Alternative antibody Fab′ fragment PEGylation strategies: combination of strong reducing agents, disruption of the interchain disulphide bond and disulphide engineering

International audiencePEGylation has been widely used as a post-production modification methodology for improving biomedical efficacy and physicochemical properties of therapeutic proteins since the first PEGylated product was approved by Food and Drug Administration in 1990. Applicability and safety of this technology have been proven by use of various PEGylated pharmaceuticals for many years. It is expected that PEGylation as the most established technology for extension of drug residence in the body will play an important role in the next generation therapeutics, such as peptides, protein nanobodies and scaffolds, which due to their diminished molecular size need half-life extension. This review focuses on several factors important in the production of PEGylated biopharmaceuticals enabling efficient preparation of highly purified PEG-protein conjugates that have to meet stringent regulatory criteria for their use in human therapy. Areas addressed are PEG properties, the specificity of PEGylation reactions, separation and large-scale purification, the availability and analysis of PEG reagents, analysis of PEG-protein conjugates, the consistency of products and processes and approaches used for rapid screening of pharmacokinetic properties of PEG-protein conjugates

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“…Deleted: until the absolute neutrophil count (ANC) has reached 10,000/mm normal performance in PK and efficacy models [49].…”

Section: Site-specific Pegylationmentioning

confidence: 99%

PEGylation of therapeutic proteins

Jevševar¹,

Kunstelj²,

Porekar

2010

Biotechnology Journal

636

481

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“…Another simple strategy is to reduce the disulphide bridges of a protein in order to expose new thiol residues. This approach proved to be very useful for the PEGylation of antibodies, since the modification of other amino acids could lead to a marked loss of the ability of antigen recognition [27].…”

Section: Chemistry Of Pegylation Of Proteinsmentioning

confidence: 99%

PEGylation of Proteins and Liposomes: a Powerful and Flexible Strategy to Improve the Drug Delivery

Milla¹,

Dosio²,

Cattel

2012

CDM

350

276

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Abstract:PEGylation is one of the most successful strategies to improve the delivery of therapeutic molecules such as proteins, macromolecular carriers, small drugs, oligonucleotides, and other biomolecules. PEGylation increase the size and molecular weight of conjugated biomolecules and improves their pharmacokinetics and pharmacodinamics by increasing water solubility, protecting from enzymatic degradation, reducing renal clearance and limiting immunogenic and antigenic reactions. PEGylated molecules show increased half-life, decreased plasma clearance, and different biodistribution, in comparison with non-PEGylated counterparts. These features appear to be very useful for therapeutic proteins, since the high stability and very low immunogenicity of PEGylated proteins result in sustained clinical response with minimal dose and less frequent administration. PEGylation of liposomes improves not only the stability and circulation time, but also the 'passive' targeting ability on tumoral tissues, through a process known as the enhanced permeation retention effect, able to improve the therapeutic effects and reduce the toxicity of encapsulated drug.The molecular weight, shape, reactivity, specificity, and type of bond of PEG moiety are crucial in determining the effect on PEGylated molecules and, at present, researchers have the chance to select among tens of PEG derivatives and PEG conjugation technologies, in order to design the best PEGylation strategy for each particular application.The aim of the present review will be to elucidate the principles of PEGylation chemistry and to describe the already marketed PEGylated proteins and liposomes by focusing our attention to some enlightening examples of how this technology could dramatically influence the clinical application of therapeutic biomolecules.

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“…Site-specific Fab functionalization can also be used for therapeutic applications since Fab fragments have been used extensively in both in vivo and in vitro studies demonstrating lower immunogenicity than intact antibodies with more efficient tissue penetration (Brogan et al, 2003;de Marco, 2011;Rao et al, 1998). Therapeutic applications of Fab fragment often benefit from the attachment of drugs, enzymes, or peptides to the antibodies to improve therapeutic efficacy (de Marco, 2011;El Khoury and Lowe, 2013;Humphreys et al, 2007;Lu et al, 1999). Therefore, utilization of UV-NBS method for functionalization of Fab fragments provides opportunities to use Fab fragments in both diagnostic and therapeutic applications while having a great potential in the design of both small-scaled sensor diagnostics and drug delivery systems.…”

Section: Resultsmentioning

confidence: 99%

Site‐specific fab fragment biotinylation at the conserved nucleotide binding site for enhanced ebola detection

Mustafaoğlu

Alves

Bilgiçer

2015

Biotech & Bioengineering

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The nucleotide binding site (NBS) is a highly conserved region between the variable light and heavy chains at the Fab domains of all antibodies, and a small molecule that we identified, indole-3-butyric acid (IBA), binds specifically to this site. Fab fragment, with its small size and simple production methods compared to intact antibody, is good candidate for use in miniaturized diagnostic devices and targeted therapeutic applications. However, commonly used modification techniques are not well suited for Fab fragments as they are often more delicate than intact antibodies. Fab fragments are of particular interest for sensor surface functionalization but immobilization results in damage to the antigen binding site and greatly reduced activity due to their truncated size that allows only a small area that can bind to surfaces without impeding antigen binding. In this study, we describe an NBS-UV photocrosslinking functionalization method (UV-NBS(Biotin) in which a Fab fragment is site-specifically biotinylated with an IBA-EG11-Biotin linker via UV energy exposure (1 J/cm(2)) without affecting its antigen binding activity. This study demonstrates successful immobilization of biotinylated Ebola detecting Fab fragment (KZ52 Fab fragment) via the UV-NBS(Biotin) method yielding 1031-fold and 2-fold better antigen detection sensitivity compared to commonly used immobilization methods: direct physical adsorption and NHS-Biotin functionalization, respectively. Utilization of the UV-NBS(Biotin) method for site-specific conjugation to Fab fragment represents a proof of concept use of Fab fragment for various diagnostic and therapeutic applications with numerous fluorescent probes, affinity molecules and peptides.

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Alternative antibody Fab′ fragment PEGylation strategies: combination of strong reducing agents, disruption of the interchain disulphide bond and disulphide engineering

Cited by 44 publications

References 23 publications

PEGylation of therapeutic proteins

PEGylation of therapeutic proteins

PEGylation of Proteins and Liposomes: a Powerful and Flexible Strategy to Improve the Drug Delivery

Site‐specific fab fragment biotinylation at the conserved nucleotide binding site for enhanced ebola detection

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