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

Tools to monitor protein aggregation

Posted By Arun Kumar
Tags: Bioprocess

The efficient large scale production of recombinant proteins requires careful handling of the protein during isolation and purification. Proteins are isolated in an environment devoid of potentially stabilizing factors. As a result, many protein samples show loss of function and reduced stability in standard buffer conditions. Stability is an important issue in protein production, since destabilized proteins are susceptible to chemical and physical alteration leading to loss of activity. Chemical alterations are related to covalent modifications such as oxidation and disulfide bond shuffling. Physical changes include protein unfolding, binding to surfaces, and aggregation. Many proteins are prone to aggregate for a variety of reasons. Protein aggregation is favored in the food industry since it imparts desirable texture and flavor. However, for pharmacological applications, protein aggregation represents a major obstacle to the development of protein-based pharmaceuticals. During drug formulation, protein aggregation can impact product quality in terms of biological activity and immunogenicity. Protein aggregation can occur at all stages in the manufacturing process including cell culture, purification, formulation, storage, shipping, and handling. Protein aggregation can either be reversible or irreversible, and aggregates can range in size from microscopic to macroscopic. Therefore, the pharmaceutical industry requires improved methods to detect, monitor, and quantify factors governing aggregation during manufacturing.


Do you like egg drop soup?

Factors that influence Protein Aggregation

Protein aggregation is an undesired interaction between protein monomers and can be caused by factors such as pH, ionic strength, mechanical agitation, protein concentration, freeze-thaw operations, temperature, and packaging materials and agents (such as glass and silicone oil). Five major mechanisms of aggregation have been known to date, namely, concentration-, conformational change-, chemical reaction-, nucleation-, and surface-induced aggregation. Surfactants are used in biotechnology to stabilize therapeutic proteins, inhibit aggregation by preventing protein adsorption on surfaces. Aggregation can be inhibited by kosmotropic salts such as ammonium or magnesium sulfate which stabilize the native protein state.

Methods to Detect Protein Aggregation

Understanding the fundamental mechanism of aggregation is valuable for identifying the factors underlying the problem and further aid in developing strategies to prevent aggregation. Several experimental methods are routinely used to determine soluble aggregates. Differential scanning calorimetry (DSC) is a non-invasive technique to evaluate protein stability, however, it is very time-consuming. The most popular of these methods is size exclusion chromatography (SEC), which requires large quantities of purified protein. Other methods like circular dichroism or dynamic light scattering are not always available in all laboratories and their results are difficult to interpret. Analytical ultracentrifugation is the most accurate, but is very expensive and none of the mentioned above possess the throughput. Native gels are much cheaper but need to be optimized for each protein and do not provide an analytical result. In summary, no single method is optimal for all aggregates. Fluorescence-based protein stability screens were developed as suitable alternative as they allow high-throughput screening of many conditions in a 96-well format. These assays rely on fluorescent dyes to probe changes of the protein’s folding state. Recently, Vandecaetsbeek and Vangheluwe described a time-dependent protein stability assay, which provides “half-life” time (t1/2) values. A protein sample is incubated with a thiol-reactive dye called CPM at a fixed elevated temperature (e.g. 42°C) and the fluorescence is measured over a period of time. The t1/2 value corresponds to the time where 50% of the maximum fluorescence is recorded. This value directly correlates with the stability of the protein. This technique allows the identification of factors capable of stabilizing a protein such as additives, detergents, ligands, and lipids.

Advantages of PROTEOSTAT® dye technology

As an alternative to time-dependent protein stability assays, environmentally-sensitive dyes, such as ANS and SYPRO® Orange, have been applied to the detection of protein unfolding in thermal shift assays, by a procedure referred to as the Thermofluor™ technique. The fluorescent dyes interact with exposed hydrophobic regions generated by unfolding of proteins when a sharp increase in fluorescence is observed which corresponds to the “melting” temperature (Tm). The dye is quenched in the aqueous solutions, but when aromatic residues in the protein are exposed due to thermal stress, the dye intercalates with exposed hydrophobic patches. Thermofluor™ is useful to assess thermostability in a systematic way as it enables to test simultaneously many conditions. Instead of monitoring protein aggregation, these dyes measure protein unfolding and are incompatible with the use of detergents. To overcome these limitations, Enzo Life Sciences developed the PROTEOSTAT® dye. It is compatible with detergents and provides an improved thermal shift approach for assessment of protein stability directly monitoring protein aggregation rather than protein unfolding. The PROTEOSTAT® Thermal shift dye is a proprietary formulation, which belongs to a class commonly known as molecular rotor dyes, which in aqueous solution is minimally fluorescent. Upon binding to the surface of aggregated proteins, the dye emits a strong red signal thus providing a homogeneous assay for the analysis of protein stability. From the thermal shift assay, a temperature at which the bulk of the protein becomes aggregated can readily be identified. The aggregation temperature is an indicator of protein stability and can be used to optimize conditions that enhance protein stability. Conditions that increase the aggregation temperature (Tagg) increase the stability of the protein. Enzo’s thermal stability assay with the PROTEOSTAT® dye has, for example, been validated to determine the stability of novel antibody-drug conjugate (ADC) ADC products, where temperature of aggregation (Tagg) was used to classify ADC products according to their propensity for aggregation (Schneider et al., 2014). The assay facilitates understanding of the underlying mechanisms impacting protein stability.

Conclusion

The same issues of protein instability and aggregation confront both basic as well as applied research, since protein aggregation results in decreased yields, failure to crystallize, and decreased specific activity. The goals of efficient protein production relies on an early and robust method of aggregation detection and elimination of aggregation during purification. Given that no clear correlation exists between protein stability and intrinsic properties such as isoelectric point, molecular weight and percentage of charged residues, thermal stability of each new protein needs to be measured for determining optimum conditions. Thermal stability assays have proved to be valuable since they are fast, simple and easy to set up at low protein cost to provide answers about factors affecting protein stability. To this effect, Enzo Life Sciences offers comprehensive tools for advancing your research in Bioprocess.

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. I. Vandecaetsbeek and P. Vangheluwe. Time-dependent protein thermostability assay. Methods Mol. Biol. (2016) 1377, 79.
  2. N. Schneider, et al. Prediction of aggregation propensity and monitoring of aggregation of antibody-drug conjugates (ADC) using PROTEOSTAT® reagents. Bioprocess Intl. (2014) 12, S.

Related Products

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 181.00 USD
 
ENZ-51023-KP002 2x96 tests 485.00 USD
Do you need bulk/larger quantities?
 

PROTEOSTAT® PDI assay kit 

Fluorescent detection | Print as PDF
 
ENZ-51024-KP050 50 tests 211.00 USD
 
ENZ-51024-KP002 2x96 tests 563.00 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 188.00 USD
 
ENZ-51027-K400 400 tests 501.00 USD
Do you need bulk/larger quantities?
 

PROTEOSTAT® Thioredoxin-1 assay kit  

Fluorescent detection | Print as PDF
 
ENZ-51033-KP002 2x96 wells 570.00 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 112.00 USD
 
ENZ-51035-K100 100 tests 308.00 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 218.00 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 385.00 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 660.00 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 660.00 USD
Do you need bulk/larger quantities?
 

Recommend this page

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

©2019 Enzo Life Sciences, Inc.,