Processing Development and Optimization: Practices and Solutions for Downstream Protein Purification Technology

Protein purification is a essential part of downstream biological technology. Mastering the basic theory and operation of protein purification technology plays an important role in the further study of students of related majors as well as their career development. The expression of recombinant proteins (especially by using bacterial vectors and hosts) is a mature technology. But the challenge lies in how to separete them in an active form.

Generally, the production of recombinant proteins based on genetic engineering technology can be divided into three parts: upstream, midstream, and downstream. Upstream technology refers to recombinant DNA technologies including gene recombination, cloning, and construction and expression of engineered bacteria, etc. Midstream technology mainly refers to the fermentation technology of engineered bacteria or cells, while downstream technology refers to the separation and purification of recombinant proteins.

Generally, proteins exist in cells in complex mixtures, and the physicochemical properties of different proteins vary greatly.

Therefore, there is no completely fixed method to separate and purify proteins from complex mixtures so far. Different recombinant proteins have different amino acid sequences and spatial structures, which leads to the differences in their physical, chemical, and biological properties. We can also design reasonable protein purification strategies based on the differences in the properties of the target protein from other proteins in lysates.

In general, protein purification can be divided into 3 successive stages: capture, intermediate purification, and polishing (as shown in the figure below). Of course, before the purification steps, it is usually preceded by extraction, clarification, and concentration of the initial cell lysate.

Purification of recombinant proteins is a crucial technology in biological research. To investigate the specific functions and structures of proteins, researchers must separate and purify recombinant proteins from organisms. The methods of protein purification are primarily based on the similarities and differences among various recombinant proteins. Non-protein substances can be removed based on the similarity between proteins, and the target recombinant proteins can be separated and purified based on their differences.

Since the types, numbers, sequences, and folding forms of amino acids that make up proteins are different, the physical and chemical properties of expressed recombinant proteins vary greatly. And the separetion and purification of recombinant protein is based on these differences in properties to separate and purify the target protein from cells or culture solutions. Properties commonly used in protein purification include: size, shape, chargeability, isoelectric point, hydrophobicity, solubility, density, ligand binding capacity, metal chelation capacity, and some specific properties(e.g., heat resistance, protease resistance), etc. Based on these properties, various methods have been developed for protein separation and purification.

Chromatography is the most widely used and reliable methods for protein separation and purification so far. It is based on the differences in the distribution of samples in the stationary phase and mobile phase, thus achieving the separation and purification of target proteins with high separation efficiency, wide applicability, and ease of scaling up. The commonly used chromatography technology mainly include five categories: size exclusion chromatography, hydrophobic interaction chromatography, cation exchange chromatography, affinity chromatography, and reverse phase chromatography. Principles of each chromatography technology as shown in the figure. In recent years, with the advancement of technology, people have developed hybrid-mode chromatography technologies that combine multiple modes based on traditional chromatography technologies toovercome some of the shortcomings of traditional chromatography. Among them, hydrophobic charge induction chromatography is the most representative type of hybrid-mode chromatography with extensive application value.

Principles of Commonly Used Chromatography Technology

The selection and combination of purification technologies is the key to a successful purification of proteins, with the ultimate goal being to achieve a high-yield, high-purity and low-cost target preteins purification processing through a reasonable design and optimization. In recent years, although some scholars have reported the possibility in using computer-based expert systems to predict and optimize purification processes, this is still difficult to apply to the actual process of protein separation and purification due to the complexity of proteins. Therefore, now, people still select and combine purification technologies based solely on experience or experiments when developing purification strategies.

Application Case - Three-Step Antiobody Purification

Sample Capture - MaXtar® ARPA Affinity Chromatography

#	Time	Peak Area	Peak Height	Peak Width	Asymmetry Factor	Peak Area%	Type
1	6.39	955.7	48.9	0.2682	0.951	2.532	BV E
2	6.944	2319.7	60.6	0.6448	0.632	6.146	W E
3	8.002	34468.1	999.9	0.5062	1.048	91.322	VB R

Intermediate Purification - MaXtar® Q Anion Exchange Chromatography

#	Time	Peak Area	Peak Height	Peak Width	Asymmetry Factor	Peak Area%	Type
1	6.369	488.9	22.7	0.299	1.108	2.089	BV E
2	6.958	1345.1	35.1	0.5474	0.614	5.747	W E
3	8.004	21572.6	636.5	0.4994	0.996	92.164	VB R

Polishing - CHT Multimode Chromatography

#	Time	Peak Area	Peak Height	Peak Width	Asymmetry Factor	Peak Area%	Type
1	6.314	140.4	6.1	0.3511	1.611	0.478	BV E
2	7.152	29207.6	1134.3	0.3864	0.738	99.522	VB R

Step	Purity	Yield
Affinity Chromatography	91.32%	102.2%
Anion Exchange Chromatography	92.16%	95.9%
Sample pH Adjustment and Filtration	95.24%	93.8%
CHT Multimode Chromatography	99.5%	86.0%
Total Yield	102.2% * 95.9% * 93.8% * 86.0% = 79.1%

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