The amount of product theoretically possible from any particular synthesis is determined by the quality of the synthesis itself, generally done on an automated synthesizer. The coupling efficiency of the synthesis is very important. At Bio-Synthesis we do our best to optimize this efficiency using modified programming and paying special attention to reagent preparation. The results of a drop in coupling efficiency of just a few percentage points on the yield can be devastating.
This is easily demonstrated by calculating the theoretical yield with the following formula: XY where "x" is the average coupling efficiency and "y" is the number of coupling.
For example, a synthesis of a 30mer (which requires 29 couplings) with an average coupling efficiency of 99%, theoretically yields 75% of product (0.9929). That same synthesis at 98% efficiency will have a maximum yield of only 55%. That one percent difference costs nearly half of the material. Consider the difficulty of making a 70 mer. Even at 99%, the best one could hope for is 50% yield. At 98% it becomes an abysmal 25% yield.
It is very difficult to maintain every machine every day at an operational efficiency of 99% or greater, despite claims to the contrary. Not only does the instrument have to be finely tuned to operate at 99%+, but other factors have to be perfect, such as the moisture content in the acetonitrile and phosphoramidite quality. The weather plays a role, as well. Extremely humid days will adversely affect the quality of synthesis by making near complete water removal almost impossible, despite using rigorous anhydrous chemistry techniques.
Modified reagents often have poor coupling efficiencies for a variety of reasons. It is not unusual for a reagent to have a coupling efficiency as low as 90%.
Some modified oligonucleotides are prepared by conjugation to an amine, thiol or carboxyl functionality. This reaction is affected by many factors as well, including the quality and age of reagent, the sequence of oligonucleotide and the quality of the buffers. Reactions can range from 10% to nearly quantitative. A mediocre conjugation can have a considerable effect on final product yield. At BSI we do our best to control the quality of all reagents through our ISO/GMP program, as well as operate with well-established SOPs and trained personnel. Despite our best efforts, time to time some reactions will still go poorly due to sequence effects.
After synthesis, the oligonucleotide is deprotected with base. With simple oligonucleotides this presents little difficulty, as long as the deprotecting reagents are fresh. However, particular modifications require deprotecting conditions so mild that the bases are not fully deprotected. In most cases, some damage occurs to the bases themselves, but particularly to dye labeled compounds. These side reactions all result in loss of yield.
Depending on the quality of the synthesis, purification can be the step where the most yield is lost. A high quality synthesis will have only a moderate amount of impurities to remove, allowing a larger cut of the product peak. Moderate and poor syntheses will have more contaminating fragments that will crowd into the product peak, requiring a tighter cut to obtain an acceptable purity. Regardless of the quality of the synthesis, the overall process of purification is costly to yield. Upwards of 50% of the theoretical yield will be consumed in many preparaions for a wide variety of reasons. Oligonucleotides that are partially deprotected and those containing degraded or damaged dyes often share enough properties with the product to make purification difficult. In some cases, the modifications on an oligonucleotide will cause the product to co-elute with shorter fragments, which is the case with unprotected primary amine modified oligonucleotides. The requested purity makes a considerable impact on yield. The difference in delivered product between a final purity of 90% and 95% can be several fold.
Here is a hypothetical synthesis to illustrate how yield is affected. We will return to the example of the 30mer described above, but add the following: it has a 5' dye and a 3' quencher. This hypothetical dye is only available as a succinimidyl ester, therefore requiring post-synthesis conjugation to a 5' amino labeled oligonucleotide. The 3' quencher in this case is a support bound reagent from which the synthesis begins, requiring no further chemistry. The oligonucleotide is further modified with three modified bases, each of which couple at 93% in this example. The amino linker couples with an efficiency of 95%.
(0.9926)(0.933)(0.95) = 0.59
The deprotection of the oligonucleotide always results in a small amount of undeprotected bases, undesired modifications, and in this case, some degradation of the quencher. It also involves transfers and filtrations that invariably result in most of the loss. Overall, a loss of another 25% of potential product due to chemical modifications, incomplete deprotections and manual manipulation is not unusual. Our yield is now at 44%, or 0.44 µmole.
The protecting group on the amino group is left on to improve the purification of the amino labeled intermediate prior to conjugation. Without it, the amino labeled oligonucleotide will elute with the failure sequences on HPLC. It is common to lose 10% of this group prior to purification during deprotection and work-up, lowering our yield to 0.40 μmole.
This product is purified, resulting in another loss of at least 25% of the product due to contamination with the before mentioned, undesired modified materials. We are now at 0.30 μmole of intermediate. The conjugation of a dye generally goes at 70-90% unless there is a sequence or reagent issue. Let us assume an efficiency of 80%.The yield is now at 0.24 μmole. This material is now re-purified to remove unconjugated material, free dye, and any side products due to damaged dyes, resulting in a further loss of 20% of the product, reducing the product to 0.19 μmole.
This is followed by a series of manipulations to desalt the sample, convert it to the proper salt and precipitate it, and then remove aliquots for final analysis prior to deliver. Unfortunately, this series of steps is more costly than appreciated, resulting in an overall loss of another 20% of the product, reducing the final yield to 0.15 μmoles. Given a 30mer with an ε of 345 (OD260 units)(mL) (μmole)-1 and a molecular weight of 10,500 g/m, or 10.5 mg/μmole, this final yield would correspond to a delivery of 52 OD260 units, 1.6 mg, of product.
We would consider this an excellent yield for a highly modified compound like this. Yields of 20-40 OD260 units for compounds of this sort are much more common. The losses described can easily be compounded by many different factors, some completely out of our control (mostly construct/sequence issues) and some within our control (level of training, routine maintenance). A compound with high Guanosine content can cause the loss of at least half of the material if it has secondary structure that makes purification difficult. Also, recall that a day with higher than usual humidity can result in the loss of a third of the product right from the onset of the synthesis by dropping coupling efficiency just one percentage point.
Almost all large-scale synthesis (50mg and larger) are ordered by set quantity, usually in units of mass. Some customers order 10 mg samples, depending on us to decide on the proper starting scale. There are cases when ordering a set quantity for smaller amounts may make sense. Many diagnostic companies require guaranteed amounts of material delivered so that they can produce the allotted number of kits, even at small quantities. This will increase your cost considerably in some cases since we may have to prepare a larger scale to ensure the yield you requested. It is best to indicate your desired final quantity need, Bio-Synthesis can decide a scale of synthesis for you.
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