Online Purchasing Account You are logged on as Guest. LoginRegister a New AccountShopping cart (Empty)
United States 

Engineering therapeutic antibodies for enhanced stability

Posted By
Tags: Bioprocess

Antibodies (Abs) are widely used in therapeutic, diagnostic, imaging, biosensor, and research applications. Antibody-based therapeutics have been highly successful clinically and represent one of the fastest growing sectors in the pharmaceutical industry. Paul Ehrlich laid the early foundation for advancing antibody therapy by postulating the concept of “magic bullets” (Strebhardt and Ulrich, 2008). Monoclonal antibodies (mAbs), through their attractive specificity, have revolutionized the field of biotechnology research by being able to pick the proverbial molecular needle from the surrounding cellular haystack. They are important tools for a broad range of applications due to their high specificity and ability to recognize virtually any target molecule. Researchers require mAbs to bind numerous important biological targets, such as lipids, metabolites of small molecule drugs, and other protein-based markers. Researchers are searching for ways to further enhance mAbs' sensitivity, selectivity, and multiplexing ability. Of the new 45 new molecular entities approved by the FDA in 2015, nine were mAbs. To be practically useful, Abs must be highly stable and bind their target antigens with high affinity. Despite their potential, few antibodies possess ideal biophysical properties. It is, therefore, pertinent that natural antibodies be tailored by a variety of methods to suit a particular therapeutic use.


Antibodies are the ultimate example of combinatorial biochemistry since each human is capable of producing on the order of one billion different antibodies, generating a library that exceeds the diversity of any product synthesized by combinatorial efforts. Some of the favorable pharmaceutical properties desired in therapeutic antibodies include high thermal stability, low aggregation propensity in order to facilitate manufacturing and storage, as well as long serum half-life. When an Ab is designed as a drug, features such as size, tissue penetration and distribution, half-life, affinity, stability, and immunogenicity should be taken into consideration and optimized accordingly. Furthermore, Abs used for diagnostic purposes and antibody-based biosensors benefit from minimal need for cold-chain storage. In view of a highly organized and differentiated molecular structure, Abs are amenable to be engineered for desired functionality and physico-chemical properties to broaden their application. A general approach consists of utilizing as a starting point, a stable IgG structural scaffold that can be engineered to improve desired features while maintaining antigen binding affinity.

The development of Ab therapeutics is an iterative process. Antibody stability engineering strategies begin by improving the residues linked to stability or using directed evolutionary strategies to identify aggregation-resistant frameworks (Dudgeon et al., 2012). By far, the most popular recombinant Ab region is the single-chain variable fragment (scFv), which represents the antigen-binding site of an antibody. Compared to other Ab fragments with more domains or interfaces, scFv can be conveniently expressed in a variety of hosts and have broad applications in medicine. Strategies to engineer scFv fragments with increased stability can be divided into rational and evolutionary approaches. For rational approaches, structure- or sequence-based knowledge is used to predict stabilizing mutations into a given scFv fragment, which can then be confirmed by site-directed mutagenesis. Grafting complementarity determining regions (CDRs) of defined specificity onto known stable frameworks is a simple and straightforward rational approach to antibody stabilization (Jung et al., 1999). Evolutionary strategies involve fine tuning consensus sequences associated with protein stability. For example, introduction of consensus residues in the candidate antibody has been demonstrated to improve stability of immunoglobulin (IgG). A disulfide bond linking N- and C-terminal ® strands of an isolated CH2 domain has been shown to significantly increase its stability. These approaches, however, have not yet been applied to the more complex, full length immunoglobulin.

McConnell and colleagues have reported antibody stabilization by combining rational and computational design to generate a human IgG with a melting temperature (Tm) of 90°C. They have provided elegant data obtained with a Thermofluor assay on the utility of CDR grafting loops to generate high-affinity antibody with enhanced biophysical characteristics. A therapeutic or diagnostic tumor-binding scFv fragment is required to retain its activity for several hours or days after injection without precipitating or being degraded. Improving protein thermostability is an important goal of protein engineering that makes Abs easier to handle. Increased thermostability offers storage options and facilitates the commercial development of therapeutic products. Systematically altering the makeup of proteins is an exciting frontier of computational biology. Applying these techniques to the development of the next generation of Ab therapies would result in many effective new treatments for cancer and an array of diseases and disorders over the next decade. Engineering human Ab technologies represents a technical tour de force that has been built through extensive knowledge of the human immunoglobulin heavy and light chains. Recent success with immunotherapy is a testimony for Ehrlich's vision and offers promise for a more efficacious “magic bullets” against cancer.

Enzo Life Sciences is a strong contributor to the Bioprocess market with tools to monitor product integrity, purity and efficiency in a high-throughput manner such as our PROTEOSTAT® Protein aggregation assay and PROTEOSTAT® Thermal shift stability assay.

Share this TechNote

Never miss a new TechNote!

Receive our TechNotes as soon as they are published.


Follow Us!

 
comments powered by Disqus

References:

  1. K. Strebhardt and A.Ullrich. Paul Ehrlich's magic bullet concept: 100 years of progress. Nat. Rev. Cancer. (2008) 8: 473.
  2. K. Dudgeon et al. General strategy for the generation of human antibody variable domains with increased aggregation resistance. Proc. Natl. Acad. Sci. U S A. (2012) 109: 10879.
  3. S. Jung, et al. Selection for improved protein stability by phage display. J. Mol. Biol. (1999) 294: 163.
  4. A.D. McConnell et al. An integrated approach to extreme thermostabilization and affinity maturation of an antibody. Protein Eng. Des. Sel. (2013) 26: 151.

Related Products

Protein A ELISA kit 

Most sensitive (9.01 pg/ml)  colorimetric ELISA for monitoring residual contamination.
ELISA, Colorimetric detection | Print as PDF
 
ADI-900-057 96 wells 460.00 USD
Do you need bulk/larger quantities?
 

PEGylated protein ELISA kit 

Validated, Complete Assay for the Sensitive Detection of a Diverse Set of PEGs in Biological Samples
ELISA, Colorimetric detection | Print as PDF
 
ADI-900-213-0001 96 wells 525.00 USD
Do you need bulk/larger quantities?
 

PROTEOSTAT® Protein aggregation assay 

Quantitative Detection of Protein Aggregates from Visible to Subvisible Particles
Flow Cytometry, Fluorescence microscopy, Fluorescent detection, Microplate | Print as PDF
 
ENZ-51023-KP050 50 tests 173.00 USD
 
ENZ-51023-KP002 2x96 tests 462.00 USD
Do you need bulk/larger quantities?
 

PROTEOSTAT® PDI assay kit 

Fluorescent detection | Print as PDF
 
ENZ-51024-KP050 50 tests 201.00 USD
 
ENZ-51024-KP002 2x96 tests 536.00 USD
Do you need bulk/larger quantities?
 

PROTEOSTAT® Thermal shift stability assay kit 

Accelerated screening for protein stability as a function of pH,  ionic strength, and concentration.
Print as PDF
 
ENZ-51027-K100 100 tests 179.00 USD
 
ENZ-51027-K400 400 tests 476.00 USD
Do you need bulk/larger quantities?
 

PROTEOSTAT® Thioredoxin-1 assay kit  

Fluorescent detection | Print as PDF
 
ENZ-51033-KP002 2x96 wells 549.00 USD
Do you need bulk/larger quantities?
 

PROTEOSTAT® Aggresome detection kit  

Robust, quantitative detection of aggresomes relevant to the study of neurodegenerative disease, liver disease and  toxicology
Flow Cytometry, Fluorescence microscopy, Fluorescent detection | Print as PDF
 
ENZ-51035-0025 25 tests 108.00 USD
 
ENZ-51035-K100 100 tests 294.00 USD
Do you need bulk/larger quantities?
 

PROTEOSTAT® Protein aggregation standards  

The only commercially available protein aggregation standards assay!
Fluorescent detection, Microplate | Print as PDF
 
ENZ-51039-KP002 2x96 wells 208.00 USD
Do you need bulk/larger quantities?
 

PROTEOSTAT® Protein refolding and aggregation sensing kit  

Identify Optimal Protein Refolding Conditions and Maximize Recovery of Functional Protein
Print as PDF
 
ENZ-51040-KP002 2x96 wells 367.00 USD
Do you need bulk/larger quantities?
 

CHO host cell protein ELISA kit 

Sensitive (10 ng/ml) ELISA  for the quantitative determination of host cell protein contamination in bulk products expressed in Chinese Hamster Ovary (CHO) expression systems.
ELISA, Colorimetric detection | Print as PDF
 
ENZ-KIT128-0001 96 wells 643.00 USD
Do you need bulk/larger quantities?
 

E. coli host cell protein ELISA kit 

Sensitive (30 ng/ml) ELISA for  the quantitative determination of host cell protein contamination in bulk products expressed in E. coli expression systems.
ELISA, Colorimetric detection | Print as PDF
 
ENZ-KIT127-0001 96 wells 643.00 USD
Do you need bulk/larger quantities?
 

BIOLIKE® Bevacizumab conformational ELISA kit 

Systematic and sensitive immunoassay to compare the structural conformation between Bevacizumab and a biosimilar mAb.
ELISA, Colorimetric detection | Print as PDF
 
ENZ-KIT118-0003 3x96 wells 964.00 USD
Do you need bulk/larger quantities?
 

BIOLIKE® Alemtuzumab conformational ELISA kit 

Systematic and sensitive immunoassay to compare the structural conformation between Alemtuzumab and a biosimilar mAb.
ELISA, Colorimetric detection | Print as PDF
 
ENZ-KIT119-0003 3x96 wells 964.00 USD
Do you need bulk/larger quantities?
 

BIOLIKE® Cetuximab conformational ELISA kit 

Systematic and sensitive immunoassay to compare the structural conformation between Cetuximab and a biosimilar mAb.
ELISA, Colorimetric detection | Print as PDF
 
ENZ-KIT120-0003 3x96 wells 964.00 USD
Do you need bulk/larger quantities?
 

BIOLIKE® Trastuzumab conformational ELISA kit 

Systematic and sensitive immunoassay to compare the structural conformation between Trastuzumab and a biosimilar mAb.
ELISA, Colorimetric detection | Print as PDF
 
ENZ-KIT121-0003 3x96 wells 964.00 USD
Do you need bulk/larger quantities?
 

BIOLIKE® Adalimumab conformational ELISA kit 

Systematic and sensitive immunoassay to compare the structural conformation between Adalimumab and a biosimilar mAb.
ELISA, Colorimetric detection | Print as PDF
 
ENZ-KIT122-0003 3x96 wells 964.00 USD
Do you need bulk/larger quantities?
 

BIOLIKE® Conserved mAb sequence conformational ELISA kit 

Systematic and sensitive immunoassay to monitor the structural conformation of a novel mAb during the development process.
ELISA, Colorimetric detection | Print as PDF
 
ENZ-KIT123-0003 3x96 wells 964.00 USD
Do you need bulk/larger quantities?
 

BIOLIKE® Infliximab conformational ELISA 

Systematic and sensitive immunoassay to compare the structural conformation between Infliximab and a biosimilar mAb.
ELISA, Colorimetric detection | Print as PDF
 
ENZ-KIT124-0003 3x96 wells 964.00 USD
Do you need bulk/larger quantities?
 

BIOLIKE® Rituximab conformational ELISA kit 

Systematic and sensitive immunoassay to compare the structural conformation between Rituximab and a biosimilar mAb.
ELISA, Colorimetric detection | Print as PDF
 
ENZ-KIT125-0003 3x96 wells 964.00 USD
Do you need bulk/larger quantities?
 

BIOLIKE® Palivizumab conformational ELISA kit 

Systematic and sensitive immunoassay to compare the structural conformation between Palivizumab and a biosimilar mAb.
ELISA, Colorimetric detection | Print as PDF
 
ENZ-KIT126-0003 3x96 wells 964.00 USD
Do you need bulk/larger quantities?
 

Recommend this page

 
For Research Use Only. Not for use in diagnostic procedures.
Keep in touch

©2017 Enzo Life Sciences, Inc.,