Genetic Engineering & Biotechnology News

AUG 2017

Genetic Engineering & Biotechnology News (GEN) is the world's most widely read biotech publication. It provides the R&D community with critical information on the tools, technologies, and trends that drive the biotech industry.

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Genetic Engineering & Biotechnology News | | AUGUST 2017 | 17 itself. ("We are not just spotting reagents on the chip, but the chip contains microchan- nels that allows us to control the flow of fluid across the array," notes Dr. Hebel.) The sec- ond technology involves a photogenerated mask. (It provides the ability to gate and control which chambers on the chip will be exposed to light.) The third technology in- volves a photogenerated acid. In conventional oligonucleotide synthe- sis, an acid-labile protecting group is gen- erally placed on each nucleotide, added to the growing nucleotide chain, and removed with acid; in synthesis on solid support, the column is flooded with acid to deprotect the nucleotide. "On arrays, we only want to remove that acid-labile group on certain features of the array at a time," advises Dr. Hebel. After the array is flooded with an acid precursor, exposure of the acid precursor to light generates the acid that deprotects the previous nucleotide only on the desired fea- ture. "That allows us to selectively deprotect and synthesize all the different sequences at one time," explains Dr. Hebel. While microarrays have become some- what less popular for certain applications in recent years, the technology allows oligo- nucleotides to be synthesized and cleaved. "Once we cleave the oligonucleotides, they may be used for quite a few different applica- tions by our customers, so even though the arrays may be somewhat obsolete for gene expression, they are still very relevant for other applications," insists Dr. Hebel. The OligoMix can also be used for se- quence capture. "In many cases, investi- gators want to perform targeted sequenc- ing, where instead of sequencing the entire genome, they need to selectively enrich for certain genomic regions," says Dr. Hebel. Several different target-capture technologies have been developed over the last few years. "The common thing between all these tech- nologies is the need for lots of primers to do the capture," notes Dr. Hebel. Sometimes, thousands or tens of thousands of oligonu- cleotides are needed to capture the regions that are of interest. Additional applications are for gene syn- thesis—when oligonucleotides synthesize gene fragments that are subsequently as- sembled—and for library construction. One of the current challenges is that the yield of the oligonucleotides in OligoMix is small because they are synthesized on the array. "We generally recommend that customers amplify the OligoMix before they use it, and while that is feasible, it adds another step, and it increases the level of complex- ity to what end-users are already trying to do," explains Dr. Hebel. Current efforts at LC Sciences are focusing on increasing the yield by improving the synthesis technology and on developing amplification technolo- gies prior to delivery of the product to the customers. "The oligonucleotide industry has changed dramatically in the past 10 years," says Mark Behlke, M.D., Ph.D., CSO at In- tegrated DNA Technologies (IDT). During the early 2000s, the main use of oligonucle- otides was for PCR primers, and at that time they were often needed in large numbers for this application. "Our largest order was over a million oligonucleotides for a single proj- ect, all of which were unmodified PCR prim- ers," recalls Dr. Behlke. The main challenge in that setting was to generate a very simple, uncomplicated product of a very high qual- ity and at a very high throughput. "While researchers still need PCR prim- ers, those products do not tend to be the ma- jor issue, and today we have instead moved toward larger scale, much more complex synthesis as the most important items," says Dr. Behlke. A crucial effort at IDT is focusing on NGS adaptors, which are indispensable for applications such as sequencing. Generat- ing these more complex products, at extreme purity and larger scale, is currently a major focus, and it requires different manufactur- ing processes and different quality control measures. Additionally, there is an increasing de- mand for complex modifications. "The frac- tion of our business that requires complex modification has increased. In addition, the modifications we are dealing with today are more complicated than the ones we dealt with a decade ago," says Dr. Behlke. Generat- ing long, highly modified, high-quality oligo- nucleotides poses a different set of challenges. "We still could incorporate those modifica- tions a decade ago. At that time, the need for oligonucleotides was much lower. Currently, we need to make them by the thousands, and the production lines have become more com- plex," says Dr. Behlke. Pharmaceutical-Grade Oligonucleotides Another recent challenge stems from the need for cGMP-quality products. "One of the goals of entering into the genomics era was transitioning research into diagnostics and therapeutics, and that is happening, but the result of that is that people working in clinical diagnostics need quality products that can receive FDA approval or CE Mark approval," says Dr. Behlke. These develop- ments reflect the success that DNA diagnos- tic applications have seen in recent years. Manufacturing these products requires cer- tain closed off, restricted access areas that operate under a different regulatory compli- ance environment. "At IDT, we have separate cGMP suites that are under ISO 13845 guidance, which enables us to sell products that can be used in FDA-approved kits and are CE Mark-ap- proved in Europe," says Dr. Behlke. Recent research advances also opened the interest for using oligonucleotides in clinical trials, with the hope for human therapeutic ap- plications. "That is a more specialized area that we have not entered into, and we refer customers to other companies that specialize in pharmaceutical-grade oligonucleotides for human administration," says Dr. Behlke. "We are primarily performing quantita- tive and qualitative analyses for oligonu- cleotide/nucleic acid and protein therapeu- tics and biomarkers," says Laixin Wang, Ph.D., vice president at NovaBioAssays, and cofounder and senior vice president at Chongqing Denali Medpharma. Scientists at NovaBioAssays provide various innova- tive analytical services for drug discovery and development applications, such as quantification and metabolite identifica- tion of small molecule drugs and large bio- pharmaceutics in biological matrices with and without automated immunoaffinity enrichment. At NovaBioAssays, a recognized oli- gonucleotide bioanalysis service provider, one of the main activities is to perform novel liquid chromatographic-based as- says to quantify and characterize oligonu- cleotide therapeutics and their metabolites in biological matrices. In addition to their sensitivity and specificity, these approaches allow multiple oligos to be quantitated in a single assay. NovaBioAssays also pro- vide in vitro stability and protein-binding evaluations. In a recent study that aimed to improve the assay sensitivity and throughout provided by liquid chromatography–mass spectrome- try (LC–MS/MS), Dr. Wang and scientists at NovaBioAssays developed a new solid-phase extraction (SPE) method that produces high- quality oligonucleotide extracts from plasma or tissue homogenate samples. In another study, to examine the performance of the three major platforms (triple quad, Q-Ex- active, and Q-Tof) that are currently being used in bioanalytical laboratories, investiga- tors at NovaBioAssays performed a side-by- side comparison to examine the platforms' strengths and limitations in performing the quantitative and qualitative bioanalysis of oligonucleotides. "With CRISPR gene-editing techniques emerging as a new modality of gene ther- apy, the characterization and quantitation of large RNA oligos (100-mer or longer) are posing challenges for analytical and bioanalytical chemists in the development of oligonucleotide/mRNA therapeutics," says Dr. Wang. Actually, four sensors are a lot better than two. If you are looking for high-performance, high-throughput SPR, we should really talk. Let's connect in real-time. www. sierrasensors .com OMICS

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