No. Our dPEG^®s are prepared de novo from pure starting materials using standard organic chemistry techniques for synthesis and purification. The only PEG in our dPEG^®s is the dPEG^® described in the name.

We offer an intermediate range of PEG linkers and spacers ranging in molecular weights from 200 to about 1,300 Daltons. This range has heretofore been unutilized or underutilized due primarily to the lack of commercially available material and functionality. Most people using PEGs have focused either on tiny PEGs （200 D or smaller）, which they synthesize themselves or on the very large polydisperse conventional PEGs （2,000-3,400 D up to 50,000 D or higher）. Quanta has been making these compounds for only the past 5 years, and we are in a l earning process, starting with the smaller units （nn = 4） and working to the higher/larger sizes （nn = 8, 12 and 24）.

There are a number of other advantages with our low and mid range MWs versus the conventional PEGs, as well as the conventional alkyl spacer containing crosslinkers （see discussions at Questions 4 and 5, below）.

Many of our customers come to us thinking that they need a large PEG in order to achieve water solubility, eliminate aggregation or obtain reduced antigenicity/immunogenicity of their target molecule. In practice, many customers find that they achieve the results they desire with our intermediate range PEGs. These smaller MWs have not been available to explore all of the options or to find an optimal substitution/modification MW. It is not necessary in many cases to give a molecule, even a large biological （e.g., an antibody）, a high MW PEG, to eliminate aggregation issues.

We also do not yet offer very large dPEG^® compounds because of the learning curve required to develop processes for synthesizing these large molecules. Numerous chemical and physical properties come into play with the larger molecules that make synthesis of very long chain PEGs challenging. The process development for these molecules is underway, but it is incomplete. Our first foray into very large dPEG^® molecules likely will come in the form of branched dPEG™s. These dPEG^®>s will use shorter chains （4, 8, or 12 ethylene oxide units）, but will contain 3 to 9 branches, and potentially 27, providing our customers with high molecular weight PEGs as discrete single compounds.

Conventional PEGs present some serious issues and challenges when used to solve drug design problems or to optimize drug design. Foremost is the fact that conventional PEGs, being polymers, are offered as substantial mixtures and therefore come as a large range of MW compounds. The MW given is simple an average MW. These mixtures are also intractable mixtures of compounds.

In addition, those underivatized base materials reproducibly available for development start at MWs above 2,000 D. This lower limit of commercial availability limits the range of applications for conventional PEGs. Further, as mentioned above, the critical issue for conventional PEGs is polydispersity. For example, a polymer of MW 2,000 conservatively contains a mixture of 30-50 compounds. Polydispersity creates numerous problems in the collection of conventional drug data, including critical pharmacokinetics. Mixtures create production and reproduction nightmares inherent in working with and chemically manipulating such mixtures, which is so vital in both therapeutic and diagnostic testing and approval. Which compound is causing the effects … positive or negative-gamma T he advantages of implementing pharmaceutical product development and modification using a single compound become obvious.

Most commercial applications of conventional PEGs have to date been directed towards using the conventional PEG as a drug carrier to increase blood circulation times, thus requiring the use of high MW compounds. This approach, though successful, has entailed much expense in dealing with the challenges of controlling a very complex mixture throughout the production and scale up of these drugs. Difficulties in finding reliable, reproducible supplies of the starting, underivatized （generally methoxy-terminated） conventional, polymeric PEGs have aggravated the problem, i.e., the reproducibility of the original polymeric process.

Another significant PEG application is drug conjugation. With conventional PEGs, several problems arise. First, the range and mixture of conjugates formed is huge. Second, knowing the pharmacokinetics of such a complex mixture is intrinsically problematic. Third, the shortest average commercially available conventional PEG linker is in the range of several 100s of Angstroms, and the option of looking at potentially more optimal ranges of conjugates simply has not been available. With our dPEG^® offerings of discrete MW crosslinkers, this situation is changing and will continue to improve as more and more options are available.

Quanta BioDesigns dPEG^® compounds eliminate the problems associated with polydispersity:
a. Single compound applications can now be modified with single compound dPEG^®s in order to maintain their analytical and chemical uniqueness;
b. When bifunctional dPEG^®s are used, the size and spatial properties are again unique and will generate a compound for testing and application that has unique properties and not a range of them; and,
c. Processing and scale-up is extremely simplified when having to only purify a single compound and not a complex mixture.

Introduction

While the aliphatic methylene chain spacers, X-（CH2）n-Y, have been useful for many years, they have serious limitations and drawbacks and have needed to be replaced for some time. Quanta BioDesign, Ltd. has introduced a wide range of crosslinkers and related products, e.g., biotinylation reagents, containing discrete polyethylene glycol （dPEG^®） -based spacers. With Quantas dPEG^® products, the end user now has a product that not only overcomes the drawbacks of the alkyl linkers and spacers but also provides many new options and advantages that cannot be obtained with conventional alkyl linkers and spacers.

Water solubility and hydrophilicity

Quantas dPEG^® linkers are extremely water soluble and hydrophilic, while the alkyl linkers are neither. The water solubility and hydrophilicity of our dPEG^®s opens up an unexplored range of applications. In contrast, although widely used, the opposite properties in the alkyl spacers have severely limited their actual and potential uses in biological systems. At least one company （Pierce） has commercially developed the sulfo-NHS esters, which are soluble in water, but this apparent solubility disappears once the label or crosslinker is reacted, and the inherent hydrophobic properties return, and the disadvantages become apparent. Unfortunay, the inexperienced user is fooled into thinking this apparent hydrophilicity is inherent to the reagents use; it is not. The hydrophobic characteristics of the alkyl linkers and spacers are most often manifest through increased aggregation and precipitation in the modified or crosslinked products in which they are used and incorporated. With Quantas dPEG^®-containing compounds, this trend is compley reversed. The dPEG^®s presence adds water solubility and hydrophilicity not in the original compound, or enhances whatever is inherent in the biological compound or drug being reacted with the dPEG^® derivative. The extent depends on many variable, including the size of the dPEG^® reacted.

For example, the biotinylation reagents using the LC linker （amino caproic acid） compared to our biotinylation reagent containing the dPEG^®4 spacer offer a startling contrast. Where the LC linker has been used, once the biological compound is biotinylated, the LC-biotin with the linker will seek hydrophobic regions in the protein and hide in them, making it less available to the streptavidin. Moreover, LC-biotin compounds have serious and very short term agglomeration and precipitation problems. Pierce generated some agglomeration data that compares the sulfo-NHS-LC-biotin （the most popular biotinylation reagent on the market） with our NHS-dPEG^®4 biotin, which has the dPEG^®4 spacer （the length of 2x LC）. The data show that human IgG biotinylated with the sulfo-NHS-LC biotin precipitates within a couple of weeks, while human IgG biotinylated NHS-dPEG^®₄ biotin （PN 10200） shows no sign of agglomeration at three weeks. The results are dramatic and surprising considering the sizes. As more customers use our products, we expect to hear many similar outcomes.

In addition to being water soluble, the dPEG^® linkers are organic soluble and can be used in organic media when this is desirable or necessary. This is true, for example, with some of the more reactive NHS esters and the like.

Application Note: Because some of our crosslinkers are viscous, we often recommend our customers initially dissolve the compound in an organic solvent. With Quantas peptide synthesis dPEG^® reagents, as well as with many of the modification reagents, the application is already going into an organic medium, so this property becomes essential.

Immunogenicity

Quantas dPEG^®-linked compounds are essentially non-immunogenic, while alkyl linkers containing more than two or three methylene groups are highly immunogenic. This is a huge advantage for dPEG^®-linked compounds and a tremendous problem for alkyl linkers. Immunogenicity creates many problems for biological compounds, many of which can be solved or improved by switching to dPEG^® products. We have customers using our MAL-dPEG^®x-NHS esters in place of the well known and widely used SMCC and related heterobifunctional crosslinkers with dramatic results when conjugating antigens to carriers for antibody production, and the final （WORD?????） of the antibodies produced. Our customers can now extend the antigen away from the carrier to various distances with no immunogenicity in the spacer … none!! It will be interesting to see this generally applied. Now antibodies generated using standard carriers and our dPEG^®s have the potential to be of far superior quality.

Non-immunogenicity has been shown repeatedly for the polydisperse polymeric PEGs, usually of high molecular weight, where we would expect any manifestations to be amplified. This is a major reason our dPEG^®s have found such extensive application.

Distance/spacing and distance/spacing control

Applications incorporating conventional alkyl spacers could benefit from the ability to use longer spacers than those currently available, but serious problems develop with their inclusion. Alkyl linkers are poorly water-soluble and are immunogenic from the start. Lengthening the spacer makes the crosslinker less water-soluble （and often less soluble in organic solvents also）, more hydrophobic, and more immunogenic. That is why little to no change in the structural range of commercial available crosslinkers has occurred for more than 20 years. In fact, changes that have occurred have often been to shorter, not longer, chain lengths of the methylene chain spacers.

In contrast, however, dPEG^® spacers are extremely water soluble, hydrophilic and non-immunogenic. These properties offer no restriction to lengthening the linkers. In addition, since we are able to make the dPEG^®s, which are single compounds, of any length starting with dPEG^®2, with the recent introduction of our dPEG^®24 product line, the chains are now approaching 90 Angstroms （90 Å） in length （linear）. Quantas customers can now select spacer chain lengths from 10 to 90 Å. Researchers developing new drugs, as well as other targeting （often diagnostic） molecules, are relying more often on modeling techniques where they can predict the optimal distances for making chemical modifications. Giving them this range of options makes Quantas dPEG^®s additionally attractive and valuable. We believe this is the wave of the future for crosslinking, labeling, and chemical modification. Modification reagents

Quanta BioDesign offers an expanding line of commercial products, which has no counterpart with alkyl chains as above. These are specifically designed to be chemically bonded to a drug, protein, or other biological compound with the objective of （a） increasing its water solubility and/or （b） decreasing its immunogenicity, antigenicity, or toxicity. We have several products which are methoxy-terminated over a MW range of about 300 to over 1,200 D that incorporate the NHS ester. Moreover, we are expanding that line by adding two options with the NHS-carbonate activated linkage, which offer the potential to be released （e.g., as a pro-drug）. We also offer methoxy-terminated products that can bond to acids, aldehydes, and sulfhydryls. The latter are of growing interest as molecular engineers can introduce the sulfhydryl almost at will using site-directed mutagenesis, and most peptides and oligos can be sulfhydryl terminated or modified. In late 2005, we plan to introduce our first branched products for use in chemical modification. This will give researchers the options of higher molecular weight dPEG^®s, as well as some unique dPEG^®s not previously commercially available in any format.

Note: The different physical properties of the dPEG^®-containing crosslinkers and modification reagents are initially perceived to be a drawback. Many of the lower MW materials are viscous liquids that can be difficult to handle. However, we find that with a little education and the initial results, our customers adapt very rapidly and creatively. Once they become accustomed to these physical properties, they can use them to their advantage by proper use of solvents and solvent systems.

Summary

For most crosslinking and labeling applications where a spacer is desired or required, the properties of the dPEG^®s outlined above should cause them to displace most applications with the aliphatic spacers. Furthermore, given the longer and multiple spacer options available for the dPEG^®s, end users have new options and new extensions of applications available to them. In addition, Quantas low MW dPEG^®s open up applications not available to the higher MW polydisperse PEGs offered by companies like Nektar Therapeutics （the lowest MWs are typically 2,000 or 3,400, average n about = 45 and 75, respectively）. Moreover, polydisperse PEGs are complex polymer mixtures, while Quantas discrete PEGs are single compounds, giving the end-user tremendous advantages over polydisperse PEGs at all steps of the application or process.

Quanta BioDesign, Ltd. is committed to developing highly cost effective, high purity, and proprietary processes for making the entire range of useful dPEG^® compounds and their derivatives for application to the widest range of therapeutic, diagnostic, and molecular engineering applications. Our compounds often open doors that have never been opened before due to the absence of the proper molecular tools. These tools are now offered and are being developed by Quanta BioDesign, Ltd. Finally, we are extremely interested in getting suggestions and feedback from our customers about new options in making other valuable dPEG^® products.