Frequently Asked Questions About dPEG® Products
To find our answers to the questions we are frequently asked about our company or about our dPEG® products, please click on the linked questions below:
Both Quanta BioDesign’s dPEG® products and monodisperse PEGs have no dispersity (Đ = 1),. See Figure 1. Monodisperse PEGs are typically separated from polymerization mixtures by various modes of chromatography to provide a single chain length and molecular weight, thereby avoiding complications resulting from dispersity. While some commercially available monodisperse PEGs have sizes up to 28 ethylene oxide units (MW ~1,250), the vast majority of monodisperse PEGs have 12 or fewer units (MW ~500)., In our patented production process, dPEG® products are prepared via standard organic chemistry methodology and not by polymerization techniques, thereby providing a single molecule with a defined and specified chain length, molecular weight, and purity., This easily scalable process is routinely applied to provide linear dPEG® products up to 2 kDa and branched dPEG® products up to 16 kDa.
The terms monodisperse PEG and discrete PEG overlap in meaning, but there are significant differences between the two terms. These differences have consequences for drug development and characterization. Describing our dPEG® products as “monodisperse,” though not inappropriate, connotes incorrect concepts with respect to our compounds. “Monodisperse” implies that either:
1) The compounds are single compounds that were made in a one-pot polymerization process and then purified from the polymeric mixture, which our compounds are not; or,
2) There is only one compound of uniform functionality, size, and shape formed from a polymeric process, which is purely a theoretical concept.
We prefer the term “discrete” since our products are synthesized in a step-wise fashion as single molecular weight compounds from pure starting materials using standard organic methodology. Thus, the terminology associated with polymer chemistry is not completely accurate. Therefore, a monodisperse PEG is a single compound prepared via a polymerization process and separated from the mixture, while a dPEG® is a single molecule of specified length and molecular weight prepared via defined stepwise reactions.
Our linear products are typically named as R1-dPEG®x-R2, where R1 and R2 denote functional, reactive, or protective groups and x indicates the number of oxygen atoms in the spacer unit. We do this to simplify the naming of the compounds. Our catalog shows the exact structure of each compound.
For example, product number 10244, amino-dPEG®4-acid, has an amino group on one end, four (4) ethylene oxide units, and a carboxylic acid group on the other end. See Figure 2. Similarly, product number 10885, Fluorescein-5(6)-amido-dPEG®12-NHS ester, has an amine-reactive NHS ester on one end, 12 ethylene oxide units, and a fluorescent organic moiety on the other end. See Figure 3.
Our products are used currently for multiple applications, including the following:
- linkers for payloads/warheads in antibody-drug conjugates (ADCs) and other targeting molecules (Fab, PDCs, ),,,,,;
- performance enhancing modifiers of pharmacokinetic, pharmacodynamic, and biodistribution properties of therapeutic, diagnostic, and theranostic biomolecules and small molecule pharmaceuticals,,,,,;
- surface modification of gold,,,,,, iron,, iridium,,,,, carbon nanotubes,, and other biocompatible surfaces,,,,,;
- assembly of both targeted and non-targeted micelles, liposomes,,, dendrimers,,, and other nanoparticles,,,;
- functionalization and “stealthing” of primary and secondary antibodies,,,; and,
- affinity tags, fluorescent dyes,,, and radiolabels for diagnostic applications,,,,,,,.
Our dPEG® products convey the beneficial properties of traditional PEGs, such as increased water solubility, reduced aggregation, increased hydrodynamic volume, and reduced immunogenicity, but they do so while limiting the complications that arise from using disperse PEGs and allow for the rational design of dPEG® conjugates and analysis of their structure-activity relationships.
No, our dPEG® products are synthesized from high-purity starting materials using standard organic chemistry techniques in a series of multi-step reactions. Please see Figure 1, above. Each dPEG® product has a specified size, molecular weight, architecture, and functionality and is characterized in the same manner as small molecules. Thus, the only PEG in our dPEG® products is the one described in the name and number, and the purity that we report is not based on average molecular weight, but on a specific molecular weight. This was shown in a 2009 study by Alister C. French, et al., where a dPEG®24 product from Quanta BioDesign was tested and found to have Đ= 1.000058 at 96% purity.
Conversely, traditional PEGs prepared via polymerization contain other homologs, and PEG used in the past typically had a Đ of up to 1.2. Years of research and development have driven this value lower so that the currently accepted standard for PEG reagents is Đ of 1.01 – 1.05 for mPEGs less than 5 kDa in size to more than 1.1 for PEGs greater than 50 kDa in size.,,, Moreover, there is no way to guarantee that the size and molecular weight distribution profile of one lot of dispersed PEG will match the profile of subsequent lots of dispersed PEG.
As the requirements for the approval of new PEG conjugates become more stringent, the trend towards a narrower range of molecular weights is expected to continue.,,. For instance, the first two PEG conjugates brought to the market in the early 1990s, Adagen® (pegademase bovine) and Oncaspar® (pegaspargase), needed only demonstrate the reproducibility of conjugation. In the case of both drugs, the PEG was treated “as an excipient that contributed to the pharmacokinetic properties of the molecule.” When Pegasys® (Peginterferon alfa-2a) and Peg-Intron® (Peginterferon alfa-2b) were introduced ten years later, regulatory agencies required more thorough characterization of each product, including the number of PEGs conjugated to the protein, characterization of each isomer, specific conjugation sites, and stability of the conjugated protein, among many other items. Although the conjugation sites of these therapeutic products were characterized, they still employed disperse PEGs. This gave rise to a population of drug conjugates with different biological properties among the members of the population. By contrast, the use of a monodisperse PEG was shown clearly to simplify the characterization of three model proteins as compared to results achieved with PEGylating the model proteins with disperse PEGs. As further research demonstrates the impact the PEG size and shape have on both chemistry and biology of PEG-conjugates, monodisperse and discrete PEGs will become increasingly important.,,,
To protect our clients’ interests, Quanta BioDesign patented the processes for the production of dPEG® constructs (see US Patents #7,888,5366 and 8,637,711) allowing them freedom to operate. We also have several patents pending on compositions of matter related to dPEG® constructs. Numerous companies worldwide distribute our products, but we manufacture all of our products in our facilities in Plain City, Ohio. For more information, please see the question on our capabilities.
Quanta BioDesign’s dPEG® Products
- Each dPEG® product is a single compound.
- When a dPEG® is conjugated to another molecule, there is a single reaction product.
- Because there is a single reaction product, analysis of the final product purity is simplified.
- A simplified analysis of the final product purity saves time and uses fewer resources, thus saving money.
Traditional, disperse PEG products
- Each traditional PEG product is a complex mixture of PEG chains.
- When a traditional PEG is conjugated to another molecule, there are many different reaction products arising from PEG dispersity.
- Because there are multiple PEG chain lengths in the final product, the analysis is complex and time-consuming;
- A complex, time-consuming analysis can be quite expensive in terms of labor and resources.
Many of our customers come to us thinking that they need a large PEG in order to improve water solubility, eliminate aggregation, reduce non-specific binding, or impart reduced antigenicity/immunogenicity to their target. The prevailing thought in the bioconjugation community has been that “bigger is better.” This is not always the case, though. Rather the optimal PEG size depends on the specific application. Some case studies are illustrative.
Pegasys® (Peginterferon alfa-2a; Schering-Plough) and Peg-Intron® (Peginterferon alfa-2b; Hoffman-LaRoche) are two PEGylated protein (cytokine) drugs developed from Interferon α2A (Pegasys®) and Interferon α2B (Peg-Intron®), both of which are approximately 19 kDa proteins, differing by only one amino acid at position 23 of the sequence. Both Pegasys® and Peg-Intron® effectively treat Hepatitis C, a potentially life-threatening infection caused by the Hepatitis C virus (HCV). In developing both drugs, initial screening was conducted using disperse 5 kDa PEG. In both drugs, the isoforms generated using the 5 kDa PEG were found to be unsatisfactory in comparison to the unmodified protein.
During the development of Pegasys®, the 40 kDa conjugate showed substantially modified PK parameters resulting in dramatically improved clinical efficacy compared to the unmodified IFN α2a. Although the mono-PEGylated conjugate only retained 7% of the parent’s in-vitro activity, the improved in-vivo profile provided a blockbuster drug and first-line treatment for hepatitis C.,
In the case of Peg-Intron®, however, a mono-PEGylated IFN-a2B for the treatment of hepatitis C achieved the desired effect with a linear 12 kDa disperse PEG. In this case, the conjugate retained 28% of the unmodified IFN-a2B in vitro activity, and although the increase in systemic exposure is modest when compared to the 40 kDa conjugate, the balance between PK and PD provided another blockbuster drug for the treatment of hepatitis C.
An example of an even smaller disperse PEG conveying the desired properties can be seen with Somavert®, a PEGylated form of the 22 kDa human growth hormone for the treatment of acromegaly. In this case conjugation of four to six 5 kDa disperse PEGs resulted in a 28-fold decrease in binding affinity, but this was offset by a 400-fold increase in serum half-life, providing a second-line treatment for acromegaly.
These demonstrated successes with 40 kDa, 12 kDa, and 5 kDa disperse PEGs illustrate the point that PEG size alone is not as important as the proper balance of PK and PD in order to achieve the desired clinical effect, as well as reproducibility of manufacturing processes. In fact, while a 5 kDa PEG failed with Pegasys® it provided the desired profile with Somavert®.
It should be noted that in the development of Pegasys® (INF-α2a) and PEG-Intron® (INF-α2b), a branched 40 kDa PEG was found to provide the optimum therapeutic properties for Pegasys®, while a linear 12 kDa PEG provided optimum therapeutic properties for PEG-Intron®, thereby demonstrating that large linear PEGs are not always the best choice for development.79,81
In addition, a branched dPEG® from Quanta BioDesign (M.W. 2,420 Da) was found to improve the pharmacokinetics and immunogenicity profiles of the enzyme organophosphorus hydrolase. See Organophosphorus Hydrolase Pharmacokinetics and Immunogenicity are Improved by Branched dPEG®.
In another example, a three-branched dPEG® with a molecular weight of 4,473 Da with three negative charges (from the ends of the three branches) was conjugated to an antibody fragment (Fab’) and labeled with 124I for tumor imaging. This construct (see, Figure 5, below) was found to provide superior tumor retention and imaging quality and opened up the possibility for simpler analytical testing in drug discovery studies.52
For small-molecule drugs, even smaller PEG conjugates provide dramatic improvement. One striking example is the radical modification of properties that occurs when a small, discrete PEG of only eight (8) ethylene glycol units is attached to the water-insoluble, toxic antifungal drug Amphotericin B. This development is discussed in detail here.
Briefly, Amphotericin B, a polyene macrolide antifungal drug, is the first-line, “gold standard” treatment for fungal infections and for the disease known as leishmania. Nevertheless, Amphotericin B is poorly soluble in water, is difficult to administer, and exhibits long-term systemic toxicity., Liposomal Amphotericin B delivery systems, with and without PEG, are used clinically, but systemic toxicity remains. PEG-Amphotericin B conjugates improve the water solubility of Amphotericin B, but until recently, relatively large PEGs of 5 kDa or above have been used, and the results were not impressive.,,
In 2016, two papers87, demonstrated that short, dPEG® products conjugated to Amphotericin B substantially alter the physical and pharmacological properties of the drug, including increased water solubility and decreased toxicity, without loss of antifungal efficacy. In both papers, the dPEG® was linked to the single free amine of Amphotericin B. The paper by Tan, et al.,91 used a methoxy-terminated dPEG®4 (PN10211, m-dPEG®4-NHS ester; molecular weight, 333.3 Daltons; dPEG® molecular weight 220.3, Daltons). In contrast, Halperin and colleagues used Quanta BioDesign’s product PN10995, Fmoc-dPEG®8-NHS ester; molecular weight, 760.8 Daltons; dPEG® molecular weight, 441.5 Daltons), followed by removal of the Fmoc protecting group to expose the terminal amine.
The research by Tan, et al., allowed the formation of a liposome-like suspension that remained dispersed in water even at high concentrations. In vitro toxicity was reduced considerably, and the product remained efficacious in killing two species of fungus that are pathogenic to humans.91 In the paper by Halperin, et al., after removal of the Fmoc protecting group from the dPEG®8, the Amphotericin B-dPEG®8 conjugates demonstrated water solubility, sharply reduced in vivo toxicity in mice, and efficacious in vitro and in vivo killing of pathogenic fungi.87 Taken together, these two papers show that it is possible to develop a clinically useful therapeutic product using only small, discrete PEG products. Moreover, because the dPEG® moiety is a single molecular weight compound, the conjugated product can be characterized fully. With a dispersed PEG, the characterization of the several conjugates formed in the reaction would be a time-consuming challenge.
In the areas of bioconjugation, ADCs, diagnostics, and surface modification the focus is not on large PEG size as much as on hydrophilic linkers of defined and consistent lengths. Both hetero- and homobifunctional dPEG® linkers with as few as four ethylene oxide units have been shown to impart beneficial properties to the resulting conjugates, and the ability to employ varying dPEG® lengths has allowed the optimization of the desired properties.
For example, researchers at Immunogen used hydrophilic likers for the construction of antibody-drug conjugates. They found that dPEG® linkers with as few as four ethylene oxide units were able to provide antibody-maytansinoid conjugates that doubled typical drug-antibody ratios (DARs), were much more potent than lower DARs, did not aggregate, and retained antibody affinity.
James R. Prudent and co-workers at Centrose Pharma developed a new class of antibody-drug conjugates (ADCs) called an “extracellular antibody-drug conjugate.” (EDC) to deliver a toxic payload to a specific extracellular site that is proximal to an extracellular target of an antibody. This research is discussed more fully at this link. The researchers evaluated linkers between the antibody and the drug. The linkers contained 0, 2, 12, 24, and 36 ethylene glycol units in the dPEG® chain. They found that the dPEG® linkers all enhanced delivery of the cardiac glycoside scillarenin β-L-aminoxyloside to tumor cells expressing dysadherin, a protein marker associated with metastatic cancer, that was targeted by the monoclonal antibody to which the payload was conjugated. The EDC with the dPEG®36 linker (approximately 1.9 kDa linear dPEG® from Quanta BioDesign, Ltd.) worked both in vitro and in vivo in mice. The in vivo performance of the EDC exceeded the performance of Rituximab. This work also shows that effective delivery of a cytotoxic payload with reduced hydrophobicity does not require a large PEG such as a 5 or 10 kDa (or larger) PEG. Instead, the highly specific therapeutic effect was delivered using a linker that is less than 2.2 kDa in size.
In another example, researchers at the NIH synthesized a series of molecular beacons for video imaging of protease expression in a matrix metalloprotease-overexpressing tumor-bearing mouse model. In order to achieve true, real-time imaging and superior signal-to-noise ratios a probe must have the proper balance between in vivo stability and sensitivity. The researchers conjugated a series of dPEG® products (n = 4, 12, 24, 48) to their probe to study this. While no significant in vitro differences were observed, the dPEG®12 conjugate showed significant in vivo enhancements including the onset of activation, signal-to-noise ratio, and tumor selectivity. This suggests that targeting of specific proteases can be tested and optimized by conjugating low molecular weight dPEG® products to various probes.
Two additional examples can be cited to show the utility of short-chain, single molecular weight PEG products.
For the first example, in 2010 and 2011, researchers at Vanderbilt University in the lab of David E. Cliffel showed that the substitution of short-chain thiol-PEG®4-alcohol or thiol-dPEG®4-acid provided superior, non-immunogenic protection of gold nanoparticles against opsonization in vivo.20,. See also Superior Surface Protection of Gold Nanoparticles With Short-Chain PEG.
In this example, Quanta BioDesign’s product number 10339, m-dPEG®24-acid, was site-specifically conjugated to a derivative of the neuropeptide galanin, which has analgesic effects in the peripheral nervous system. This prevented galanin from crossing the blood-brain barrier, but it concomitantly enhanced the peptide’s analgesic effects in the peripheral nervous system. See also, PEGylated Galanin Shows Enhanced Analgesic Effects in PNS. Please note that product number 10339 has been discontinued and replaced by product number 11289, m-dPEG®25-acid.
A major reason for using PEG in the development of biotherapeutic agents is that it is classified as non-immunogenic. Indeed, it has long been known that PEG shields molecules from the immune system.57,, This is called the “stealth effect” of PEG.
In recent years, some reports in the scientific literature claim to observe immune system responses – including the formation of anti-PEG antibodies – to PEGylated biomolecules. For example, large dispersed PEG products have been shown to be immunogenic when conjugated to highly immunogenic proteins.. These reports have not gone unchallenged, because there remain many unanswered questions about this phenomenon.
Research has shown that the size and type of terminal group of a traditional PEG make a difference in the immune system response to PEG-biomolecule conjugates.,, Additionally, PEG architecture, chain length, and surface coating density may influence the potential immune response.,, Furthermore, recruitment of certain so-called “stealth” proteins to the PEGylated surface appears to suppress immune system responses to PEGylated biomolecules has been demonstrated.,,
Quanta BioDesign’s dPEG® products have been shown to prevent opsonization by the immune system, thus demonstrating the stealth effect of PEG.20 Moreover, an immunogenic response to biomolecules modified with our dPEG® products has not been demonstrated. Possibly, the short, discrete chain length of dPEG® molecules possess suitable architecture and provide sufficient surface coating density to enable dPEG®-modified biomolecules to evade immune system responses.
Although the historical scientific literature demonstrates the utility of high molecular weight PEGs, we do not offer comparable linear dPEG® products (beyond dPEG®48). Negative chemical and physical properties are observed with the larger linear PEGs. For example, large linear, dispersed PEGs have been shown to greatly diminish the potency and efficacy of the biomolecules to which they are conjugated (e.g., antibodies, enzymes).,, Furthermore, in PEGylated lipid nanoparticles (LNPs) a phenomenon exists that is known as the “PEG dilemma.” The PEG dilemma is this: as the amount of surface PEGylation with high molecular weight PEG increases on an LNP, the stability and serum half-life of the LNP increase, but the binding affinity, potency, and efficacy of the conjugated biomolecule decrease. The PEG dilemma’s effects include (but are not limited to) the following:
- Poor cell internalization;
- Poor endosomal escape;
- Reduced targeting efficiency; and,
- Diminished potency and efficacy of conjugated biomolecules.,,
Quanta BioDesign’s dPEG® products sharply limit or even avoid the negative effects that arise from large, linear, traditional PEGs. First, our linear PEG products are much smaller than traditional PEG products. As the case studies above demonstrate, in many cases, short, linear, dPEG® linkers and spacers perform better than traditional, large, linear PEG linkers and spacers. Second, for higher molecular weight dPEG® products, we circumvent the intrinsic, negative properties of large, linear, traditional PEGs by synthesizing branched dPEG® constructs. These dPEG® products use shorter chains (4 to 24 ethylene oxide units per chain) containing 3 to 9 branches that are assembled into a variety of architectures. These unique constructs provide our customers with high molecular weight dPEG® products (>15 kDa) as single molecular weight compounds with discrete chain lengths.
Thus, we recommend dPEG® products over traditional PEG products for three reasons.
- The single molecular weight and discrete chain length of dPEG® products give our customers complete control over the design and assembly of macromolecular architectures. See Figure 1, above.
- The single molecular weight and discrete chain length of dPEG® products (again, see Figure 1) simplifies the analysis of products that incorporate dPEG®
- Our dPEG® products avoid the problems associated with large, linear, traditional PEGs, including loss of binding affinity, loss of potency and efficacy, and the “PEG dilemma” by using short, linear, dPEG® products or by using high-molecular-weight branched dPEG® In many published studies, dPEG® products outperform traditional PEG products.
These reasons are “the dPEG® difference” that cause our products to stand out over traditional, linear PEG products. To see a positive difference in your research or product development, incorporate our dPEG® constructs in your diagnostic, therapeutic, or theranostic products.
Our dPEG® products are amphiphilic, which means that they are soluble in both water and some organic solvents. The amphiphilicity of any given dPEG® product may shift depending on the reactive, protective, or functional groups that sit at the ends of the dPEG® chains. Thus, some dPEG® products may prefer organic solvent over water, while others may prefer water over organic solvent. However, PEG chains hydrogen-bond three (3) water molecules per oxygen atoms in the chain. Thus, a dPEG® product always has some water solubility, though it may be rather small.
“Green” and sustainable chemistry increases in importance to scientists yearly. Numerous reviews address green and sustainable chemistry. Table 1, below, summarizes the solvents that Quanta BioDesign knows and uses for dissolving our dPEG® products.
Table 1: Useful Solvents for dPEG® Products
Most dPEG® products dissolve readily in acetonitrile. The notable exceptions are the amino-dPEG®-acids from dPEG®12 and smaller. Product numbers 10244, 10067, and 10277 are insoluble in acetonitrile. Product number 10287, amino-dPEG®12-acid, is sparingly soluble in acetonitrile. For these products, methanol and water are the preferred solvents.
Acetonitrile dissolves dPEG® products containing active esters (NHS esters and TFP esters). However, acetonitrile readily absorbs water from the atmosphere, and the presence of water in the solvent may hydrolyze the active esters. Quanta BioDesign strongly recommends drying acetonitrile by standing it over 3Å molecular sieves for 24 – 48 hours before use.
Acetonitrile is water-miscible. Reactions with biologics such as proteins, peptides, glycans, and nucleic acids preferentially take place in aqueous buffer. For dPEG® products that hydrolyze or have limited solubility in water, dry acetonitrile can be used to make a stock solution of those dPEG® products. An aliquot of the stock solution is added to the reaction buffer at the time of the reaction.
CAS number: 75-05-8
PubChem CID: 6342
ChemSpider ID: 6102
Acetone dissolves many dPEG® products and is relatively benign for environmental health and safety (EHS) considerations. However, it is incompatible with amines (reacts to form a Schiff base) and oxyamine-containing products (reacts to form an oxime). Acetone is highly hygroscopic and should be dried over molecular sieves before using it.
CAS number: 67-64-1
PubChem CID: 180
ChemSpider ID: 175
Dimethylacetamide and Dimethylformamide
Dimethylacetamide (DMAC) and dimethylformamide (DMF) dissolve most dPEG® products. DMAC and DMF are water-miscible and dry readily upon standing for 24 – 48 hours over 3Å molecular sieves. These two solvents readily absorb water from the atmosphere. Consequently, drying over molecular sieves is necessary before using these solvents with dPEG® products containing active esters.
DMF tends to degrade over time and form free amines that can react, for example, with active esters of dPEG® acids. When using with dPEG® products, DMF should be of the highest purity and “fresh” (out of a newly opened bottle) or recently distilled. DMAC does not degrade over time. Therefore, we at Quanta BioDesign prefer DMAC over DMF.
Unfortunately, DMF and DMAC have relatively high boiling points (153°C for DMF, and 165°C for DMAC at 101.3 kPa/760 Torr), which makes complete removal of the solvent challenging for heat-labile products. Moreover, DMAC and DMF are associated with potential reproductive toxicity problems. Thus, they may not be acceptable solvents for countries subject to the European Union’s “Registration, Evaluation, Authorization, and Restriction of Chemicals” (REACH) Act.
CAS number: 127-19-5
PubChem CID: 31374
ChemSpider ID: 29107
CAS number: 68-12-2
PubChem CID: 6228
ChemSpider ID: 5993
Dimethylsulfoxide (DMSO) is a well-known, widely used green solvent. When pure, DMSO is a clear, colorless, water-miscible, polar aprotic solvent. It dissolves nearly all of Quanta BioDesign’s dPEG® products. DMSO has a high boiling point (189°C at 101.3 kPa/760 Torr) and, thus, is difficult to remove from a reaction mixture. Stock solutions of dPEG® products in DMSO find frequent use in reactions with biological products in aqueous reaction media. When used to dissolve dPEG® compounds containing active esters, DMSO must be dried over 3Å molecular sieves before use and then handled under dry conditions because it is highly hygroscopic.
CAS number: 67-68-5
PubChem CID: 679
ChemSpider ID: 659
Ethanol is a relatively environmentally friendly compound that can be a useful solvent for many dPEG® products. However, ethanol is unsuitable for dissolving products containing NHS or TFP active esters. The active esters exchange with ethanol in solution, resulting in a non-functional product. Ethanol is hygroscopic; therefore, drying ethanol over molecular sieves before use may be necessary.
CAS number: 64-17-5
PubChem CID: 702
ChemSpider ID: 682
Ethyl acetate has limited usefulness with dPEG® products. Nevertheless, for dPEG® products of size dPEG®8 or smaller ethyl acetate may prove useful, especially when the end functional groups are hydrophobic. Ethyl acetate is relatively benign to the environment.
CAS number: 141-78-6
PubChem CID: 8857
ChemSpider ID: 8525
Methanol dissolves most dPEG® products. However, like ethanol, do not use methanol with dPEG® compounds containing NHS or TFP active esters. Moreover, unlike ethanol, methanol has well-known toxicity issues. Methanol is the preferred organic solvent for amino-dPEG®-acids ≤dPEG®12 (see the text under acetonitrile for more information).
CAS number: 67-56-1
PubChem CID: 887
ChemSpider ID: 864
The solvent 2-propanol (also called isopropanol or isopropyl alcohol) has limited use with dPEG® products. It is a somewhat viscous, hygroscopic solvent. Although it does not have a high boiling point compared to DMAC, DMF, or DMSO, it still can be challenging to remove all 2-propanol from a reaction mixture.
CAS number: 67-63-0
PubChem CID: 3776
ChemSpider ID: 3644
Tetrahydrofuran (THF) is another solvent with limited usefulness. If anhydrous conditions are required, THF alone is not able to completely dissolve products that are longer than a dPEG®12 chain. For these longer dPEG® products, up to 30 volume% water may be required with THF to get the product to dissolve fully.
CAS number: 109-99-9
PubChem CID: 8028
ChemSpider ID: 7737
Water is the ultimate green solvent, and water-solubility is one of the characteristics for which dPEG® products are renowned. Nevertheless, two cautions apply when using water as a solvent for dPEG® products. First, users should be aware that water hydrolyzes active esters to carboxylic acids. The hydrolysis occurs at and above pH 7, with the hydrolytic rate increasing with pH. Second, dPEG® products with strongly hydrophobic functional groups on the dPEG® chain have poor-to-no solubility in water. For example, product number 11362, DBCO-dPEG®4-TFP ester, has a hydrophobic functional group on each end of the dPEG® chain. Consequently, it has very poor water solubility; DMAC, DMSO, and acetonitrile are recommended alternatives to water for this product.
CAS number: 7732-18-5
PubChem CID: 962
ChemSpider ID: 937
Are there other useful solvents?
Undoubtedly, more solvents than the ones listed above dissolve our dPEG® products. The solvents listed above are the ones that Quanta BioDesign’s scientists use on a semiregular-to-regular basis. They know these solvents and their limitations, and these are the ones that we recommend to our customers. If you find a solvent outside of this list that you think we should put on the list, please contact us and let us know. We love to hear our customers’ thoughts and suggestions on how better to use our dPEG® products.
Quanta BioDesign has been making dPEG® products for more than 20 years. During this time, we have greatly expanded our capabilities and product lines. We offer a broad range of highly pure dPEG® modifiers, linkers, and spacers ranging in molecular weights from 200 to about 2,300 Daltons for linear compounds and up to 16,000 Daltons for branched compounds. The market has hitherto been limited to smaller monodisperse PEGs (~1200 Da or smaller) or medium-sized conventional disperse PEGs (2,000-3,400 Da up to 20,000 Da or higher). Even these have not been sufficiently exploited, mostly due to a lack of commercial availability stemming from synthetic and purification challenges, the limited functionality of the PEGs, and irreproducible lot-to-lot variability.
Our dPEG® products are prepared via a robust and highly reproducible process and can incorporate a large range of functionalities. Thus, we have the flexibility to produce any dPEG® product (including custom products) on scales ranging from milligrams (mg) to multi-Kilograms (Kg) with purity suitable for research, diagnostic, and therapeutic applications. Our commercial-scale reactors allow us to achieve these larger quantities of our products. While we do not operate a cGMP facility we have cGMP manufacturing partners to support both commercial cGMP and non-cGMP production. We are available for customer audits, with appropriate policies and procedures in place to guarantee reproducible results. From initial product development through market release and beyond, we help our customers produce a safe, reliable, superior product.
For a complete list of our product offerings, please take a look at our online catalog. We also offer custom syntheses to develop molecules tailored to specific needs, so if you do not see what you are looking for please contact us and allow us to leverage our experience in the field to assist you.
 “Dispersity” was formerly known as the “polydispersity index” or “PDI”. In 2009, the International Union of Pure and Applied Chemistry (IUPAC) deprecated the terms polydispersity index and PDI in favor of the term “dispersity” represented by the symbol Ð. See, Gilbert, R. G.; Hess, M.; Jenkins, A. D.; Jones, R. G.; Kratochvíl, P.; Stepto, R. F. T. Dispersity in polymer science (IUPAC Recommendations 2009). Pure Appl. Chem. 2009, 81, 351-353. https://doi.org/10.1351/PAC-REC-08-05-02 See also, Stepto, R. F. T. Erratum. Pure Appl. Chem. 2009, 81, 779. https://doi.org/10.1351/PAC-REC-08-05-02_erratum
 Veronese, F. M.; Mero, A.; Pasut, G. Protein PEGylation, Basic Science and Biological Applications. In PEGylated Protein Drugs: Basic Science and Clinical Applications; Veronese, F. M., Ed.; Milestones in Drug Therapy; Birkhäuser Basel: Basel, 2009; pp 11-31. https://doi.org/10.1007/978-3-7643-8679-5_2.
 Zhao, R. Y.; Wilhelm, S. D.; Audette, C.; Jones, G.; Leece, B. A.; Lazar, A. C.; Goldmacher, V. S.; Singh, R.; Kovtun, Y.; Widdison, W. C.; et al. Synthesis and Evaluation of Hydrophilic Linkers for Antibody-Maytansinoid Conjugates. J. Med. Chem. 2011, 54 (10), 3606-3623. https://doi.org/10.1021/jm2002958.
 Davis, P. D.; Crapps, E. C. (Quanta BioDesign, Ltd.). Selective and Specific Preparation of Discrete PEG Compounds. U.S. Patent 7,888,536, February 15, 2011.
 Quiles, S.; Raisch, K. P.; Sanford, L. L.; Bonner, J. A.; Safavy, A. Synthesis and Preliminary Biological Evaluation of High-Drug-Load Paclitaxel-Antibody Conjugates for Tumor-Targeted Chemotherapy. J. Med. Chem. 2010, 53(2), 586-594. https://doi.org/10.1021/jm900899g.
 Tiberghien, A. C.; Levy, J-N.; Masterson, L. A.; Patel, N. V.; Adams, L. R.; Corbett, S.; Williams, D. G.; Hartley, J. A.; and Howard, P. W. Design and synthesis of Tesirine, a clinical antibody-drug conjugate pyrrolobenzodiazepine dimer payload. ACS Med. Chem. Lett. 2016, 7(11), 983-987. https://doi.org/10.1021/acsmedchemlett.6b00062
 Burke, P. J.; Hamilton, J. Z.; Jeffrey, S. C.; Hunter, J. H.; Doronina, S. O.; Okeley, N. M.; Miyamoto, J. B.; Anderson, M. E.; Stone, I. J.; Ulrich, M. L.; Simmons, J. K.; McKinney, E. E.; Senter, P. D.; and Lyon, R. P. Optimization of a PEGylated glucuronide-monomethylauristatin E linker for antibody-drug conjugates. Mol. Cancer Ther. 2017, 16(1), 116-123. DOI: 10.1158/1535-7163.MCT-16-0343
 Zhang, L.; Klein, B. D.; Metcalf, C. S.; Smith, M. D.; McDougle, D. R.; Lee, H.-K.; White, H. S.; Bulaj, G. Incorporation of Monodisperse Oligoethyleneglycol Amino Acids into Anticonvulsant Analogues of Galanin and Neuropeptide Y Provides Peripherally Acting Analgesics. Mol. Pharmaceutics 2013, 10(2), 574-585. https://doi.org/10.1021/mp300236v.
 Metcalf, C. S.; Klein, B. D.; McDougle, D. R.; Zhang, L.; Smith, M. D.; Bulaj, G.; White, H. S. Analgesic Properties of a Peripherally Acting and GalR2 Receptor-Preferring Galanin Analog in Inflammatory, Neuropathic, and Acute Pain Models. J Pharmacol Exp Ther 2015, 352(1), 185-193. https://doi.org/10.1124/jpet.114.219063.
 Kaminskas, L. M.; Boyd, B. J.; Karellas, P.; Krippner, G. Y.; Lessene, R.; Kelly, B.; Porter, C. J. H. The impact of molecular weight and PEG chain length on the systematic pharmacokinetics of PEGylated poly L-lysine dendrimers. Mol. Pharmaceutics 2008, 5(3), 449-463. https://doi.org/10.1021/mp7001208
 Novikov, B. N.; Grimsley, J. K.; Kern, R. J.; Wild, J. R.; Wales, M. E. Improved pharmacokinetics and immunogenicity profile of organophosphorus hydrolase by chemical modification with polyethylene glycol. J. Contr. Rel. 2010, 146(3), 318-325. https://doi.org/10.1016/j.jconrel.2010.06.003
 Quiles, S.; Raisch, K. P.; Sanford, L. L.; Bonner, J. A.; Safavy, A. Synthesis and preliminary biological evaluation of high-drug-load paclitaxel-antibody conjugates for tumor-targeted chemotherapy. J. Med. Chem. 2010, 53(2), 586-594. https://doi.org/10.1021/jm900899g
 Halperin, A.; Shadkchan, Y.; Pisarevsky, E.; Szpilman, A. M.; Sandovsky, H.; Osherov, N.; Benhar, I. Novel water-soluble Amphotericin B-PEG conjugates with low toxicity and potent in vivo efficacy. J. Med. Chem. 2016, 59(3), 1197-1206. https://doi.org/10.1021/acs.jmedchem.5b01862
 Arosio, D.; Manzoni, L.; Araldi, E. M. V.; Scolastico, C. Cyclic RGD functionalized gold nanoparticles for tumor targeting. Bioconjugate Chem. 2011, 22(4), 664-672. https://doi.org/10.1021/bc100448r
 Simpson, C. A.; Agrawal, A. C.; Balinski, A.; Harkness, K. M.; Cliffel, D. E. Short-chain PEG mixed monolayer protected gold clusters increase clearance and red blood cell counts. ACS Nano, 2011, 5(5), 3577-3584. https://doi.org/10.1021/nn103148x
 Oh, E. , Fatemi, F. K., Currie, M. , Delehanty, J. B., Pons, T. , Fragola, A. , Lévêque‐Fort, S. , Goswami, R. , Susumu, K. , Huston, A. L. and Medintz, I. L. (2013), PEGylated Luminescent Gold Nanoclusters: Synthesis, Characterization, Bioconjugation, and Application to One‐ and Two‐Photon Cellular Imaging. Part. Part. Syst. Charact., 2013, 30(5), 453-466. DOI: 10.1002/ppsc.201200140
 Smith, C. A.; Simpson, C. A.; Kim, G.; Carter, C. J.; Feldheim, D. L. Gastrointestinal bioavailability of 2.0 nm diameter gold nanoparticles. ACS Nano. 2013, 7(5), 3991-3996. https://doi.org/10.1021/nn305930e
 Sultan, D.; Ye, D.; Heo, G. S.; Zhang, X.; Luehmann, H.; Yue, Y.; Detering, L.; Komarov, S.; Taylor, S.; Tai, Y-C.; Rubin, J. B.; Chen, H.; Yongjian, L. Focused ultrasound enabled trans-blood brain barrier delivery of gold nanoclusters: Effect of surface charges and quantification using positron emission tomography. Small 2018, 1703115, 1-9. https://doi.org/10.1002/smll.201703115.
 Smolensky, E. D.; Park, H-Y. E.; Berquo, T. S.; Pierre, V. C. Surface functionalization of magnetic iron oxide nanoparticles for MRI applications: effect of anchoring group and ligand exchange protocol. Contrast Media Mol. Imaging Journal. 2010, 6(4), 189-199. August 2010. DOI:10.1002/cmmi.417.
 Xu, C.; Zhenglong, Y.; Kohler, N.; Kim, J.; Chung, M. A.; Sun, S. FePt nanoparticles as an Fe reservoir for controlled Fe release and tumor inhibition. J. Am. Chem. Soc. 2009, 13(42), 15346-15351. https://doi.org/10.1021/ja905938a
 Li, S. P.-Y.; Liu, H.-W.; Zhang, K. Y.; Lo, K. K.-W. Modification of Luminescent Iridium(III) Polypyridine Complexes with Discrete Poly(Ethylene Glycol) (PEG) Pendants: Synthesis, Emissive Behavior, Intracellular Uptake, and PEGylation Properties. Chemistry – A European Journal 2010, 16 (28), 8329-8339. https://doi.org/10.1002/chem.201000474.
 Li, S. P.; Tsai, J. L.; Lo, K. K. Luminescent Iridium(III) Polypyridine PEG Complexes: Synthesis, Photophysical, and Biological Properties. In 2010 IEEE International Conference on Nano/Molecular Medicine and Engineering; 2010; pp 66-71. https://doi.org/10.1109/NANOMED.2010.5749807.
 Lo, K. K.-W.; Li, S. P.-Y.; Zhang, K. Y. Development of Luminescent Iridium(III) Polypyridine Complexes as Chemical and Biological Probes. New J. Chem. 2011, 35 (2), 265-287. https://doi.org/10.1039/C0NJ00478B.
 Lo, K. K.-W.; Zhang, K. Y.; Li, S. P.-Y. Design of Cyclometalated Iridium(III) Polypyridine Complexes as Luminescent Biological Labels and Probes. Pure and Applied Chemistry 2011, 83(4), 823-840. https://doi.org/10.1351/PAC-CON-10-08-20.
 Lo, K. K.-W. Luminescent Rhenium(I) and Iridium(III) Polypyridine Complexes as Biological Probes, Imaging Reagents, and Photocytotoxic Agents. Acc. Chem. Res. 2015, 48(12), 2985-2995. https://doi.org/10.1021/acs.accounts.5b00211.
 Kameta, N.; Matsuzawa, T.; Yaoi, K.; Masuda, M. Short polyethylene glycol chains densely bound to soft nanotube channels for inhibition of protein aggregation. RSC Advances, 2016, 6(43), 36744-36750. https://doi.org/10.1039/C6RA06793J
 Kameta, N.; Ding, W.; Dong, J. Soft nanotubes derivatized with short PEG chains for thermally controllable extraction and separation of peptides. ACS Omega. 2017, 2(9), 6143-6150. https://doi.org/10.1021/acsomega.7b00838
 Kohli, N.; Vaidya, S.; Ofoli, R. Y.; Worden, R. M.; Lee, I. Arrays of Lipid Bilayers and Liposomes on Patterned Polyelectrolyte Templates. Journal of Colloid and Interface Science 2006, 301 (2), 461-469. https://doi.org/10.1016/j.jcis.2006.05.048.
 Kidambi, S.; Chan, C.; Lee, I. Tunable Resistive M-DPEG Acid Patterns on Polyelectrolyte Multilayers at Physiological Conditions: Template for Directed Deposition of Biomacromolecules. Langmuir 2008, 24 (1), 224-230. https://doi.org/10.1021/la702925r.
 Anderson, A. S.; Dattelbaum, A. M.; Montaño, G. A.; Price, D. N.; Schmidt, J. G.; Martinez, J. S.; Grace, W. K.; Grace, K. M.; Swanson, B. I. Functional PEG-Modified Thin Films for Biological Detection. Langmuir 2008, 24 (5), 2240-2247. https://doi.org/10.1021/la7033438.
 Anderson, A. S.; Dattelbaum, A. M.; Mukundan, H.; Price, D. N.; Grace, W. K.; Swanson, B. I. Robust Sensing Films for Pathogen Detection and Medical Diagnostics. In Frontiers in Pathogen Detection: From Nanosensors to Systems; International Society for Optics and Photonics, 2009; Vol. 7167, p 71670Q. https://doi.org/10.1117/12.809383.
 Mehrotra, S.; Lee, I.; Liu, C.; Chan, C. Polyelectrolyte Multilayer Stamping in Aqueous Phase and Non-Contact Mode. Ind. Eng. Chem. Res. 2011, 50 (15), 8851-8858. https://doi.org/10.1021/ie102011m.
 Wolfenden, M. L.; Sakamuri, R. M.; Anderson, A. S.; Prasad, L.; Schmidt, J. G.; Mukundan, H. Determination of Bacterial Viability by Selective Capture Using Surface-Bound Siderophores. 2012, 2012. https://doi.org/10.4236/abc.2012.24049.
 Zhao, Y.; Duan, S.; Zeng, X.; Liu, C.; Davies, N. M.; Li, B.; Forrest, M. L. Prodrug Strategy for PSMA-Targeted Delivery of TGX-221 to Prostate Cancer Cells. Mol. Pharmaceutics 2012, 9(6), 1705-1716. https://doi.org/10.1021/mp3000309.
 Kale, A. A.; Torchilin, V. P. Environment-Responsive Multifunctional Liposomes. In Liposomes: Methods and Protocols, Volume 1: Pharmaceutical Nanocarriers; Weissig, V., Ed.; Methods in Molecular Biology; Humana Press: Totowa, NJ, 2010; pp 213-242. https://doi.org/10.1007/978-1-60327-360-2_15.
 Stefanick, J. F.; Ashley, J. D.; Kiziltepe, T.; Bilgicer, B. A Systematic Analysis of Peptide Linker Length and Liposomal Polyethylene Glycol Coating on Cellular Uptake of Peptide-Targeted Liposomes. ACS Nano 2013, 7 (4), 2935-2947. https://doi.org/10.1021/nn305663e.
 Csizmar, C. M.; Petersburg, J. R.; Hendricks, A.; Stern, L. A.; Hackel, B. J.; Wagner, C. R. Engineering Reversible Cell-Cell Interactions with Lipid Anchored Prosthetic Receptors. Bioconjugate Chem. 2018, 29 (4), 1291-1301. https://doi.org/10.1021/acs.bioconjchem.8b00058.
 Kaminskas, L. M.; Boyd, B. J.; Karellas, P.; Krippner, G. Y.; Lessene, R.; Kelly, B.; Porter, C. J. H. The Impact of Molecular Weight and PEG Chain Length on the Systemic Pharmacokinetics of PEGylated Poly L-Lysine Dendrimers. Mol. Pharmaceutics 2008, 5 (3), 449-463. https://doi.org/10.1021/mp7001208.
 Kaminskas, L. M.; McLeod, V. M.; Ascher, D. B.; Ryan, G. M.; Jones, S.; Haynes, J. M.; Trevaskis, N. L.; Chan, L. J.; Sloan, E. K.; Finnin, B. A.; et al. Methotrexate-Conjugated PEGylated Dendrimers Show Differential Patterns of Deposition and Activity in Tumor-Burdened Lymph Nodes after Intravenous and Subcutaneous Administration in Rats. Mol. Pharmaceutics 2015, 12(2), 432-443. https://doi.org/10.1021/mp500531e.
 Mehta, D.; Leong, N.; McLeod, V. M.; Kelly, B. D.; Pathak, R.; Owen, D. J.; Porter, C. J. H.; Kaminskas, L. M. Reducing Dendrimer Generation and PEG Chain Length Increases Drug Release and Promotes Anticancer Activity of PEGylated Polylysine Dendrimers Conjugated with Doxorubicin via a Cathepsin-Cleavable Peptide Linker. Mol. Pharmaceutics 2018, 15(10), 4568-4576. https://doi.org/10.1021/acs.molpharmaceut.8b00581.
 Vrolijk, M. Silica Nanoparticles for the Delivery of DNA and RNAi in Cancer Treatment. Graduate College Dissertations and Theses 2017.
 Ma, K.; Wiesner, U. Modular and Orthogonal Post-PEGylation Surface Modifications by Insertion Enabling Penta-Functional Ultrasmall Organic-Silica Hybrid Nanoparticles. Chem. Mater. 2017, 29 (16), 6840-6855. https://doi.org/10.1021/acs.chemmater.7b02009.
 Ziaei, P.; Geruntho, J. J.; Marin-Flores, O. G.; Berkman, C. E.; Grant Norton, M. Silica Nanostructured Platform for Affinity Capture of Tumor-Derived Exosomes. J Mater Sci 2017, 52 (12), 6907-6916. https://doi.org/10.1007/s10853-017-0905-0.
 Kao, T.; Kohle, F.; Ma, K.; Aubert, T.; Andrievsky, A.; Wiesner, U. Fluorescent Silica Nanoparticles with Well-Separated Intensity Distributions from Batch Reactions. Nano Lett. 2018, 18 (2), 1305-1310. https://doi.org/10.1021/acs.nanolett.7b04978.
 Ding, H.; Carlton, M. M.; Povoski, S. P.; Milum, K.; Kumar, K.; Kothandaraman, S.; Hinkle, G. H.; Colcher, D.; Brody, R.; Davis, P. D.; et al. Site Specific Discrete PEGylation of 124I-Labeled MCC49 Fab′ Fragments Improves Tumor MicroPET/CT Imaging in Mice. Bioconjugate Chem. 2013, 24(11), 1945-1954. https://doi.org/10.1021/bc400375f.
 Popov, J.; Gilabert-Oriol, R.; Bally, M. B. Unique Therapeutic Properties and Preparation Methodology of Multivalent Rituximab-Lipid Nanoparticles. European Journal of Pharmaceutics and Biopharmaceutics 2017, 117, 256-269. https://doi.org/10.1016/j.ejpb.2017.04.024.
 Sano, K.; Nakajima, T.; Ali, T.; Bartlett, D. W.; Wu, A. M.; Kim, I.; Paik, C. H.; Choyke, P.; Kobayashi, H. Activatable Fluorescent Cys-Diabody Conjugated with Indocyanine Green Derivative: Consideration of Fluorescent Catabolite Kinetics on Molecular Imaging. JBO 2013, 18 (10), 101304. https://doi.org/10.1117/1.JBO.18.10.101304.
 Sano, K.; Nakajima, T.; Miyazaki, K.; Ohuchi, Y.; Ikegami, T.; Choyke, P. L.; Kobayashi, H. Short PEG-Linkers Improve the Performance of Targeted, Activatable Monoclonal Antibody-Indocyanine Green Optical Imaging Probes. Bioconjugate Chem. 2013, 24 (5), 811-816. https://doi.org/10.1021/bc400050k.
 Zheng, Y.; Ji, S.; Czerwinski, A.; Valenzuela, F.; Pennington, M.; Liu, S. FITC-Conjugated Cyclic RGD Peptides as Fluorescent Probes for Staining Integrin Αvβ3/Αvβ5 in Tumor Tissues. Bioconjugate Chem. 2014, 25 (11), 1925-1941. https://doi.org/10.1021/bc500452y.
 Greg T. Hermanson, PEGylation and Synthetic Polymer Modification. In Bioconjugate Techniques, 3rd Edition. Academic Press, imprint of Elsevier: Waltham, MA 02451 (USA), 2013; pp 787-838.
 Dijkgraaf, I.; Liu, S.; Kruijtzer, J. A. W.; Soede, A. C.; Oyen, W. J. G.; Liskamp, R. M. J.; Corstens, F. H. M.; Boerman, O. C. Effects of Linker Variation on the in Vitro and in Vivo Characteristics of an 111In-Labeled RGD Peptide. Nuclear Medicine and Biology 2007, 34(1), 29-35. https://doi.org/10.1016/j.nucmedbio.2006.10.006.
 Shi, J.; Kim, Y.-S.; Zhai, S.; Liu, Z.; Chen, X.; Liu, S. Improving Tumor Uptake and Pharmacokinetics of 64Cu-Labeled Cyclic RGD Peptide Dimers with Gly3 and PEG4 Linkers. Bioconjugate Chem. 2009, 20(4), 750-759. https://doi.org/10.1021/bc800455p.
 Li, L.; Turatti, F.; Crow, D.; Bading, J. R.; Anderson, A.-L.; Poku, E.; Yazaki, P. J.; Williams, L. E.; Tamvakis, D.; Sanders, P.; et al. Monodispersed DOTA-PEG-Conjugated Anti-TAG-72 Diabody Has Low Kidney Uptake and High Tumor-to-Blood Ratios Resulting in Improved 64Cu PET. Journal of Nuclear Medicine 2010, 51(7), 1139-1146. https://doi.org/10.2967/jnumed.109.074153.
 Li, L.; Crow, D.; Turatti, F.; Bading, J. R.; Anderson, A.-L.; Poku, E.; Yazaki, P. J.; Carmichael, J.; Leong, D.; Wheatcroft, M. P.; et al. Site-Specific Conjugation of Monodispersed DOTA-PEGn to a Thiolated Diabody Reveals the Effect of Increasing PEG Size on Kidney Clearance and Tumor Uptake with Improved 64-Copper PET Imaging. Bioconjugate Chem. 2011, 22(4), 709-716. https://doi.org/10.1021/bc100464e.
 Jamous, M.; Tamma, M. L.; Gourni, E.; Waser, B.; Reubi, J. C.; Maecke, H. R.; Mansi, R. PEG Spacers of Different Length Influence the Biological Profile of Bombesin-Based Radiolabeled Antagonists. Nuclear Medicine and Biology 2014, 41(6), 464-470. https://doi.org/10.1016/j.nucmedbio.2014.03.014.
 Valverde, I. E.; Vomstein, S.; Mindt, T. L. Toward the Optimization of Bombesin-Based Radiotracers for Tumor Targeting. J. Med. Chem. 2016, 59(8), 3867-3877. https://doi.org/10.1021/acs.jmedchem.6b00025.
 Maschauer, S.; Einsiedel, J.; Hübner, H.; Gmeiner, P.; Prante, O. 18F- and 68Ga-Labeled Neurotensin Peptides for PET Imaging of Neurotensin Receptor 1. J. Med. Chem. 2016, 59(13), 6480-6492. https://doi.org/10.1021/acs.jmedchem.6b00675.
 Fee, C.; Damodaran, V. B. Protein PEGylation: An overview of chemistry and process considerations. Eur. Pharm. Rev. 2010, 15(1), 18-26.
 Povoski, S. P.; Davis, P. D.; Colcher, D.; Martin, Jr., E. W. Single molecular weight discrete PEG compounds: emerging roles in molecular diagnostics, imaging, and therapeutics. Expert Rev. Mol. Diagn. 2013, 13, 315-319. https://doi.org/10.1586/erm.13.19
 French, A. C.; Thompson, A. L.; Davis, B. G. High-Purity Discrete PEG-Oligomer Crystals Allow Structural Insight, Angew. Chem. Int. Ed. 2009, 48, 1248-1252. See footnote 19 in the paper and Figure S1 in the supplemental information. https://doi.org/10.1002/anie.200804623.
 Turecek, P. L.; Bossard, M. J.; Schoetens, F.; Ivens, I. A. PEGylation of Biopharmaceuticals: A Review of Chemistry and Nonclinical Safety Information of Approved Drugs. J Pharm Sci 2016, 105, 460-475. https://doi.org/10.1016/j.xphs.2015.11.015
 Fliervoet, L. A. L.; Najafi, M.; Hembury, M.; Vermonden, T. Heterofunctional Poly(ethylene glycol) (PEG) Macroinitiator Enabling Controlled Synthesis of ABC Triblock Copolymers. Macromolecules 2017, 50, 8390-8397. https://doi.org/10.1021/acs.macromol.7b01475
 Xuan, S.; Gupta, S.; Li, X.; Bleuel, M.; Schneider, G. J.; Zhang, D. Synthesis and characterization of well-defined PEGylated polypeptoids as protein-resistant polymers. BioMacromolecules 2017, 18, 951-964. https://doi.org/10.1021/acs.biomac.6b01824
 Viegas, T. X.; Veronese, F. M. Regulatory Strategy and Approval Processes Considered for PEG-Drug Conjugates and Other Nanomedicines. In PEGylated Protein Drugs: Basic Science and Clinical Applications; Veronese, F. M., Ed.; Milestones in Drug Therapy; Birkhäuser Basel: Basel, 2009; pp 273-281. https://doi.org/10.1007/978-3-7643-8679-5_16.
 See also, Table 1 and references therein at reference 66, above.
 Ibid., page 274.
 Ibid., pages 274-275.
 Mero, A.; Spolaore, B.; Veronese, F. M.; Fontana, A. Transglutaminase-Mediated PEGylation of Proteins: Direct Identification of the Sites of Protein Modification by Mass Spectrometry Using a Novel Monodisperse PEG. Bioconjugate Chem. 2009, 20 (2), 384-389. https://doi.org/10.1021/bc800427n.
 Davis, P. D.; Crapps, E. C. (Quanta BioDesign, Ltd.). Selective and Specific Preparation of Discrete PEG Compounds. U.S. Patent 8,637,711, January 28, 2014.
 Gull, I.; Samra, Z. Q.; Aslam, M. S.; Athar, M. A. Heterologous Expression, Immunochemical and Computational Analysis of Recombinant Human Interferon Alpha 2b. Springerplus 2013, 2. https://doi.org/10.1186/2193-1801-2-264.
 Hepatitis C http://www.who.int/news-room/fact-sheets/detail/hepatitis-c (accessed Nov 20, 2018).
 Pasut, G. PEGylated α Interferons: Two Different Strategies to Achieve Increased Efficacy. In PEGylated Protein Drugs: Basic Science and Clinical Applications; Veronese, F. M., Ed.; Milestones in Drug Therapy; Birkhäuser Basel: Basel, 2009; pp 205-216. https://doi.org/10.1007/978-3-7643-8679-5_12. See specifically pages 207-211.
 Ibid., Table 1, page 212.
 Bailon, P.; Palleroni, A.; Schaffer, C. A.; Spence, C. L.; Fung, W.-J.; Porter, J. E.; Ehrlich, G. K.; Pan, W.; Xu, Z.-X.; Modi, M. W.; et al. Rational Design of a Potent, Long-Lasting Form of Interferon: A 40 KDa Branched Polyethylene Glycol-Conjugated Interferon α-2a for the Treatment of Hepatitis C. Bioconjugate Chem. 2001, 12(2), 195-202. https://doi.org/10.1021/bc000082g.
 See references 79 and 80, above.
 Finn, R. F. PEGylation of Human Growth Hormone: Strategies and Properties. In PEGylated Protein Drugs: Basic Science and Clinical Applications; Veronese, F. M., Ed.; Milestones in Drug Therapy; Birkhäuser Basel: Basel, 2009; pp 187-203. https://doi.org/10.1007/978-3-7643-8679-5_11.
 Novikov, B. N.; Grimsley, J. K.; Kern, R. J.; Wild, J. R.; Wales, M. E. Improved Pharmacokinetics and Immunogenicity Profile of Organophosphorus Hydrolase by Chemical Modification with Polyethylene Glycol. Journal of Controlled Release 2010, 146 (3), 318-325. https://doi.org/10.1016/j.jconrel.2010.06.003.
 Halperin, A.; Shadkchan, Y.; Pisarevsky, E.; Szpilman, A. M.; Sandovsky, H.; Osherov, N.; Benhar, I. Novel Water-Soluble Amphotericin B-PEG Conjugates with Low Toxicity and Potent in Vivo Efficacy. J. Med. Chem. 2016, 59(3), 1197-1206. https://doi.org/10.1021/acs.jmedchem.5b01862.
 Conover, C. D.; Zhao, H.; Longley, C. B.; Shum, K. L.; Greenwald, R. B. Utility of Poly(Ethylene Glycol) Conjugation To Create Prodrugs of Amphotericin B. Bioconjugate Chem. 2003, 14(3), 661-666. https://doi.org/10.1021/bc0256594.
 Sedlák, M.; Buchta, V.; Kubicová, L.; Šimůnek, P.; Holčapek, M.; Kašparová, P. Synthesis and Characterisation of a New Amphotericin B-Methoxypoly(Ethylene Glycol) Conjugate. Bioorganic & Medicinal Chemistry Letters 2001, 11(21), 2833-2835. https://doi.org/10.1016/S0960-894X(01)00532-7.
 Sedlák, M.; Pravda, M.; Staud, F.; Kubicová, L.; Týčová, K.; Ventura, K. Synthesis of PH-Sensitive Amphotericin B-Poly(Ethylene Glycol) Conjugates and Study of Their Controlled Release in Vitro. Bioorganic & Medicinal Chemistry 2007, 15(12), 4069-4076. https://doi.org/10.1016/j.bmc.2007.03.083.
 Tan, T. R. M.; Hoi, K. M.; Zhang, P.; Ng, S. K. Characterization of a Polyethylene Glycol-Amphotericin B Conjugate Loaded with Free AMB for Improved Antifungal Efficacy. PLOS ONE 2016, 11(3), e0152112. https://doi.org/10.1371/journal.pone.0152112.
 Marshall, D. J.; Harried, S. S.; Murphy, J. L.; Hall, C. A.; Shekhani, M. S.; Pain, C.; Lyons, C. A.; Chillemi, A.; Malavasi, F.; Pearce, H. L.; et al. Extracellular Antibody Drug Conjugates Exploiting the Proximity of Two Proteins. Molecular Therapy 2016, 24(10), 1760-1770. https://doi.org/10.1038/mt.2016.119.
 Zhu, L.; Xie, J.; Swierczewska, M.; Zhang, F.; Quan, Q.; Ma, Y.; Fang, X.; Kim, K.; Lee, S.; Chen, X. Real-Time Video Imaging of Protease Expression In Vivo. Theranostics 2011, 1, 18-27. https://doi.org/10.7150/thno/v01p0018.
 Simpson, C. A.; Huffman, B. J.; Gerdon, A. E.; Cliffel, D. E. Unexpected Toxicity of Monolayer Protected Gold Clusters Eliminated by PEG-Thiol Place Exchange Reactions. Chem. Res. Toxicol. 2010, 23(10), 1608-1616. https://doi.org/10.1021/tx100209t.
 Zhang, L.; Klein, B. D.; Metcalf, C. S.; Smith, M. D.; McDougle, D. R.; Lee, H.-K.; White, H. S.; Bulaj, G. Incorporation of Monodisperse Oligoethyleneglycol Amino Acids into Anticonvulsant Analogues of Galanin and Neuropeptide Y Provides Peripherally Acting Analgesics. Mol. Pharmaceutics 2013, 10(2), 574-585. https://doi.org/10.1021/mp300236v.
 Suk, J. S.; Xu, Q.; Kim, N.; Hanes, J.; Ensign, L. M. PEGylation as a Strategy for Improving Nanoparticle-Based Drug and Gene Delivery. Advanced Drug Delivery Reviews 2016, 99, 28-51. https://doi.org/10.1016/j.addr.2015.09.012.
 Owens, D. E.; Peppas, N. A. Opsonization, Biodistribution, and Pharmacokinetics of Polymeric Nanoparticles. International Journal of Pharmaceutics 2006, 307(1), 93-102. https://doi.org/10.1016/j.ijpharm.2005.10.010.
 Cui, J.; Björnmalm, M.; Ju, Y.; Caruso, F. Nanoengineering of Poly(Ethylene Glycol) Particles for Stealth and Targeting. Langmuir 2018, 34(37), 10817-10827. https://doi.org/10.1021/acs.langmuir.8b02117.
 Armstrong, J. K. The Occurrence, Induction, Specificity and Potential Effect of Antibodies against Poly(Ethylene Glycol). In PEGylated Protein Drugs: Basic Science and Clinical Applications; Veronese, F. M., Ed.; Milestones in Drug Therapy; Birkhäuser Basel: Basel, 2009; pp 147-168. https://doi.org/10.1007/978-3-7643-8679-5_9.
 Schellekens, H.; Hennink, W. E.; Brinks, V. The Immunogenicity of Polyethylene Glycol: Facts and Fiction. Pharmaceutical Research 2013, 30(7), 1729-1734. https://doi.org/10.1007/s11095-013-1067-7.
 Yang, Q.; Lai, S. K. Anti-PEG Immunity: Emergence, Characteristics, and Unaddressed Questions. Wiley Interdisciplinary Reviews: Nanomedicine and Nanobiotechnology 2015, 7(5), 655-677. https://doi.org/10.1002/wnan.1339.
 “The anti-PEG immune response depended on the immunogenicity of proteins, the extent of PEGylation, and the Mw of mPEG. In contrast, branching of mPEG had an insignificant effect on the anti-PEG immune response to the PEGylated proteins.” From the abstract of Wan, X.; Zhang, J.; Yu, W.; Shen, L.; Ji, S.; Hu, T. Effect of Protein Immunogenicity and PEG Size and Branching on the Anti-PEG Immune Response to PEGylated Proteins. Process Biochemistry 2017, 52, 183-191. https://doi.org/10.1016/j.procbio.2016.09.029.
 Wang, C.; Cheng, X.; Sui, Y.; Luo, X.; Jiang, G.; Wang, Y.; Huang, Z.; She, Z.; Deng, Y. A Noticeable Phenomenon: Thiol Terminal PEG Enhances the Immunogenicity of PEGylated Emulsions Injected Intravenously or Subcutaneously into Rats. European Journal of Pharmaceutics and Biopharmaceutics 2013, 85(3, Part A), 744-751. https://doi.org/10.1016/j.ejpb.2013.10.002.
 Shimizu, T.; Abu Lila, A. S.; Fujita, R.; Awata, M.; Kawanishi, M.; Hashimoto, Y.; Okuhira, K.; Ishima, Y.; Ishida, T. A Hydroxyl PEG Version of PEGylated Liposomes and Its Impact on Anti-PEG IgM Induction and on the Accelerated Clearance of PEGylated Liposomes. European Journal of Pharmaceutics and Biopharmaceutics 2018, 127, 142-149. https://doi.org/10.1016/j.ejpb.2018.02.019.
 Ozer, I.; Tomak, A.; Zareie, H. M.; Baran, Y.; Bulmus, V. Effect of Molecular Architecture on Cell Interactions and Stealth Properties of PEG. Biomacromolecules 2017, 18(9), 2699-2710. https://doi.org/10.1021/acs.biomac.7b00443.
 Pozzi, D.; Colapicchioni, V.; Caracciolo, G.; Piovesana, S.; Capriotti, A. L.; Palchetti, S.; Grossi, S. D.; Riccioli, A.; Amenitsch, H.; Laganà, A. Effect of Polyethyleneglycol (PEG) Chain Length on the Bio-Nano-Interactions between PEGylated Lipid Nanoparticles and Biological Fluids: From Nanostructure to Uptake in Cancer Cells. Nanoscale 2014, 6(5), 2782-2792. https://doi.org/10.1039/C3NR05559K.
 Li, S.-D.; Huang, L. Stealth Nanoparticles: High Density but Sheddable PEG Is a Key for Tumor Targeting. Journal of Controlled Release 2010, 145(3), 178-181. https://doi.org/10.1016/j.jconrel.2010.03.016.
 Papi, M.; Caputo, D.; Palmieri, V.; Coppola, R.; Palchetti, S.; Bugli, F.; Martini, C.; Digiacomo, L.; Pozzi, D.; Caracciolo, G. Clinically Approved PEGylated Nanoparticles Are Covered by a Protein Corona That Boosts the Uptake by Cancer Cells. Nanoscale 2017, 9(29), 10327-10334. https://doi.org/10.1039/C7NR03042H.
 Schöttler, S.; Becker, G.; Winzen, S.; Steinbach, T.; Mohr, K.; Landfester, K.; Mailänder, V.; Wurm, F. R. Protein Adsorption Is Required for Stealth Effect of Poly(Ethylene Glycol)- and Poly(Phosphoester)-Coated Nanocarriers. Nature Nanotechnology 2016, 11(4), 372-377. https://doi.org/10.1038/nnano.2015.330.
 da Silva Freitas, D.; Abrahão-Neto, J. Biochemical and Biophysical Characterization of Lysozyme Modified by PEGylation. International Journal of Pharmaceutics 2010, 392(1), 111-117. https://doi.org/10.1016/j.ijpharm.2010.03.036.
 Hsieh, Y.-P.; Lin, S.-C. Effect of PEGylation on the Activity and Stability of Horseradish Peroxidase and L-N-Carbamoylase in Aqueous Phases. Process Biochemistry 2015, 50(9), 1372-1378. https://doi.org/10.1016/j.procbio.2015.04.024.
 Hatakeyama, H.; Akita, H.; Harashima, H. A Multifunctional Envelope Type Nano Device (MEND) for Gene Delivery to Tumours Based on the EPR Effect: A Strategy for Overcoming the PEG Dilemma. Advanced Drug Delivery Reviews 2011, 63(3), 152-160. https://doi.org/10.1016/j.addr.2010.09.001.
 Hatakeyama, H.; Akita, H.; Harashima, H. The Polyethyleneglycol Dilemma: Advantage and Disadvantage of PEGylation of Liposomes for Systemic Genes and Nucleic Acids Delivery to Tumors. Biological and Pharmaceutical Bulletin 2013, 36(6), 892-899. https://doi.org/10.1248/bpb.b13-00059.
 Kurimoto, S.; Yoshinaga, N.; Igarashi, K.; Matsumoto, Y.; Cabral, H.; Uchida, S. PEG-OligoRNA Hybridization of mRNA for Developing Sterically Stable Lipid Nanoparticles toward In Vivo Administration. Molecules 2019, 24(7), 1303. https://doi.org/10.3390/molecules24071303.
 (a) Anastas, P. T.; Warner, J. Charles. Green Chemistry : Theory and Practice; Oxford University Press: Oxford [England]; New York, 1998. (b) Capello, C.; Fischer, U.; Hungerbühler, K. What Is a Green Solvent? A Comprehensive Framework for the Environmental Assessment of Solvents. Green Chem. 2007, 9(9), 927-934. https://doi.org/10.1039/B617536H. (c) Anastas, P.; Eghbali, N. Green Chemistry: Principles and Practice. Chem. Soc. Rev. 2009, 39(1), 301-312. https://doi.org/10.1039/B918763B. (d) Tobiszewski, M.; Mechlińska, A.; Namieśnik, J. Green Analytical Chemistry – Theory and Practice. Chem. Soc. Rev. 2010, 39(8), 2869-2878. https://doi.org/10.1039/B926439F. (e) Clarke, C. J.; Tu, W.-C.; Levers, O.; Bröhl, A.; Hallett, J. P. Green and Sustainable Solvents in Chemical Processes. Chem. Rev. 2018, 118(2), 747-800. https://doi.org/10.1021/acs.chemrev.7b00571. (f) Houlton, Sarah. Enhancing solvents’ sustainability. Chemistry World website. https://www.chemistryworld.com/news/enhancing-solvents-sustainability/3010810.article (accessed Mar 11, 2020).
 Byrne, F. P.; Jin, S.; Paggiola, G.; Petchey, T. H. M.; Clark, J. H.; Farmer, T. J.; Hunt, A. J.; Robert McElroy, C.; Sherwood, J. Tools and Techniques for Solvent Selection: Green Solvent Selection Guides. Sustain Chem Process 2016, 4(1), 7. https://doi.org/10.1186/s40508-016-0051-z.