Bioanalytical Techniques Applied on the Downstream Protein Purification Process

A study of the main bioanalytical techniques that are applied at different stages of the downstream protein purification process to ensure overall product quality control.
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Abstract 
This paper aims to identify and evaluate bioanalytical techniques applied in the downstream protein (DSP purification process) to get high-quality product quality control. There is a review of past research on bioanalytical techniques for downstream protein product quality control. The approach helps identify and collect relevant data and information on bioanalytical techniques and DSP processes. Data obtained from secondary sources is synthesized based on relevance to the research topic. Centrifugation, chromatography, and filtration methods are the main bioanalytical techniques in the harvest and filtration, the release of intracellular products, buffer exchange and concentration, purification, and formulation stages of the downstream protein purification process. Utilizing the most efficient techniques in each phase of downstream protein is essential to getting high-quality protein products.
Introduction 
Downstream processing (DSP) is required to separate, (isolate), refine, and purify a mixture of components, and there is a need to overcome bottlenecks. There has been advancement in protein production and purity, focusing on optimizing high yield production and purity in less costly means. In DSP, there is a focus on controlling product quality and purification, and it is helpful to differentiate the product and process-related contaminants (Rolinger, Rüdt & Hubbuch, 2020, p. 2). Protein purification often depends on the size, form, solubility, and surface charge of protein molecules, including biospecific affinity. A multistage downstream process is required to eliminate impurities and get the concentration of the required product. 
Obtaining the target protein product in a purified form. Effective downstream bioprocessing enhances protein purity, but there is also a decrease in process volume that needs to be addressed to avoid losing the protein products. Optimizing bioseparation is also prioritized to ensure product quality in downstream protein purification. Removing impurities is essential, so the whole downstream protein purification process achieves a high yield (Somasundaram et al., 2018, p. 2895). In biotherapeutic manufacturing, the DSP is the last stage where the target protein is harvested and purified. The research focuses on bioanalytical techniques in protein purification in the downstream processing stages and elaborates how the techniques are used.
Research Question: What bioanalytical techniques are applicable in the downstream protein purification processes to guarantee high product quality and control ?
Literature Review
Downstream Process Development
 Downstream protein purification is a multistep process that eliminates contaminants to ensure the protein product is pure and homogeneous. DSP is the last phase where the protein gets harvested and purified to achieve the desired product quality. The DSP steps for recombinant protein preparation follow similar steps but are also influenced by the expression host system (Tripathi, and Shrivastava, 2019, p.16). Furthermore, an efficient downstream process is required to isolate products and achieve high-purity products (Kruschitz and Nidetzky, 2022, p. 2). Statistical methods such as principal component analysis and the Design of Experiments (DoE) approach help optimize purification in the DSP and improve efficiency (Li and Venkatasubramanian, 2016, p.354). Continuous downstream processing is more efficient than the traditional batch processing of biosynthetic products, and there are innovative downstream processing techniques to promote continuous DSP.
Figure 1: Bioprocessing workflow of downstream 
Source: Carvalho et al., 2019: 82
Bioanalytical techniques for downstream protein purification processes
Bioanalytical techniques have been developed and validated for the downstream protein purification processes. The main steps in the downstream protein purification processes are solid-liquid separation, the release of intracellular products, concentration, purification, and formulation.
* Solid-Liquid separation (harvest and filtration): The downstream protein purification process begins with separating the large insoluble contaminants from the harvest solution. Some of the contaminants include cells and cell debris. Clarification, which is the mechanical form of separation often requires depth filtration, is one of the techniques for removing large-sized particles. The size and morphology of cells influence the method of solid-liquid separation. Protein’s water and fat solubility influences extraction from biomaterials when water or organic solvents are used (Liu et al., 2020, p. 2).
* Release of Intracellular Products: It is necessary to release biotechnological products such as enzymes and vitamins within the cells before facilitating further processing. Identifying and separating cells or microorganisms before purifying protein ensures there are fewer contaminants. Cell disruption is based on physical, chemical, or enzymatic methods. 
* Buffer Exchange and Concentration: Separating the concentrated from the particles is undertaken before removing water to achieve the desired products concentration.
* Purification: The purification process often relies on chromatography, but there are different methods of separating compounds and removing residual impurities. Chromatography techniques combined with other separation and purification methods are necessary to separate molecules and purify molecules in a complex mixture (Babaie, 2020). The goal of purification is to achieve product purity at a high yield. 
* Formulation: The last step ensures that biotechnological products are in the desired form and they are stable. Batch ultrafiltration/infiltration is commonly used for most recombinant protein products, and to improve efficacy and product quality, there is a need to focus on continuous ultrafiltration and diafiltration processes (Zydney, 2016, p. 8). Ultrafiltration focuses on product concentration, and diafiltration focuses on buffer exchange. Proteins tend to lose their biological activity, and there may be an addition of stabilizers to proteins, which increases protein shelf life and makes it easier to handle, store, or distribute. 
Methods
There is a review of past research to identify bioanalytical techniques for downstream protein product quality control stages. Synthesizing and appraising research will also highlight updated information (Brämer et al., 2019, p. 5). When synthesizing the information from different electronic databases, locating relevant research information and interpreting the results is necessary. The reviews focus on present evidence, research findings, and conclusions and how these are relevant to the research question. The number of published literature on DSP and product quality has increased in the past two decades, and relevant and recent research is used as a source of information and evidence. 
Results and Discussion
Solid-Liquid separation (harvest and filtration) 
Depth Filtration and Hollow Fiber
Performing solid-liquid separation takes time substantial time but is the first step in harvesting that often utilizes depth filtration and hollow fiber ultrafiltration. Depth filtration focuses on a mixture pumped through a membrane or interface, and this separates the debris. Hollow fiber removes the supernatant and traps some of the biomass in the hollow fiber filter membrane. The solid-liquid separation often requires a series of filters, and there is a focus on removing the cell, impurities, and cell debris (Carvalho et al., 2019, p. 84). Optimizing the hollow fiber membrane performance requires considering products recovery and the maximum load capacity. However, there are different impurities that affect filitration. Depth filtration using an efficient membrane filter removes most impurities, but the pore sizes affect the rate of impurity removal.
Centrifugation
Harvest clarification and filtration can involve centrifugation, depth filtration, and sterile filtration. The separation principle for centrifugation is density difference and thermal denaturation is the main damage to proteins. Centrifugation or filtration technologies are used to achieve the desired clarification with a focus on the removal of large particles and then sub-micron particles in secondary centrifugation (Besnard et al., 2016, p 3). Centrifugation facilitates the removal of cells and cell debris removal through sedimentation, and centrifugation has been used for the high solid loads (Besnard et al., 2016, p. 3).
Membrane filters 
Membrane filters ought to have high detention efficiency, but the pore sizes influence the impurities removed, and the filters retain particles based on size (Besnard et al., 2016, p 5). Membrane filters have defined pore size ranges, are scalable, and retain contaminants within the filter structure. Filtration technologies are essential in clarification, and they separate fluids from particulates both depth filters and membrane filtering are used to filter the required volume.
Flocculation and Precipitation
The flocculation and precipitation process allows separation very small suspended solids and colloids that have not been separated in other processes, and flocculation and precipitation are used before filtration or centrifugation. To break these suspensions and produce agglomeration of particles, the coagulation-flocculation treatment is used and these are two differentiated processes (Singh and Herzer, 2017, p. 120). Coagulation is the destabilization of colloidal particles, which can be achieved especially through the neutralization of their electrical charges with the addition of a coagulant (chemical reagent). Coagulants are chemical products that in solution provide an electrical charge opposite to that of the colloid. Flocculation is the grouping of the discharged particles when they come into contact with each other. 
Release of Intracellular Products: 
High pressure homogenization
The high-pressure homogenization (HPH) approach can modify protein functionality. The technique focuses on forcing cell suspension at high pressure through a homogenization valve or orifice. HPH is a mechanical method that is scalable and can handle large volume processes and is one of the effective cell disruption methods (Zhang et al., 2019). There is the disaggregation of particles after liquid dispersions that breaks cells, and here is reduced particle size of the liquid dispersions. There is also an increase in the total surface area of particles after homogenization, and this leads to better product physical stability.
Impingement
Among the physical methods of cell disruption is impingement, which is one of the options for cell disruption with pressure homogenizers. However, care should be exercised as there is a risk of sheer denaturation of the protein because of the high shear forces when there are high-velocity streams in impingement. Cell-disruption technologies are essential in optimizing protein product recovery, and high-throughput (HT) technologies are now utilized in DSP, including cell disruption, as they are more efficient than traditional laboratory-scale techniques (Tripathi and Shrivastava, 2019, p.15). In the impingement technique, a stream of suspended cells at high pressure and velocity hits other stream of suspended cells or hits a stationary surface. At the point of contact...