1. Introduction
  2. Custom Analytical Service
  3. Custom Analytical Service for Antisence oligonucleotide
  4. MALDI-TOF QC System
  5. HPLC
  6. GC
  7. NMR
  8. Test of Heavy Metal content
  9. Test of water content
  10. Bioburden test
  11. Endotoxin test
Introduction

At Bioneer, quality control is fundamental to our manufacturing processes and guarantees high quality product.

Bioneer’s Quality Control for Oligonucleotides can be divided into three distinct areas:

1. Bioneer’s Oligo Ordering Systems:

Orders from customers are gathered on a main production server system prior to synthesis. To eliminate re-entry errors, on-line and e-mail orders are preferred.

Orders are automatically distributed (batched) into an appropriate synthesizer according to the length of oligo, the type of modification, and users’ plate choice. Every lot to be synthesized is labeled with its own Barcode ID, which is used for identifying the oligonucleotide plate through the synthesis process. Bioneer’s Quality Assurance Staff can monitor all procedures from synthesis to aliquoting using our proprietary Automatic Oligonucleotide Production System (AOPSystem).

2. Automatic Maldi-TOF QC:

Bioneer employs multiple Maldi-TOF mass spectrometers that are fully automated from loading to mass determination. The mass spectrometry data for each sample is automatically inserted into the oligo information sheet. Bioneer is one of the few oligo producers that checks all oligonucleotides (single or high throughput orders) by MALDI-TOF and provides mass data with each oligo, free of charge.

Typical Oligo Datasheet with MALDI-TOF information

Examples of a typical 30mer and 23mer oligo spectrum, in this case employing a Kratos Maldi-Tof system.

Example of High Throughput Plate QC Analysis

The mass spectrum and result of 92 (plate) oligonucleotides – Bioneer can also provide “spectrochecked” oligo QC data for users of the Sequenom SNP Analysis System.

3. QC of Large Scale, Antisense and Decoy oligos:

The high quality oligonucleotides used in gene therapy and other therapeutic-type applications obviously require more stringent QC. Bioneer has developed specialized QC methods for large scale, antisense and decoy oligos. These specialized QC procedures comprise HPLC, CE, gel electrophoresis, NMR and additional specific tests such as endotoxin, bioburden, moisture content etc. Bioneer’s wide array of Q.C. procedures satisfies all of our users' specific QC requirements. The array of QC steps available from Bioneer is as follows:

1.

Ion-Exchange HPLC analysis

2.

Reverse Phase HPLC analysis

3.

Capillary Electrophoresis

4.

NMR analysis

5.

Moisture content analysis

6.

Sodium content analysis

7.

Heavy metal content analysis

8.

Solvent content analysis

9.

Endotoxin test

10

Bioburdent test

Bioneer can also supply reports of API stability testing under the contract period.

For information on pricing and ordering, as well as additional information on any of Bioneer’s specialized QC procedures, please e-mail order.usa@bioneer.com

 
Custom Analytical Service

(USD)     
Service Price in USD
Items 50nmole scale 200nmole scale 500nmole scale 1umole scale Gram ~ Kg scale Kg<scale
I-E HPLC 25 25 25 25 50 50
RP HPLC 20 20 20 20 40 40
Capillary Electrophoresis 30 30 30 30 30 30
Maldi-Tof analysis, Spectrocheck *1 1 1 1 1 2 2
Maldi-Tof analysis, Other mass system Free Free Free Free 2 2

 *1 : For those users who ordered more than 96 oligos, QC will be $100 for each plate. If the oligos were ordered less than 96 oligos in separate tubes, please refer to the price above in the table.

Data will be provided on CD or paper; however, if the oligos are more than 96, it will be provided in CD format.

 

Custom Analytical Service for Antisence oligonucleotide
Service Price
Items 50nmole scale 200nmole scale 500nmole scale 1umole scale Gram ~ Kg scale Kg<scale
I-E HPLC 25 25 25 25 50 50
RP HPLC 20 20 20 20 40 40
Capillary Electrophoresis 30 30 30 30 30 30
NMR analysis         20 20
PAGE analysis 15 15 15 15 15 15
Moisture content analysis         35 35
Metal content analysis         50 50
Solvent content analysis         35 35
Endotoxin test(LAL)         50 50
Bioburden test         100 100
Maldi-Tof analysis,
Other mass system 		2 2 2 2 2 2

 

The Use of Matrix-Assisted Laser Desorption / Ionization Time of Flight Mass Spectrometry of Synthetic Oligonucleotide QC

At Bioneer MALDI-TOF (Matrix Assisted Laser Desorption-Ionization Time of Flight) is the technology used extensively for failure detection and other problems that cannot be resolved by other methods. Bioneer’s fully automated, high throughput QC systems allow the company to provide superior, high quality product superior to that of our competitors. The QC systems installed at Bioneer currently can check the quality of 35,000 synthetic oligonucleotides per day. Each and every oligo is supplied with an oligo data sheet that includes Maldi-Tof mass spectrum.

Interpreting Maldi-Tof mass QC for Oligonucleotide

A Maldi-Tof mass spectrometer accurately measures molecular weight of a sample. The technique is most useful because it compares the theoretical mass calculated on the basis of oligonucleotide sequence to actual measured data. Maldi–Tof can also be used to check for sequence errors that may occur while inputting sequences. Such a QC method is an absolute requirement for sequence dependent experiments, such as PCR, cloning and sequencing. It can also be used to check whether an oligo has been modified correctly. CE or HPLC analysis cannot be used to check modifications. Maldi-Tof is also used to check for the presence of truncated oligos and salt contamination.

A Maldi-Tof mass system is most suitable for the QC of oligonucleotides less than 50 bases long. Longer oligonucleotides (> 50 mer) cannot be ionized effectively (100%) by the laser, therefore they can not be easily detected and therefore will show a poor detection signal that may fail QC. At Bioneer any oligo greater than 50 bases in length are checked for quality by PAGE. PAGE QC data sheets are provided with each oligo >50 bases.

The Bioneer QC system

The customer order data is initially saved on the main server system and then transferred to Synthesis. Following synthesis, oligos are spotted on Maldi-Tof mass plates using a proprietary, fully automated, 384 well sample OD quantification/ dispensing robot developed by Bioneer. The Maldi plates are then transferred to QC Division.

Any transfer of oligo samples and plates between different Divisions and/or equipment requires the bar-code on each sample racks to be checked by a production specialist to confirm the oligo data. Bar coding ensures compliance and allows related Divisions to easily retrieve important oligo data from the main server system.

After receiving the oligos and all the related information, the QC department checks the quality of the oligos. The mass spectrum of each oligo is saved and the QC program checks whether the oligos have been synthesized appropriately. Upon completion the final QC data is transferred to the main server.

The Bioneer QC Program is also used to confirm oligo contaminants (including truncated oligos) present in the Maldi spectra. Another key advantage of Bioneer’s QC system is its ability to automatically insert a mass spectra (0.6X mass ~ 1.3X mass) of each oligo into the oligo data sheet. Mass spectrum for all ordered oligos will be provided to customers at no extra charge.

Please note that since the Sequenom Spectrocheck system which is applicable for some SNP users employs its own program for oligo QC, $100 USD will be charged for every 96 samples if this QC system is required. Mass spectrum data of examined samples can be provided on a CD if required.

Maldi data is delivered from QC to the main server, and subsequently all the related information is delivered to Packaging so that the correctly synthesized samples will be delivered in the appropriate format as requested by the customer. Bioneer delivers oligos in a selection of different tubes, 96-well plates, or 384 well plates as per the customers' preferences. After packaged completely, all the oligos will be shipped by FedEx or UPS, and via their tracking systems, customers may monitor the exact place where the ordered oligos are in transportation.

In QC, data on all failed samples is automatically returned to Synthesis, and the re-synthesis of the failed oligos proceeds whilst QC examines the failed oligo further. This rapid exchange of related information is key to Bioneer’s rapid oligo turnaround time.

 

HPLC Analysis of Oligo Purity

Reverse Phase HPLC

At Bioneer Reverse Phase HPLC is mostly used to QC of intermediates or single stranded DNA produced in the oligo synthesis process. It is a simple QC technique for modified oligo with hydrophobic groups. Reverse Phase is faster and cheaper than Ion Exchange methods and requires less sample.

Fig1) Example of oligo (26mer) purity analysis using a Reverse Phase (C-18) Column.


Purity analysis using Ion-exchange chromatography method (using Anion-exchange column)

HPLC, Equipped with a DIONEX’ s DNAPac column, is used to QC of oligos, in particular - Decoy oligos. The high resolution capability of Ion exchange can easily separate single strand DNA and double strand DNA. At Bioneer ion exchange chromatography is commonly used to QC Decoy oligos, and plays a key role in QC confirmation with strict QC standards required for gene therapy.


Fig 2) Double strand DNA (DS DNA) test. Double stranded oligos are used for Decoy or EMSA experiments. Prior to any experiments, the formation of DS DNA must be checked. For Decoy experiments used in the development of new drugs, it is necessary to check the ratio of DS DNA in the Decoy. In order to guarantee the medical efficacy, the medicine should be formed in Decoy like API from the start of drug development. DS DNA confirmation is an FDA requirement.

 

Product purity test under Gas Chromatograph

Gas Chromatograph (GC) is used to QC for solvent content in Decoy oligos and S-oligos used in Gene Therapy. Prior to administering any oligo based drug to humans it is vitally important to check for the presence of residual organic solvents that may remain after synthesis and purification. Solvent content may compromise efficacy and cause unwanted side effects. The types of residual organic solvents that may be present include acetonitrile, pyridine and toluene etc. Concentrations should be minimally less 0.1%.

Fig1)  Standard solvent data.


Fig 2) Solvents confirmation data in antisence oligonucleotide

 

NMR - Spectroscopy

Nuclear Magnetic Resonance (NMR) spectrometer plays a very important role in understanding 3-dimensional structures of molecules. With increasing interests in the structure of biological materials, the use of NMR spectrometer is expanding into new areas, such as drug development, DNA analysis, human genomic and proteomic research and so on. NMR is commonly used to determine physical structure at the molecular level.

At Bioneer NMR is used for 31P-NMR measurement to compare typical frequency values for phosphates present in DNA backbones. By comparing actual frequencies with theoretical it is possible to check the state and purity of phosphates in synthetic oligos.

 

Heavy Metal Testing

For antisence and Decoy oligos that are directly injected into animals or humans as medicines in the pre-clinical or clinical phase, it is necessary for check for heavy metal groups that may influence the efficacy or may cause side effects. The types of heavy metals that require QC may differ in each oligo. Inductively Coupled Plasma-Optical Emission Spectrometers (I.C.P), Atomic Absorbance Spectrophotometers(AAS) and I.C.P Mass Spectrometers are routinely employed to QC oligos for heavy metal groups. Upon request, Bioneers oligos can be checked quantitatively/qualitatively for metals such as Lead, Nickel and Fe etc.

 

Water Content Analysis

Bioneer can also QC oligos for water content. A Sartorius’ Water Content Measurement instrument (MA-30) is employed to measure water contents that may remain in synthesized antisense oligo following the final drying step of the oligo purification process.

 

Bioburden Testing

Bioneer confirms the sterility of an aseptic oligo production environment by routinely conducting microbial testing of the water used in the synthesis process and final aliquoting steps. Susceptible areas of potential microbial contamination in the synthesis process and the environment, including operators are also checked periodically. Prevention ensures that the final oligos will be proven to be safe and free from microbial contaminations.

 

Endotoxin test

Bioneer also utilzes a Kinetic Chromogenic Analysis (KCA) method to confirm that the oligos are free of any exothermic materials. Generally exothermic materials present in injectable therapeutics are endotoxins from microbial contamination, especially from Gram negative bacteria contamination and must be avoided.

Kinetic Chromogenic Analysis (KCA) is based on an enzyme linked color reaction (limulus Amoebocyte Lysate reaction). The presence of endotoxins is quantified by measuring color of the reaction against known standards. Many samples can be quantified simultaneously using a standard micro-plate reader. KCA is a fast, cost-effective and short measurement. With such a method, Bioneer only provides oligos with less than 0.25 EU/ml for therapeutic applications.