The importance of quantitation in protein purification and expression optimization

Biomolecule quantitation is fundamental to multiple biological research protocols and in the production of pharmaceutical products. Confirming the purification of biomolecules such as antibodies and proteins is desirable to characterize biological processes and ensure starting material quality.^1.2

Quantitation applications are critical to a range of research and therapeutic protocols.

Protein purification

With ever-growing biotechnology capabilities, protein engineering is an increasingly important aspect of research and biological therapies, giving researchers confidence in protein purification methodologies before protein studies.³

Similarly, quantitation applications ensure that proteins intended for pharmaceutical use comply with rigorous quality parameters to ensure patient safety. 

Expression optimization

Many molecular biology experiments start with cloned DNA, which is then transferred to E.coli or a similar expression vehicle to obtain the protein of interest.

To maximize the expression of the required protein, the DNA construct must be designed to enhance the efficacy of expression by the vehicle selected. Moreover, the purity of the DNA must be guaranteed through quantitation applications to increase the output of the target protein.

Adeno-associated virus (AAV) titer determination

AAV is widely used as the vector to introduce functional genes to humans with a disorder that stems from a genetic aberration. Strict regulatory standards, including vector purity and potency specifications, control such gene therapy.

Accurate determination of therapeutic AAV dose is vital for the gene therapy process. Quantitation applications demonstrate the purity of AAV vectors and confirm their dose.⁴

Serological testing

Determining antigen-specific antibodies in plasma samples is a vital diagnostic tool and provides important serosurveillance information during pandemic infections.⁵

Analytic tests that provide quantitative results usually require complex and labor-intensive immunoassays. Thus, there is a need for a rapid, highly sensitive technique for antibody quantitation applications.

Biolayer interferometry

Biolayer interferometry (BLI) presents a novel label-free solution for multiple quantitation applications.^6.7.8 This technique measures the interference pattern of white light reflected from a biosensor’s surface, indicating the presence of biomolecular interactions.

Combined with a standard curve of known concentrations, this technique is a highly efficient and suitable tool for rapid data acquisition over various quantitation applications. Suitable for crude and purified samples, it is ideal for complex matrices applications.

BLI technology is suitable for quantitation applications involving complex matrices. It can be used for quantitation applications in samples that are crude supernatants, cell lysates, and purified proteins. 

High sensitivity is achieved across numerous quantitation applications, from small molecules to large biologic complexes. Furthermore, measurements are made in real-time, so changes in biomolecule concentration can be monitored with high precision.

Only target biomolecules are measured, making BLI a powerful tool for many quantitation applications. 

Gator products for BLI quantitation applications

Gator Bio offers a range of BLI solutions for quantitation applications such as protein purification. GatorPrime^TM is a robust, maintenance-free instrument for label-free analysis of multiple sample types.⁹ With automated biosensor loading and automated regeneration of biosensors, it offers a hands-free alternative to labor-intensive ELISA methodologies for quantitation applications. 

Eight independent spectrometers are incorporated in GatorPrime, enabling the simultaneous quantitation of eight samples. One hundred sixty-eight samples per run can be performed in assays, making it an efficient solution for quantitation applications. Quick-start modes make it easy for non-specialist operatives to use.

GatorPlus is a high-throughput device suited to quantitation, kinetics determination, and epitope binning. In addition, Gator Bio provides specialized solutions which further facilitate quantitation applications.¹⁰

For instance, Gator^TM AAVX allows for the direct capture and quantitation of AAVs in column eluates, crude lysates, cell culture supernatants, and cell lysates using the CaptureSelect^™ anti-AAVX ligand. Rapid and continuous His-tagged proteins without the need for Ni²⁺ recharging is enabled by the Gator^TM Ni-NTA Kit.

Software provided with Gator BLI systems complies with CFR Title 21¹¹, which is critical for researchers applying quantitation applications to the development of monitoring of pharmaceutical drugs.

All data acquired is time-stamped and traceable, and its enhanced audit trails, including recorded user sessions, comply with FDA guidance. The Gator system is thus suitable for use in environments where adherence to good manufacturing/laboratory practice is paramount, and analyses must be CFR21-compliant. 

References and further reading

1. Westbrook JA, et al. Proteomics Clin Appl. 2015 Apr;9(3-4):295-300. Doi: 10.1002/prca.201400120.
2. Heegaard NH, et al. Electrophoresis. 1999 Oct;20(15-16):3122-33.
3. Lillehoj EP, Malik VS. Adv Biochem Eng Biotechnol. 1989;40:19-71.
4. Jungmann A, et al. Hum Gene Ther Methods. 2017; 28(5):235-246.
5. Dzimianski JV, et al. Sci Rep. 2020;10(1):21738.
6. Qiao SP, et al. Chembiochem. 2021 Jun 2;22(11):1974-1984.
7. Noy-Porat T, et al. STAR Protoc. 2021 Sep 15;2(4):100836.
8. Dysinger M, et al. J Immunol Methods. 2012 May 31;379(1-2):30-41.
9. Gator. https://www.gatorbio.com/products/gatorprime
10. Gator. https://www.gatorbio.com/products/biosensors-and-reagents
11. Gator. https://www.gatorbio.com/products/software

About Gator Bio, Inc.

Gator Bio is a world-leading biosensor company headquartered in Palo Alto, CA. At Gator Bio, we provide researchers the tools and instrumentation to advance their research. From antibody engineering to small molecule drug discovery to basic research, Gator Bio can be used to bring meaning to the unknown. From the original inventors of label-free biolayer interferometry (BLI), Gator Bio provides the next generation of BLI technology.

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