research scale. Therefore,
new manufacturing technologies to ensure highscalability at a lower cost is incredibly important.
The Research Institute of Biosocience Products andFine Chemicals, a R&D arm of Ajinomoto Co., Inc.,is working on a novel RNA producing system basedon microbial fermentation of Corynebacteriumglutamicum. Ajinomoto Co., Inc. has a long history
and vast experience using C. glutamicum in various
bio-manufacturing applications.
In this application, C. glutamicum is used as arecombinant RNA producing host strain. This fastgrowing, robust bacterium offers high productivityof target RNA, is non-pathogenic and nonendotoxic, has a clarified genome sequence andlow endogenous RNase cellular activity. Using
microbial fermentation, C. glutamicum provides anovel RNA producing system for the bio-productionof various types of long chain RNA (> 100 mer) withhigh scalability and yield at a low cost with highpurity profiles.

FUTURE ADVANCEMENTS
Overall, the future of oligonucleotide synthesis
and the ability to successfully develop large scalequantities of oligonucleotides that can be used indrug therapies are both dependent on a CDMO’s
ability to effectively scale up oligonucleotides tomanufacture at a lower cost, while maintaining ahigh yield and purity.

FROM RESEARCH TO COMMERICAL QUANTITIES: A SEAMLESS SUPPLY OF OLIGONUCLEOTIDES

Ajinomoto Bio-Pharma Services has invested
significant resources to build manufacturingplatforms that meet the needs of theoligonucleotide market, from research (µg – mg)
to commercial (g – 20 kg) quantities and everything
in between. Our goal is to provide clients with theconfidence that they can cost-effectively, easily,and quickly develop, scale up, manufacture
and aseptically fill their oligonucleotide frombeginning to end. By selecting a CDMO thatpossesses extensive oligonucleotide experience,a structure that meets your stage of production,and the range of service capabilities to meet yourneeds, you will find yourself more prepared toseamlessly move your oligonucleotide-based drug
therapeutic out of the lab and into the hands ofpatients, who need it most.

Ajinomoto Bio-Pharma Services Oligonucleotide API Manufacturing Center at the Osaka, Japan campus.

info@US.AjiBio-Pharma.com
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