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Figure 1: Structure of the DOPE-PEG12-LPE/LPK lipidated oligopeptides used to study liposome fusion.

SNARE-Mediated Membrane Fusion and dPEG®, Part 2

Part 2: Controlling Liposome Fusion Using SNARE Protein Mimics

For Part 1 of this three-part series, click here. For part 3, click here.

Introduction

Continuing earlier work, researchers at the laboratory of Alexander Kros examined the parameters that affect membrane fusion in liposomes composed of the minimal model developed in their earlier paper (1). In this paper (2), the research group demonstrated that, using the model system previously developed, "the rate and extent of fusion and the number of fusion rounds can be manipulated by adjusting the fusogen and liposome concentrations" (quote from the abstract).

The SNARE protein model for membrane fusion developed by the research group contained 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) connected to one of two oligopeptides (LPE or LPK) via a dPEG®12 linker derived from PN10283, Fmoc-N-amido-dPEG®12-acid, where the PEG linker served as a means to provide a flexible spacer between the DOPE and the oligopeptides. See Figure 1, below. The coiled-coil forming oligopeptides each contain three-heptad repeats, and when populations of liposomes containing the LPE and LPK constructs are mixed, they associate and lead cleanly to liposome fusion of the two different liposome populations. As discussed in the earlier post on this subject, the model system developed by Kros and colleagues is an accurate, minimalist model of SNARE-mediated liposome fusion.

 

Figure 1: Structure of the DOPE-PEG12-LPE/LPK lipidated oligopeptides used to study liposome fusion.
Figure 1: Structure of the DOPE-PEG12-LPE/LPK lipidated oligopeptides used to study liposome fusion.

For this research, the authors characterized the influence on liposome fusion of lipopeptide concentration, lipid concentration, and lipids of positive curvature. The authors also described how liposome fusion in the model system varies with liposome collision, docking, and lipid mixing rates.

 

 

How Liposome Fusion Works

The group first studied whether liposome fusion was targeted (that is, molecular recognition between two different fusogens) was required for fusion to occur or non-targeted (no molecular recognition required). This analysis was carried out by mixing populations of liposomes not modified with either LPE or LPK with populations of liposomes carrying either LPE or LPK. In these experiments, no liposome fusion occurred. However, in experiments in which LPE-modified liposomes were mixed with LPK-modified liposomes, liposome fusion occurred. This indicated that molecular recognition between the LPK and LPE peptides was required for liposome fusion to occur; hence, the fusion was targeted.

Next, the researchers examined the effect of concentration of the two fusogens on liposome fusion. By mixing decreasing proportions (mole%) of the lipopeptide fusogens in a fixed amount of total lipids, the scientists were able to determine the lower limit and optimal amount of fusogens required for liposome fusion. By this method, it was determined that 0.05 mole% lipopeptide was the lower limit of fusogens required for at least one round of liposome fusion to occur. The optimal amount of fusion was found to be 0.75 mole% LPE or LPK incorporated into the liposome membranes. Circular dichroism data demonstrated that the lipidated oligopeptides (LPE and LPK) were most likely distributed evenly across the membrane surface at 0.75 mole%.

 

The general trend of increasing membrane fusion rates with increasing proportions of fusogens arises because two liposomes that diffuse into close proximity are more likely to be displaying complementary peptides in the correct orientation for binding in the approaching area, and hence are more likely to dock and undergo fusion. (2) at page 1048.

 

Above 0.5 mole% lipopeptide concentration, the researchers found that completely lipid mixing occurred rapidly. Large particles ("giant liposomes") and clusters of docked and fused particles formed, and the particles sedimented in a matter of hours. Conversely, at 0.25 mole% or less lipopeptide concentration, the particles that form from liposome fusion are initially small, increase in hydrodynamic diameter size, then decrease again over time before plateauing "at the size corresponding to the fusion of two liposomes in which the volume is conserved rather than the outer surface area" (2, p. 1050). This indicates that the SNARE mimetic liposome fusion is "clean," meaning that no loss of content occurs during fusion.

The research group analyzed whether lipid concentration affected membrane fusion, and if so, by how much. Using FRET experiments, the group found that complete lipid mixing was more efficient if there were more fusogens (the DOPE-PEG12-oligopeptides) on fewer liposomes than if there were fewer fusogens on more liposomes. A lower limit of 0.025 mM lipids (0.25 µM lipopeptide) was established as the lower limit of lipid concentration at which liposome fusion occurred efficiently.

Finally, the group examined the effect of changing lipid curvature on liposome fusion. Using the positive curvature lipid lysophosphatidylcholine (LPC) instead of DOPE altered the shape of the liposomal surface and prevented formation of the so-called "stalk intermediate" that is required in SNARE-mediated fusion (the stalk intermediate requires a negative curvature in order to form). With as little as 15 mole% LPC incorporated into liposomes, the researchers found that lipid mixing decreased dramatically; however, docking (the first step in liposome fusion) was not inhibited, but hemifusion and full fusion were prevented from occurring.

The research performed provided detailed understanding of how the reduced SNARE model works to promote liposome fusion. This detailed degree of understanding is important for developing applications of liposome fusion (or membrane fusion) such as targeted delivery of drugs to cells and controlled mixing of reagents at nanoliter volumes, such as would occur in a lab-on-a-chip application.

 

dPEG® Role in This Research

As mentioned in an earlier post, the DOPE-PEG12-LPE/LPK constructs used PN10283, Fmoc-N-amido-dPEG®12-acid to create the flexible extension between the lipid membrane surface and the oligopeptides. If you need to add PEG to your supramolecular construct, why not consider dPEG®? There are numerous advantages to adding PEG to your peptide or protein construct. First, PEG is amphiphilic. Second, PEG reduces aggregation and precipitation of biomolecules. Third, PEG diminishes the immunogenicity of molecules to which it is conjugated. Conventional PEG, though, is polydispersed, meanign that it is composed of numerous PEG molecules. The "weight" of a conventional PEG is expressed as an average molecular weight across the range of sizes. Quanta BioDesign's dPEG® constructs — designed, patented, and manufactured in the United States — are single molecular weight PEG molecules functionalized with various chemical moieties. Whereas conventional PEGs confound analysis by presenting an intractable mixture of different size molecules, dPEG® constructs enhance analysis by being a single, discrete molecule that can be accurately characterized using ordinary analytical chemistry tools.

We encourage you to look at all our products and find what you need for your next research project. You'll be pleasantly surprised at what you can do with dPEG®!

 

References

(1) Hana Robson Marsden, Nina A. Elbers, Paul H. H. Bomans, Nico A. J. M. Sommerdijk, and Alexander Kros. A Reduced SNARE Model for Membrane Fusion. Angew. Chem. Int. Ed (2009), 48, 2330-2333. DOI: 10.1002/anie.200804493

(2) Hana Robson Marsden, Alexander V. Korobko, Tingting Zhen, Jens Voskuhl, and Alexander Kros. Controlled liposome fusion mediated by SNARE protein mimics. Biomaterials Science (2013), 1, 1046-1054. DOI: 10.1039/c3bm60040h

 

About the Author

Robert H. Woodman, Ph.D. is a Senior Product Development Scientist and the QC Manager for Quanta BioDesign, Ltd. He is on LinkedIn at https://www.linkedin.com/in/roberthwoodman, on Twitter at @RobertHWoodman and @QuantaBioDesign.

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Chemical structure of PN10283, Fmoc-N-amido-dPEG®12-acid

SNARE-Mediated Membrane Fusion and dPEG®, Part 1

Part 1: A Reduced SNARE Model for Membrane Fusion

The cells of all living things depend on membrane fusion for intra- and intercellular transport of molecules. In both cellular membrane fusion and intracellular vesicle fusion, the fusion process is controlled and guided by SNARE proteins. SNARE is an acronym for Soluble NSF Attachment Protein Receptor. NSF stands for N-ethylmaleimide-Sensitive Factor. Reviews of SNARE protein structure and function can be found here, here, here, and here. An example of a SNARE protein is synaptobrevin. Click part 2 and part 3 to read the other pieces in this series. Figure 1 shows a diagram of the process of membrane fusion. Figure 2 shows detail of two membranes fusing with SNARE proteins involved in the process. Both images are from Wikipedia and have a creative commons license.

FIgure 1 shows the process of lipid bilayer fusion such as is found in cell membrane fusion and vesicle fusion.
Figure 1: Lipid Bilayer Fusion Process (image from Wikipedia; image is not altered from the original)

Lipid bilayer fusion by MDougM. Licensed under Public Domain via Creative Commons

Figure 2 shows the exocytosis machinery in membrane fusion.
Figure 2: The Exocytosis Machinery Showing How Membrane Fusion Works (image from Wikipedia.com via a Creative Commons License; image is not altered from the original).

Exocytosis-machinery by Danko Dimchev Georgiev, M.D. - http://en.wikipedia.org/wiki/Image:Exocytosis-machinery.jpg. Licensed under CC BY-SA 3.0 via Creative Commons

Membrane Fusion Modeling

It is critical to model membrane fusion in order to understand and control it. A good model of membrane fusion via SNARE proteins helps scientists understand the fusion process and may permit them to conduct membrane fusion under highly controlled conditions to carry out advanced applications in medical diagnostics and therapeutics. To create a good model for membrane fusion, researchers must find the minimal biochemical machinery needed to reproduce membrane fusion fully in a test system.

In 2009, researchers at the lab of Alexander Kros, Leiden Institute of Chemistry, Leiden University, The Netherlands, took a significant step toward that goal(1). In the process, they used Quanta BioDesign's product PN10283, Fmoc-N-amido-dPEG®12-acid as part of the model system(2). Starting with 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE), Kros' group coupled PN10283 to DOPE. The DOPE-PEG construct was then coupled to one of two lipidated oligopeptides, designated LPE and LPK. Each peptide contains a three-heptad repeat (designated E and K) that are the shortest known coiled-coil pair to form a stable heterodimer. See Figure 3, below.

Figure 3: (a) Space-filling model of the two lipidated oligopeptides, LPE and LPK. (b) The two lipidated oligopeptides are spontaneously incorporated into lipid bilayers resulting in liposomes with either LPE or LPK at the surface. When a population of liposomes carrying LPE is mixed with a population of liposomes carrying LPK, the liposomes spontaneously fuse. (c) Diagram showing the fusion of liposomes with the minimal SNARE model of the paper (left) and the SNARE-based protein model (right).
Figure 3: (a) Space-filling model of the two lipidated oligopeptides, LPE and LPK. (b) The two lipidated oligopeptides are spontaneously incorporated into lipid bilayers resulting in liposomes with either LPE or LPK at the surface. When a population of liposomes carrying LPE is mixed with a population of liposomes carrying LPK, the liposomes spontaneously fuse. (c) Diagram showing the fusion of liposomes with the minimal SNARE model of the paper (left) and the SNARE-based protein model (right).

The purpose of this design was to mimic the essential functions of a SNARE protein complex but to minimize the number of different elements needed to direct membrane fusion. The DOPE portion of the construct thus spontaneously anchors the LPE and LPK oligopeptides into the membrane. The dPEG® linker provides a short, flexible spacer that extends the oligopeptides away from the membrane. The two peptides themselves provided the driving force to bring the two membranes together, allowing for membrane fusion.

Using circular dichroism and fluorescence resonance energy transfer (also called Förster resonance energy transfer, known popularly by the acronym FRET) assays with liposome constructs, the researchers were able to distinguish and analyze three distinct stages in membrane fusion. Each stage required an energy input in order to proceed. In the first stage, the liposomes were brought into close proximity. A FRET assay using nitrobenzofuran and lissamine rhodamine dyes attached to the lipid bilayer surfaces showed that the LPE/LPK coiled coil complex that forms spontaneously when the liposomes are in close proximity provides sufficient energy to bring the liposomes together and initiate lipid mixing of the outer lipid layers (known as hemifusion). Further FRET experiments showed that the fusion process initiated by the LPE/LPK coiled coil complex provided enough energy to drive the fusion process to the point of mixing both the outer and inner lipid layers. Furthermore, once the lipid layers were mixed, the contents of the liposomes were observed to mix also.

Conclusions

The approach taken in this paper allowed the research group to build a minimal model of membrane fusion. Indeed, the authors concluded,

"The reduced SNARE model presented herein has been shown to meet all of the characteristics of native membrane fusion, and this similarity combined with the ease of use makes the system a true minimal model for SNARE mediated membrane fusion." (1) at page 2333

Quanta BioDesign Helps You Build Complex Constructs

This research shows the power of modeling even for complex systems such as membrane fusion. Moreover, this research demonstrates the flexible utility of dPEG® products, in this case PN10283, Fmoc-N-amido-dPEG®12-acid.

If you need to add PEG to your supramolecular construct, why not consider dPEG®? There are numerous advantages to adding PEG to your peptide or protein construct. First, PEG is amphiphilic, providing enhanced solubility in both water and organic solvents. Second, PEG has been proven to reduce aggregation and precipitation of biomolecules. Third, PEG reduces immunogenicity of molecules to which it is conjugated.

Dispersity is the key advantage of using Quanta BioDesign's dPEG® products instead conventional PEG products. Conventional PEG is dispersed. Quanta BioDesign's dPEG® products are single molecular weight compounds. That is, rather than having a single discrete molecular size and weight, a conventional PEG is composed of numerous PEG molecules. The "weight" of a conventional PEG is expressed as an average molecular weight across the range of sizes. Quanta BioDesign's dPEG® constructs — designed, patented, and manufactured in the United States — are single molecular weight PEG molecules functionalized with various chemical moieties. Whereas conventional PEGs confound analysis by presenting an intractable mixture of different size molecules, dPEG® constructs enhance analysis by being a single, discrete molecule that can be accurately characterized using ordinary analytical chemistry tools.

Check out our latest products online. You'll be pleasantly surprised at what you can do with dPEG®!

References

(1) Hana Robson Marsden, Nina A. Elbers, Paul H. H. Bomans, Nico A. J. M. Sommerdijk, and Alexander Kros. A Reduced SNARE Model for Membrane Fusion. Angew. Chem. Int. Ed (2009), 48, 2330-2333. DOI: 10.1002/anie.200804493

(2) The dPEG® was purchased from Iris Biotech GmbH, one of Quanta BioDesign's highly valued distributors. Iris Biotech is located in Marktredwitz, Germany.

About the Author

Robert H. Woodman, Ph.D. is a Senior Product Development Scientist and the QC Manager for Quanta BioDesign, Ltd. He is on LinkedIn at https://www.linkedin.com/in/roberthwoodman, on Twitter at @RobertHWoodman and @QuantaBioDesign.

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Chart showing the analgesic activities of various galanin analogues used in a formalin pain assay.

PEGylated Galanin Shows Enhanced Analgesic Effects in PNS

Galanin is a naturally occurring neuropeptide in the human body that facilitates communication between cells to balance a myriad of physiological functions. Neuropeptides are biosynthesized molecules used by the human body for everything from neurogenesis to cell communication.  Galanin’s main receptor sites reside in the central nervous system (CNS), and it normally crosses the blood brain barrier; however, the peripheral nervous system (PNS) also reacts directly to galanin and its receptors in sites of pain mediation.1 Galanin’s role as a pain inhibitor is a peripheral one, which means that, as a drug, its analgesic role would be secondary.  Peripheral effects of peptides are overshadowed by their effectiveness in carrying out primary functions and limiting concentration in low receptor site areas. Peripheral roles of neuropeptides can play just as important of a role as the primary roles in regulating changes in the nervous system.

 

Structure of the galanin neuropeptide found in humans. The peptide sequence is GWTLNSAGYLLGPHAVGNHRSFSDKNGLTS
Figure 1: Structure of the galanin neuropeptide found in humans. The peptide sequence is GWTLNSAGYLLGPHAVGNHRSFSDKNGLTS

 

Common methods of altering drug permeability include lipidization, counterionization, poly-disperse PEGylation, carrier protein conjugation, and many more. The modification is a means to justify an end, in this case the efficacy of the drug. A research team from the University of Utah’s College of Pharmacology and Toxicology and NeuroAdjuvants, Inc., in Salt Lake City studied PEGylation of galanin in response to their research done on other methods of alteration to the neuropeptide. Results of previous research by the group showed increased penetration across the blood brain barrier by galanin. The analgesic properties of galanin are restricted when allowed to permeate unrestricted from blood to brain. The group hypothesized that “dPEGylation will restrict penetration of the neuropeptide analogues into the brain, while their activities in the PNS would be retained”2.

 

Figure 2: Octanol/Water Partition Coefficients (logD) of Galanin Analogues. Y axis: LogD=Log([peptide in octanol)/(peptide in water)]
Figure 2: Octanol/Water Partition Coefficients (logD) of Galanin Analogues. Y axis: LogD=Log([peptide in octanol)/(peptide in water)]
 

Figure 2 shows data for the availability of an altered galanin (Gal-B2) with a discrete PEG in solution between octane and water2. The data shows that the PEGylated Gal-B2 favors the aqueous phase instead, which hypothetically limits its permeability across the blood brain barrier.
The testing of the peptides was carried out by studying the effects both in vitro and in vivo testing methods. The in vitro method included testing the effectiveness of the peptide to bind to its receptor by measuring fluorescence in a competitive binding assay. After PEGylation the neuropeptides retained nanomolar affinities for their receptor sites2. The in vivo testing included formalin injections into the dermal layer of mice paws to trigger a pain response. The mouse’s response to the pain, quantified by licking the wound, was compared across a series of unmodified and modified peptides and a control.

 

Figure 3: Chart showing the analgesic activities of various galanin analogues used in a formalin pain assay.
Figure 3: Analgesic activities of various galanin analogues used in a formalin pain assay.

 

Figure 4: Table showing the anticonvulsant activity of various galanin analogues.
Figure 4: Anticonvulsant activity of various galanin analogues.

Figures 3 and 4 show some surprising results relative to the group's hypothesis. The PEGylated peptide retained similar analgesic activity as the unmodified peptide for acute pain (Figure 3, phase I), and was better than the unmodified peptide in controlling inflammatory pain (Figure 3, phase II). Figure 4 shows that the PEGylated peptides (Gal-B2-dPEG24 and Gal-R2-dPEG24) had reduced anticonvulsant protective effects when administered to mice via intracerebroventricular (icv) injections and had no anticonvulsant protective effects when administered via intraperitoneal (ip) injections. This is most clearly shown by comparing the area under the curve (AUC) of the unmodified Gal-B2 against the AUC of each modified peptide.

This research is significant in that it shows that PEGylation of a biologically active peptide can change both the biodistribution and bioactivity profile of the peptide. This gives promise for “further exploration of monodisperse oligoethyleneglycol residues toward more neuroactive peptides that mediate their actions in the central and peripheral nervous systems”2. As research into this field continues, access to high quality, diverse PEGylation reagents will be a cornerstone.

Our Role in This Research

In this research the role of discrete PEG was important, because polydisperse PEGylation forms a diverse array of peptides3. Quanta BioDesign's patented discrete PEG (dPEG®) technology produces PEGylation reagents that are single molecules, not an intractable mixture of different PEG linker lengths each with a different molecular weight. Using our m-dPEG®24-acid reagent to modify Gal-B2 through a lysine side chain resulted in specific single molecules (Gal-B2-dPEG24 and Gal-R2-dPEG24) that could be fully characterized.

An amide bond between the dPEG® and the peptide was the end goal and we offer myriad products with free acids, active esters, and amines to help create that bond. To create other types of linkages, we offer products with different functional groups, including hydrazides (to form hydrazone bonds), oxyamines (oxime bonds), aldehydes (secondary amines, oximes), thiols (disulfides), and many more. In addition to our linear dPEG® products, Quanta Biodesign also offers dPEG® reagents with multiple arms to better modify your small molecule, peptide, or protein without the cost of extra reaction sites.

 

References on Galanin and PEGylation:

1. Lang, R.; Gundlach, A. L.; Kofler, B. The galanin peptide family: receptor pharmacology, pleiotropic biological actions, and implications in health and disease. Pharmacol. Ther. 2007, 115, 177−207.

2. Zhang L, Klein BD, Metcalf CS, Smith MD, McDougle DR, Lee HK, White HS, Bulaj G. Incorporation of monodisperse oligoethyleneglycol amino acids into anticonvulsant analogues of galanin and neuropeptide Y provides peripherally acting analgesics. Mol. Pharm. (2013), 10(2): 574-85

3. Gaberc-Porekar, V.; Zore, I.; Podobnik, B.; Menart, V. Obstacles and pitfalls in the PEGylation of therapeutic proteins. Curr. Opin. Drug Discovery Dev. 2008, 11, 242−50.

 

Example Linear, Amine-Reactive dPEG® Products (also available as NHS esters and 2,3,5,6-tetrafluorophenyl esters)

m-dPEG®2-acid

m-dPEG®4-acid

m-dPEG®12-acid

m-dPEG®24-acid (used in this study to modify galanin)

m-dPEG®37-acid

m-dPEG®48-CO(CH2)3-acid

 

Example Branched Amine-Reactive dPEG® Products (also available as NHS esters and 2,3,5,6-tetrafluorophenyl esters)

Carboxyl-dPEG®₄-(m-dPEG®₄)₃

Carboxyl-dPEG®₄ -(m-dPEG®₈)₃

Carboxyl-dPEG®₄-(m-dPEG®₁₁)₃

 

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dPEG®-Modified Diabodies Improve Tumor Imaging

Researchers in Australia and the United States have shown that dPEG®-modified diabodies improve positron emission tomography (PET) imaging of tumors by reducing kidney uptake of diabody and extending diabody half-life in the bloodstream, which thus allows more diabody to be taken up by the tumor.1 These findings suggest that better tumor imaging can be achieved using less material, because more of the diabody that targets the tumor gets to the tumor and less of it is excreted by the kidney.

Tumor Imaging and Diabodies

Tumor imaging in mammals requires matching blood clearance and tumor uptake with the half-life of the engineered antibody. Whole antibodies with molecular weights greater than 70,000 Daltons (abbreviated 70KDa) are blocked by the glomerular filtration barrier. Smaller antibodies have lower molecular weights; thus, they pass through the barrier into the kidney and are excreted. Whole antibodies, though, can be much more immunogenic than smaller antibody fragments, and the Fc region of a whole antibody can sometimes cause mis-targeting of antibodies. For this reason, antibody fragments are preferred over whole antibodies for applications such as tumor imaging.

Dimeric antibody fragments, known as "diabodies," are designed single-chain Fv (scFv) fragments consisting  of a heavy chain variable domain (VH) connected to a light-chain variable domain (VL) by a peptide linker that is too short to permit the VH and VL fragments to associate with each other, thus forcing them to associate with complementary domains of another chain, creating two antigen binding sites. See the original paper here. For more information, see here (note: some of the information at this link is behind a paywall). Diabodies can be designed to be bivalent and bispecific. For reviews on bispecific antibodies clichere and here.

In this paper,1 diabodies against tumor-associated glycoprotein 72 (TAG-72) were used. TAG-72 is a mucin-like glycoprotein that is found on the surfaces of breast, ovarian, colon, and pancreatic cancer cells. The anti-TAG-72 diabodies that were studied clear rapidly from blood (t1/2=30-60 minutes), and they have good uptake by tumors expressing TAG-72, due in part to the bivalent nature of the diabodies. Because these diabodies have a molecular weight of about 50 KDa, they are also rapidly cleared (t1/2=12 hours) from the body through kidney uptake of the diabody, which is problematic for diagnostic imaging and therapeutic applications. If the diabodies could be kept from passing through the glomerular filtration barrier, they would stay in the bloodstream longer, thus giving them more time to find and bind tumor-associated TAG-72. (Please click here for more information on glomerular filtration.)

One way to keep smaller molecules such as diabodies from passing through the glomerular filtration barrier is to increase the hydrodynamic volume of the molecule and, hence, increase the apparent molecular weight of the molecule. Molecules with molecular weights greater than 70KDa do not pass easily, if at all, through the barrier. PEGylation increases the hydrodynamic volume of peptides and proteins. (Click here for more information.) PEG molecules are amphiphilic, and in aqueous solution, PEG will hydrogen bond extensively with water, increasing the hydrodynamic volume and causing the molecule to which the PEG is attached to behave as if it is a molecule with a much higher molecular weight. This makes the PEGylated molecule much more difficult to remove from the body by glomerular filtration

.

Diabodies Have Improved Biodistribution When dPEG®ylated

Research by Li Lin et. al., affiliated with Beckman Research Institute of City of Hope, Duarte, California and Avipep Pty Ltd. of Parkville Victoria, Australia shows that conjugating diabodies to discrete polyethylene glycol (dPEG®) derivatives increases the apparent molecular weight of the diabody disproportionately to the size of the dPEG® derivative, thus reducing kidney uptake of the diabody, without adversely affecting the tumor-to-blood (T/B) ratio of diabody. Starting with Quanta BioDesign's Fmoc-NH-dPEG®n-COOH derivatives, Lin and coworkers synthesized DOTA-NH-PEGn-Cys-VS derivatives, where n=0, 12, 24, or 48 ethylene oxide repeats in the dPEG® linker (VS stands for vinyl sulfone). The synthetic scheme is shown in Figure 1.

 

Figure 1: Synthetic Scheme for the dPEG® DOTA derivatives used to modify diabodies.

Figure 1: Synthetic Scheme for the dPEG® DOTA derivatives used to modify diabodies.

 

These compounds were then site-specifically conjugated to an anti-TAG-72 diabody that had been engineered to have a surface disulfide moiety that could be reduced to form a free thiol for conjugation. Using 111In labeling of the DOTA-PEG constructs, the biodistribution of the derivatived diabodies was mapped in athymic mice. The results (see Figure 2, below) showed that as the PEG linker increased in length the rate of kidney uptake and blood clearance of the diabody decreased, while at the same time the amount of labeled diabody on the tumor increased. With the n=0 construct conjugated to the anti-TAG-72 diabody, the apparent molecular weight of the diabody was about 50KDa, and the blood clearance half-life was 30 minutes. With increasing PEG linker length, the apparent molecular weight of the diabodies (as determined by size exclusion chromatography) increased to  60kDa (n=12), 70kDa (n=24), and 80kDa (n=48). For the largest (n=48) derivative, the blood clearance half-life had increased to 6 hours. This increase in apparent molecular weight was much larger than the weight of the linker added to the diabody.

 

Biodistribution over time of DOTA-PEGn-AVP04-50 diabody conjugates in mice bearing LS174-T xeonografts.
Figure 2: Biodistribution over time of DOTA-PEGn-AVP04-50 diabody conjugates in mice bearing LS174-T xeonografts. Yellow=kidney; dark blue=tumor; red=blood; green=liver.

 

Labeling the DOTA-NH-PEGn-Cys-avibody derivatives with 64Cu (half life of 12 hours), the researchers used positron emission tomography (PET) to image tumors expressing TAG-72 antigen in athymic mice at different time points. The results are shown in Figure 3.

 

Tumor Imaging using PET with 64Cu-labeled DOTA-NH-PEGn-AVP04-50
Figure 3: PET imaging of LS174-T xenografts in athymic mice with 64Cu-labeled DOTA-NH-PEGn-AVP04-50. Red=blood; green=liver; yellow=kidney; turquoise=tumor. Pow A shows the PEG0 construct. Row B is the PEG12 construct. Row C is the PEG24 construct. Row D is the PEG48 construct.

 

Figure 2 shows that as the dPEG® linker length grows from 0 to 48 tumor uptake increases by approximately 20%, while the kidney uptake drops by about 90%. Figure 3 provides visual imaging of tumors within the test animals using 64Cu-labeled DOTA. It is clear that as the dPEG® increases, the kidney uptake decreases, and the tumor uptake increases.  The authors note, however, that the T/B ratios of the n=48 conjugate vary and are not ideal for imaging. One way to possibly avoid this problem is to have more than one DOTA on the dPEG® itself. Figure 4 shows one of our novel side-arm loaded dPEG® constructs using a tyrosine core. This could prove useful for conjugating more than one DOTA and one diabody onto the dPEG®.

 

PN11567; MAL-(NH-dPEG4-Tyr(CH2-CO2H))3-NH-m-dPEG24
Figure 4: PN11567; MAL-(NH-dPEG®4-Tyr(CH2-CO2H))3-NH-m-dPEG®24

 

One of the interesting findings in this paper is that the authors of this paper were able to achieve a large effect (an increase of hydrodynamic volume equivalent to a 30KDa increase in mass) with a relatively small PEG linker (48 ethylene oxide units is about 2.1KDa). Moreover, the linker used is a discrete molecule, not a dispersed polymer with mixture of sizes, nor a monodispersed molecule that was purified out of a dispersed mixture. With Quanta BioDesign's technology, we synthesize discrete PEG molecules starting with very high purity materials and build the linker in progressive steps. A dPEG® provides increased water solubility, greater hydrodynamic volume, decreased immunogenicity, ease of characterization (because it is a single molecule, not a mixture), prevention of aggregation, longer circulation time in the body, and so forth. The disadvantages of polymer PEGs, including difficulty characterizing the final product from a dispersed mixture of sizes and tremendous loss of activity when very large PEGs are attached to small molecules, proteins, or peptides, are not found in dPEG® products.

Quanta BioDesign invented dPEG® technology, and with it, we are reinventing PEGylation. We are always creating new dPEG® constructs. In addition, if you look through our catalog and do not see what you want, we can synthesize the dPEG® construct specific to your needs. You can also call or E-mail us with any questions that you may have about our products!

 

References

1 Li L, Crow D, 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 Chemistry (2011), 4, 709–716. doi: 10.1021/bc100464e.

2 Virginia G. Johnson, et al. Analysis of a Human Tumor-associated Glycoprotein (TAG-72) Identified by Monoclonal Antibody B72.3. Cancer Research (1986), 46, 850-857.

3 Donald G. Sheer, Jeffrey Schlom, and Herbert L. Cooper.  Purification and Composition of the Human Tumor-associated Glycoprotein (TAG-72) Defined by Monoclonal Antibodies CC49 and B72.3. Cancer Research (1988), 48. 6811-6818.

 

About the Author

Ian Hotham, B.S., received his Bachelor of Science degree in Chemistry from The Pennsylvania State University in Spring of 2013. Ian is a Process Development Chemist involved in synthesizing new dPEG® and in improving and scaling up synthetic processes. You can connect with Ian on LinkedIn at www.linkedin.com/pub/ian-hotham/51/b28/a08

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Label monoclonal antibodies site specifically with ETAC reagents

ETAC and labeling monoclonal antibodies

Monoclonal antibodies and their small fragments (Fabs, scFv, diabodies etc.) are intriguing objects for creation of protein-based medicines. These proteins can be site-specifically modified with ETAC-dPEG® ("ETAC" abbreviates "Equilibrium Transfer Alkylation Cross-link"; "dPEG®" is the registered trade name for "discrete Poly(Ethylene Glycol)") reagents. Using ETAC, a three-carbon bridge is formed linking the two cysteine sulfur atoms. The dPEG® attached to the ETAC reduces the protein's immunogenicity and prevents rapid clearance of the protein from the bloodstream. This, in turn, helps to maintain a desired therapeutic concentration between doses, thereby reducing the risk of loss of efficacy. The structure of ETAC-reagent and generation of the dPEG®-monosulfone which undergoes a site-specific conjugation with a Fab are outlined below in Figure 1. For details, see, for example, "Comparative binding of disulfide-bridged PEG-Fabs", Bioconjugate Chemistry (2012), 23, 2262-2277; and "Disulfide bridge based PEGylation of proteins", Advances in Drug Delivery Reviews (2008), 60, 3-12.

 

Monoclonal antibodies can be site specifically labeled using the ETAC-dPEG® reagent shown here, after generation of the monosulfone as depicted in the scheme.
Figure 1: Structure of the ETAC-dPEG® reagent and generation of the monosulfone to label monoclonal antibodies site specifically.

 

An accessible disulfide bond can be selectively reduced under mild conditions with DTT or TCEP (Quanta BioDesign product number PN10014) without destroying the tertiary structure of the monoclonal antibody or antibody fragment. Once an accessible disulfide is reduced, the two free cysteine sulfur atoms become available for reaction with the ETAC-reagent (See "Disulfide bridge based PEGylation of proteins"). The PEG-bis-sulfone eliminates the sulfinic anion and generates PEG-monosulfone which then undergoes conjugation, particularly with Fabs. Conjugation occurs by the formation of a three-carbon bridge linking the two cysteine sulfur atoms with PEG attached through the middle carbon of the bridge. Conjugation of PEG at this site normally has minimal impact on the binding properties of the monoclonal antibody or Fab. Figure 2, below, shows a generalized scheme for this process.

General scheme showing how monoclonal antibodies or other disulfide-containing proteins can be site-specifically labeled using an ETAC-dPEG® reagent.
Figure 2: General scheme showing how monoclonal antibodies or other disulfide-containing proteins can be site-specifically labeled using an ETAC-dPEG® reagent.

 

Potential problems with PEGylated monoclonal antibodies as drugs

Having high molecular weight dPEG® in solution can stabilize the native and compact structure of human albumin, does not obscure the protein’s active surface, and folds independently of the protein. The majority of clinically used PEGylated medicines are heterogeneous mixtures that have been produced by non-specific and inefficient PEG-conjugation reaction to different nucleophilic sites on the protein ( See "Comparative binding of disulfide-bridged PEG-Fabs").

Several monoclonal antibodies marketed as drugs use maleimide to conjugate PEG to thiol. Examples of PEGylated monoclonal antibodies manufactured this way include the drug certolizumab pegol (tradename Cimzia) and the now-withdrawn drug peginesatide, a PEGylated peptide.  Although, thiol conjugation is quite efficient, the addition of unpaired cysteine can result in disulfide scrambling and protein aggregation. In addition, there are data that maleimide derived reagents are labile to hydrolysis and can undergo exchange reactions in vivo. See Reversible maleimide-thiol adducts yield glutathione-sensitive poly(ethylene glycol)-heparin hydrogels. Polym Chem (2013), 4, 133-143; and Tunable degradation of maleimide-thiol adducts in reducing environments. Bioconjugate Chem (2011), 22(10), 1946-1953Therefore, development of site-specific approaches and novel reagents that address low conjugation efficiency and PEG-conjugate stability is important, especially in PEGylated monoclonal antibodies and Fab fragments that have potential to be used as therapeutic agents.

 

ETAC-dPEG® reagents offer advantages over traditional PEGylation reagents

Our novel heterobifunctional ETAC-dPEG® (linear or branched) reagents with active TFP- or NHS-groups (for example, ETAC-dPEG®24-NHS ester, PN11685, Figure 3, and ETAC-dPEG®36-TFP ester, PN11686, Figure 4) can be used for preparation of homogeneous, highly potent antibody drug conjugates. It allows to combine the unique targeting capabilities of monoclonal antibodies with therapeutic or diagnostic payload (cytotoxic drug, toxin), dPEG®- and cleavable or noncleavable linker. As a result, the targeted cell (e.g. cancerous) can be damaged either by the released cytotoxic drug, or by the complex of degraded antibody, linker, and drug.

 

Figure 3: PN11685, ETAC-dPEG®-NHS ester, can be used to label monoclonal antibodies or other proteins site specifically. For more information, please contact Quanta BioDesign, Ltd.
Figure 3: PN11685, ETAC-dPEG®24-NHS ester, is a single molecule (not polydispersed) PEGylation reagent that can be used to label monoclonal antibodies or other proteins site specifically. For more information, please contact Quanta BioDesign, Ltd.

Figure 4: PN11686, ETAC-dPEG®36-TFP ester, is a single molecule (not polydispersed) PEGylation reagent that can be used to label monoclonal antibodies site specifically. For more information, please contact Quanta BioDesign, Ltd.
Figure 4: PN11686, ETAC-dPEG®36-TFP ester, is a single molecule (not polydispersed) PEGylation reagent that can be used to label monoclonal antibodies site specifically. For more information, please contact Quanta BioDesign, Ltd.

 

It is important to note that a dPEG® is a single molecular species. (See "What is dPEG®?") Traditional PEGylation reagents are dispersed polymer mixtures. Working with a traditional, polydispersed PEG makes complete characterization of the final product (whether small molecules, peptides, monoclonal antibodies, or other proteins) difficult, because the complex heterogeneity of the polydispersed PEG makes it intractable to analysis.

 

PEGylation reagents for all applications

More than just ETAC reagents, Quanta BioDesign, Ltd., offers PEGylation reagents for almost every need in bioconjugation, therapeutics, diagnostics, theranostics, nanotechnology, bionanotechnology, and many other fields. Our functional groups are diverse, and our chemistry allows us to make a broad range of dPEG® linkers from 1-49 ethylene glycol units as single molecular entities. Look through our catalog today! If you cannot find the linker chemistry you want for your specific application, please feel free to call us and ask. We offer custom synthesis of dPEG® reagents to our customers. We will be happy to speak with you and discuss your needs.  Just call us today!

 

Links to additional PEGylation reagents of interest

TCEP - an effective reagent for reducing disulfides to thiols.

Biotinylation Reagents - biotinylate almost anything with one of our Biotin-dPEG® reagents.

Crosslinking reagents with a variety of chemistry options, including thiol-reactive maleimide and SPDP.

Fluorescent and dye labels - label monoclonal antibodies or other proteins.

 

Victor D. Sorokin, Ph.D. – Received his Ph.D. in Organic Chemistry from the Moscow State University in Russia and completed his postdoctoral training at the University of Texas (1993-1996) where he started his industrial career. He has recently joined our company as a Sr. Product Development Scientist. He has over 15 years of synthetic organic chemistry experience with chemical and pharmaceutical companies developing the synthesis of complex molecules (including natural chiral compounds) on mg/g scale, allowing scale-up to multi-kg quantities. For the last 5 years he has been working on the synthesis of various nucleosides (both DNA, RNA) and oligonucleotides in solution phase using H-phosphonate and phosphoramidate chemistry approach. You can contact Victor on LinkedIn at www.linkedin.com/pub/victor-sorokin/11/417/34b.

 

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Superior Surface Protection of Gold Nanoparticles With Short-Chain PEG

Surface protection of gold nanoparticles is improved by using short-chain, alcohol-terminated dPEG® linkers rather than (2-mercaptopropanoyl)glycine (tiopronin) or mercapto-undecyl-tetraethyleneglycol, according to research findings from the lab of David E. Cliffel, Department of Chemistry, Vanderbilt University. Short-chain dPEG®s increase water solubility, are non-toxic, and show no immune response to anti-PEG antibodies at low concentrations.(1)

Surface protection and opsonization

Tiopronin and mercapto-undecyl-tetraethyleneglycol (Figure 1) have been used for monolayer surface protection of gold nanoparticles, but both have problems associated with their use. Using tiopronin as the monolayer for a gold nanoparticle surface above 40μM causes severe renal damage that ultimately kills test animals.(2) Mercapto-undecyl-tetraethyleneglycol has been shown to have poor water solubility when added to the cluster as the monolayer. To reduce the damage caused by tiopronin in vivo mercapto-undecyl-tetraethyleneglycol is added to the monolayer in high concentrations.(2) These high concentrations create anti-PEG antibodies to attack the cluster and make it unreactive by the mechanism known as opsonization.(1) Opsonization (Figure 2) occurs when anti-PEG antibodies react with the monolayer and render the cluster unreactive and is removed from the body.

 

Chemical structures of tiopronin and mercapto-undecyltetraethyleneglycol used in surface protection of gold nanoparticles.
Figure 1: Tiopronin (left) and mercapto-undecyltetraethyleneglycol (right) used in surface protection of gold nanoparticles

 

Process of Opsonization. Picture copyright 2011 by The Board of Trustees of the University of South Carolina. Used with permission.
Figure 2: Process of Opsonization. Picture copyright 2011 by The Board of Trustees of the University of South Carolina. http://www.microbiologybook.org/bowers/immune%20cells.htm Used with permission.

Short-chain dPEG® compounds enhance surface protection while avoiding opsonization

Short-chain, thiol-dPEG® compounds used in a mixed monolayer with tiopronin increase the water solubility of the tiopronin short-chain monolayer as compared to a mixed monolayer of tiopronin and mercapto-undecyltetraethyleneglycol. This increase in water solubility is attributed to the elimination of the akyl chain of the mercapto-undecyltetraethyleneglycol. A mixed monolayer containing 10% short-chain, alcohol-terminated dPEG® (Figure 3a) on the gold nanoparticle showed no renal damage or other toxicity.(1) The short-chain dPEG® apparently shields the cluster from opsonization and allows for fluid movement of the monolayer, which is thought to be the mechanism that thwarts opsonization.(1) Note that Quanta BioDesign sells the S-acetyl protected version of this alcohol as product number 10156 (see also Figure 3c).

 

Compounds used for surface protection studies. A. Thiol-PEG4-alcohol. B. Thiol-dPEG®4-acid (Quanta BioDesign PN10247). C. S-acetyl-thiol-dPEG®4-alcohol, which was not used in the study but is the S-acetyl-protected version of 3a. 3c is Quanta BioDesign product number 10156.
In studying surface protection of gold clusters, Thiol-PEG4-alcohol and Thiol-dPEG®4-acid were compared. A. Thio-PEG4-alcohol. B. Thiol-dPEG®4-acid (Quanta BioDesign PN10247). C. S-acetyl-dPEG®4-alcohol (Quanta BioDesign PN10156) which is the S-acetyl-protected version of 3a). This product was not used in this study, but it can be used for future, similar applications.

 

The short-chain dPEG® mixed monolayer showed no immune response in vivo. At a 10% molar exchange ratio using an alcohol-terminated short-chain dPEG® mixed monolayer, no immune response occurred in animal models. Red blood cell count increased at a 65-70% molar exchange ratio using a thiol-dPEG®-carboxy-terminated short chain dPEG® (Figure 3b), but again, no immune response occurred. This is Quanta BioDesign's product number 10247. Surface protection of the gold nanoparticles was gained without the complication of anti-PEG antibodies or the serious problem of renal damage. These results favor use of short-chain dPEG® compounds in mixed monolayer with tiopronin instead of mercapto-undecyltetraethyleneglycol.(2)

 

PEG chain length affects in vivo residence time

PEG chain length directly affects residence time in the body. Short-chain dPEG®s have been shown to move through the body much faster (24 hours) than longer chains (2-4 weeks)(1) For applications where a short residence time is desirable, short-chain dPEG® compounds are a strong asset, because they provide high water solubility, no immune response, and no toxicity issues.

Short-chain dPEGs® make a difference in the biological aspect of surface protection chemistry. Quanta BioDesign is the inventor of, and world leader in, dPEG® technology with a vast range of products with varying length and terminal groups with high purity (>90%) for your convenience. If you do not see a product that you want, please call or e-mail for custom synthesis! We want to help you get the best out of your scientific application!

 

References

Simpson, C. A.; Agrawal, C. A.; 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. http://pubs.acs.org/doi/abs/10.1021/nn103148x

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, 1608–1616. http://pubs.acs.org/doi/abs/10.1021/tx100209t

 

Additional surface protection and surface modification products

Click here for general surface modification products from Quanta BioDesign.

Click here for metal surface modification products from Quanta BioDesign.

PN10156, S-acetyl-dPEG®4-alcohol

PN10160, S-acetyl-dPEG®8-alcohol

PN10939, S-acetyl-dPEG®12-alcohol

PN10247, Thiol-dPEG®4-acid

PN10183, Thiol-dPEG®8-acid

PN10850, Thiol-dPEG®12-acid

 

Lipoic acid (1,2-dithiolane-3-pentanoic acid), because of its strained 5-member dithiolane ring, provides superior dative bonding to gold surfaces and, hence, superior surface protection to gold surfaces. Quanta BioDesign, Ltd. offers several lipoic acid-functionalized dPEG® derivatives. A partial list of our line of such products is below:

PN10806, Lipoamido-dPEG®4-acid

PN10641, Lipoamido-dPEG®4-TFP ester

PN10807, Lipoamido-dPEG®8-acid

PN10642, Lipoamido-dPEG®8-TFP ester

PN10808, Lipoamido-dPEG®12-acid

PN10814, Lipoamido-dPEG®12-TFP ester

PN10811, Lipoamido-dPEG®24-acid

PN10643, Lipoamido-dPEG®24-TFP ester

 

You can see all of our lipoic acid derivatives in our Metal Surface Modification Reagents list.

Ian Hotham, B.S., received his B.S. in Chemistry from The Pennsylvania State University in Spring of 2013. Ian is a Process Development Chemist involved in process development and scale-up activities. You can connect with Ian on LinkedIn at www.linkedin.com/pub/ian-hotham/51/b28/a08

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Thiol Reactive Crosslinkers for Bioconjugation

Thiol reactive crosslinkers are one of the most common classes of crosslinkers in bioconjugation (1). The popularity of conjugation to a thiol is due in part to its presence in many proteins, but they are not as prevalent as amines, which are another site for conjugation. This will allow for greater control of the conjugation. Even greater control of the conjugation process is afforded if a thiol reactive compound is combined with an amine reactive compound to create a heterobifunctional crosslinker.

Thiol reactive crosslinkers from Quanta BioDesign, Ltd.

 

1. Maleimide crosslinkers

Quanta BioDesign offers a variety of homo- and heterobifunctional thiol reactive crosslinkers for bioconjugation with dPEG® spacers of several different lengths. Among those we offer are maleimide crosslinkers. Near neutral pH, the double bond of the maleimide reacts preferentially and very rapidly with a thiol to form a thioether bond that is not susceptible to reduction (2). Quanta BioDesign offers homobifunctional bis-maleimides as well as a few heterobifunctional maleimide bioconjugation crosslinkers with the other end being an amine reactive active ester. One of our most popular products is Mal-dPEG®4-NHS ester, product number 10214 (shown in Figure 1). It contains a 22 atom (24.8 Å) tetraethylene glycol spacer functionalized on one end with a thiol reactive maleimidopropyl group and on the other end with an amine reactive propionic acid-N-hydroxysuccinimide (NHS) ester.

 

Thiol reactive PN10214, MAL-dPEG®4-NHS ester
Figure 1: Thiol reactive crosslinker PN10214, one of Quanta BioDesign's most popular PEGylation reagents, has a thiol reactive maleimide group on one end and an amine reactive NHS ester on the other end of a tetraethylene glycol linker.

 

Quanta BioDesign also offers this product with dPEG®2, dPEG®6, dPEG®8, dPEG®12, dPEG®24, and longer dPEG® linkers. You can view them all on our website. They are listed below in this post.  Another version of this product is PN10551, where the NHS ester is replaced by the 2,3,5,6-tetrafluorophenyl (TFP) ester. In-house research by Quanta BioDesign, Ltd., demonstrates that the TFP ester is much less susceptible than the NHS ester to hydrolysis.

 

2. Pyridyl disulfide crosslinkers

Quanta BioDesign also offers pyridyl disulfide (SPDP) bioconjugation crosslinkers (see also here), and with these, the thiol reacts with the SPDP moiety to produce a new disulfide bond, as illustrated in Reaction 1. Pyridine-2-thione is generated, but it cannot react with any remaining SPDP crosslinker because it does not contain a thiol (3, 4). If desired, the newly-formed disulfide bond can be cleaved with a reducing agent. It can also be oxidized back to the disulfide bond, which provides a flexibility not available with the maleimide crosslinkers. Like the maleimides, the SPDP crosslinkers are also offered as the NHS and TFP esters.

 

The thiol reactive pyridyl disulfide (SPDP) group is used in bioconjugation.
Reaction Scheme for the thiol reactive pyridyl disulfide (SPDP) group in bioconjugation

 

Thiol reactive crosslinkers are available now from Quanta BioDesign

Both the maleimide and SPDP crosslinkers with dPEG® are available from Quanta BioDesign with a variety of PEG spacer lengths, ranging from four to twenty-four ethylene oxide units (and in some cases, even longer) Whatever the length and functionality you need for your thiol reactive crosslinking PEGylation reagent, Quanta BioDesign can provide it for you. If you do not see what you want in our catalog, contact us about a custom synthesis. We can provide you with what you are looking for.

 

References

1.  Hermanson, Greg T. Bioconjugate Techniques, 3rd Edition. Waltham, MA: Elsevier (Academic Press), copyright 2013, 1146 pages. (A copy of the 2nd edition of Greg's phenomenal work is available from Quanta BioDesign, Ltd., for $75 plus shipping, or for free with any order of $500 or more, excluding tax and shipping. Look here for more details.)

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2.  Smyth, Derek G., Blumenfeld, O. O., and Konigsberg, W. Reactions of N-ethylmaleimide with peptides and amino acids. Biochem J. (1964), 91, 589-595.

Click here to return to the text.

3.  Carlsson, J., Håkan, D., and Axén, R. Protein thiolation and reversible protein-protein conjugation. Biochem J. (1978), 173, 723-737.

Click here to return to the text.

4.  Myers, D. A., Murdoch, W. J., and Villemez, C. L. Protein-peptide conjugation by a two-phase reaction, Biochem J. (1985), 227(1), 343.

Click here to return to the text.

 

Additional Products from Quanta BioDesign, Ltd.

MAL-dPEG®2-NHS ester

MAL-dPEG®4-NHS ester

MAL-dPEG®6-NHS ester

MAL-dPEG®8-NHS ester

MAL-dPEG®12-NHS ester

MAL-dPEG®24-NHS ester

Please call or email us and ask about our MAL-dPEG®x-TFP ester derivatives. If you want a longer length dPEG® spacer than you see here, please call or email us about that also. We will be glad to discuss them with you!

Dan Dawson, M.S. received his BS in Chemistry from the University of Indianapolis in 2006, and his M.S. in Organic Chemistry from the University of Michigan in 2008. Dan is a Process Development Chemist involved in process development and scale-up activities. You can connect with Dan on LinkedIn at www.linkedin.com/pub/daniel-dawson/18/6a2/718.

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Organophosphorus Hydrolase Pharmacokinetics and Immunogenicity are Improved by Branched dPEG®

Organophosphorus hydrolase  (OPH,EC 8.1.3.1), also known as Aryldialkylphosphatase, is a remarkably stable homodimeric enzyme that can detoxify organophosphate compounds. Organophosphate compounds are the basis of numerous pesticides (e.g., malathion) and chemical warfare weapons (e.g., sarin, VX). Organophosphates act by blocking the action of the enzyme acetylcholinesterase. Overuse and misuse of organophosphate pesticides are major causes of acute pesticide poisoning and death. See also here.

Boris N. Novikov and colleagues with the Department of Biochemistry and Biophysics at Texas A&M University, reported in the Journal of Controlled Release on the effects of modifying organophosphorus hydrolase (often abbreviated OPH) with linear and branched PEG derivatives. The group chose these PEG reagents because conjugates altered by PEGs see “their immunogenicity is reduced and the renal excretion is slowed considerably, leading to prolonged half-life, reduced side effects and increased treatment efficiency,” C.S. Fishburn, The pharmacology of PEGylation: balancing PD with PK to generate novel therapeutics, J. Pharm. Sci. 97 (2008) 4167–4183.

 

 dPEG® Reagents Modifying Organophosphorus Hydrolase

PEG derived conjugates, specifically discrete linear and branched PEGs, were used to help generate data on the biochemical, biophysical, and pharmacological properties or organophosphorus hydrolase. The discrete PEGs chosen were all terminated with a methoxy group at one end of the PEG linker and functionalized with an NHS ester on the other end of the linker to react with surface-accessible lysine residues, forming an amide bond between the PEG linker and organophosphorus hydrolase. The linear, discrete PEGs had spacing lengths of 16.4Å (PEG4), 30.8Å (PEG8), and 44.9Å (PEG12), while the branched, discrete PEG chosen is shown in Figure 1, below. All of the PEG linkers used were made by Quanta BioDesign Ltd. and purchased through Quanta BioDesign distributor Thermo Scientific (formerly Pierce).

 

PN10401, NHS-dPEG®-(m-dPEG®12)3, used to modify organophosphorus hydrolase

Figure 1: The branched discrete PEG used in this research to modify organophosphorus hydrolase.

 

Attaching the PEG linkers altered the biophysical properties of organophosphorus hydrolase. Specifically, the catalytic functionality (kcat) decreased for all PEGylated species, but the enzyme retained substantial activity of 30% - 60% depending on substrate. Moreover, the substrate affinities of the modified enzymes increased (thus, kM decreased), which meant that the overall catalytic efficiencies of the unmodified and modified enzymes were comparatively close.

 

Organophosphorus Hydrolase PEGylation Altered Pharmacokinetics and Immunogenicity

In animal testing, unmodified organophosphorus hydrolase had a circulated half-life of 0.86 hr and a mean residence time of 1.1 hr. PEGylation of the enzyme even with the smallest PEG (PEG4) increased the circulated half-life and mean residence time of the modified enzyme compared to the unmodified enzyme. PEGylation with the large branched PEG (Figure 1, above) resulted in a dramatically longer circulated half-life (32.5 hr) and mean residence time (46.7 hr). See Figure 2, below.

 

Figure 2: Amount of circulating organophosphorus hydrolase (unmodified vs PEGylated) remaining in bloodstream over time
Figure 2: Amount of circulating enzyme (unmodified vs PEGylated) remaining in bloodstream over time

 

Moreover, injection of unmodified organophosphorus  hydrolase into test animals caused formation of antibodies to the enzyme. Injection of the PEG-modified enzymes into test animals slightly reduced, but did not prevent, formation of antibodies to organophosphorus hydrolase. The differences in antibody formation between the linear PEG4, PEG8, and PEG12 were not statistically significant. By contrast, though, antibodies formed to the enzyme modified with the branched PEG12 (Figure 1) were significantly less compared to the unmodified enzyme. See Figure 3, below.

 

Figure 3: Antibodies formed to organophosphorus hydrolase, comparing unmodified and PEGylated enzyme
Figure 3: Antibodies formed to organophosphorus hydrolase, comparing unmodified and PEGylated enzyme

 

In their conclusions, Novikov, et al., observed,

“The ability of the PEG modifications to both prolong the residence time in the vascular system and to lower immunogenicity of the conjugates was shown to be directly correlated with mass of the attached polymer, with maximal effect achieved with the branched PEG12 ....”

They then suggested that “... it is reasonable to expect an enhanced circulatory residence of the bacterial enzyme (OPH) conjugated with PEG12 in ... humans.”

 

Quanta BioDesign’s Role in dPEG® Research and Manufacturing

Quanta BioDesign, Ltd. is the developer and leading innovator and manufacturer of discrete PEGylation reagents. We look constantly for new collaborations and opportunities for custom synthesis in creating new dPEG® products. Our customers’ ideas drive the creation of many new products and have given us many popular products. We scientists and Quanta BioDesign are committed to developing new dPEG® constructs for all types of applications including diagnostic, therapeutic, theranostic, and nanotechnology uses. All ideas are welcomed, and we look forward to helping your research by providing the PEGylation reagents necessary for your research and manufacturing needs, whether you need milligrams or multiple kilograms.

For information about any of the PEGylation reagents we make, custom synthesis, or to order products visit us at www.QuantaBioDesign.com.

 

References:

B.N. Novikov, et al., Improved pharmacokinetics and immunogenicity profile of organophosphorus hydrolase by chemical modification with polyethylene glycol, J. Control. Release (2010), doi:10.1016/j.jconrel.2010.06.003

C.S. Fishburn, The pharmacology of PEGylation: balancing PD with PK to generate novel therapeutics, J. Pharm. Sci. 97 (2008) 4167–4183

 

 Additional dPEG® PEGylation Reagents

Carboxyl-dPEG®₄-(m-dPEG®₄)₃

Carboxyl-dPEG®₄-(m-dPEG®₁₁)₃

Carboxyl-dPEG®₄-(m-dPEG®₂₄)₃

Carboxyl-dPEG®₄-(m-dPEG®₁₂)₃

Carboxyl-dPEG®₄ -(m-dPEG®₈)₃

NHS-dPEG®₄-( m-dPEG®₁₂)₃-ester

 

Adam Fulkert, B.S. – Received his B.S. in Chemistry from The Ohio State University in the autumn of 2011. Adam is a Process Development Chemist involved in process development and scale-up activities.

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