Fab Antibody PEGylation Service

Introduction of Fab Antibody PEGylation ServiceFab

Fab fragments are less stable, and have a short half-life (minutes to hours) that can be resolved by PEGylation generally. Polyethylene glycol (PEG) is a homofunctional cross-linking agent that can improve pharmacological properties. The activated PEG derivatives can modify the cross-linking of peptides and proteins to improve the water solubility and anti-protein degradation ability.

PEGylation is the coupling of PEGylation with proteins to increase solubility and prolong half-life. The activity of PEGylation protein decreases with the increase of molecular weight of PEGylation. PEGylation is a mature and widely used technology, and there are approximately 20 FDA-approved PEGylated drugs worldwide.

PEGylation can be carried out randomly (non-selectively) or at a fixed point. In the case of non-selective PEGylation, it attaches primarily to the primary amino groups of surfacing exposed lysine by activated esters or carbonate of pegylated chains. pH and PEG size had some effects on PEG attachment sites.

Fig.1 Structural representation of Fab-PEG. (Reference source: PEGylation Prolongs the Half-Life of Equine Anti-SARS-CoV-2 Specific F(ab')2 - Pu bMed (nih.gov))

The fab fragment is a complete antibody fragment composed of VH, heavy chain constant region CH1, light chain VL, and light chain constant region CL, which is connected by an interchain disulfide bond to form a heterodimer, with only one complete antigen binding site, and its molecular weight is about 1/3 of the complete IgG molecule. Fab's small size (about 50 kDa) makes it more suitable for applications requiring better tissue penetration and a short half-life.

Advantages of Fab fragment: It can penetrate tissue faster than full antibodies, reducing the production of immune responses such as antibody-dependent cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) for intraocular injection or as an antidote. Compared with whole antibodies, the serum half-life of Fab is shorter, and the treatment of the disease has certain limitations.

Fab is typically produced in a bacterial, high-yield production process and is less expensive than full antibody production. The fab fragment is a substitute for whole antibodies in clinical therapy. Antigen-binding fragment (Fab) formats, such as the approved certolizumab pegol (alpha-TNF-α), use this format.

Fig.2 Phage titration to calculate library size. (Reference: Antibody Phage Display - PubMed (nih.gov))

Alpha Lifetech Can Provide

Alpha Lifetech has a perfect antibody platform to ensure antibody production. Alpha Lifetech can also provide customized services such as Fab antibody production, antibody labeling, antibody crosslinking, and high throughput antibody screening to meet your needs.

Alpha Lifetech has been deeply involved in protein technology for many years. Alpha Lifetech established a perfect and stable protein platform that can save time in scientific research or project research and facilitate subsequent production. Alpha Lifetech can also provide customers with antibody labeling, protein expression, protein modification and detection, protein interaction services (Western blot, GST Pull-down, etc), and other protein assays services.

Fab Antibody PEGylation Service

Antibody PEGylationMethods of PEGylation

One way of PEGylation is by attaching PEG to a free-hinged cys away from the epitope-binding region. PEGylation can cause steric hindrance, which reduces the binding affinity to the receptor, but at the same time can extend the circulatory half-life, making the receptor-ligand interaction therapeutic.

Fig.3 Methods of PEGylation. (Reference source: PEGylation Prolongs the Half-Life of Equine Anti-SARS-CoV-2 Specific F(ab')2 - PubMed (nih.gov))

Antibody PEGylationMethods of PEGylation

Another method of PEGylation is that PEG specifically attaches to heavy chain and light chain Cys residues. This approach requires a highly stable Fab because of the spatial effect of PEG, which can cause the heavy and light chains to separate.

Fig.4 Methods of PEGylation. (Reference source: PEGylation Prolongs the Half-Life of Equine Anti-SARS-CoV-2 Specific F(ab')2 - PubMed (nih.gov))

Applications of Fab PEGylationApplication

(1) Improve circulatory half-life, reduce antigenicity and toxicity;

(2) Currently, there are ten FDA-approved pegylated products, one of which is a pegylated Fab fragment. Certolizumab pegol (Cimzia) for Crohn's disease, ankylosing spondylitis, and rheumatoid arthritis.

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A Case of Fab Antibody PEGylation

Nakamura H verified the effect of PEGylation on Fab through pharmacokinetics. PEGylation was shown by ELISA to increase the half-life, demonstrating that Fab mutants with a new interchain SS bond and a free cysteine at the C-terminal could be pegylated without changing function.

Fig.5 Wild-type Fab and Fab PEGylation. (Reference: C-Terminal Cysteine PEGylation of Adalimumab Fab with an Engineered Interchain SS Bond)

Fab Antibody PEGylation Service Workflow

Steps	Service Content	Timeline
Step1: PEG Molecular Screening	Screening suitable PEG materials	1 day
Step2: Bioactivity Screening	Select the appropriate reaction group	2-3 days
Step3: Reaction System Screening	Buffer system, pH, time	2-3 days
Step4: Protein Purification	Methods of purification of target protein: Affinity chromatography, ion exchange, density gradient centrifugation, molecular sieve	2-3 days
Step5: Remove Impurities	Select appropriate separation methods to remove protein residues	1 day