skip to Main Content
My Account  Search by Structure    

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


Read More

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.



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.)

Click here to return to the text.

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

Read More
Back To Top