Impact of Freezing, Thawing, and Downstream Processing Cycles on Protein Products

 A study of the impact of freezing and thawing cycles on protein products during the downstream processing cycles and the technologies minimize the impact damage to the protein products
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Abstract 
There is an analysis of information on the impact of freezing and thawing cycles on protein products and the impact of downstream processing cycles on the protein products. The information includes technologies minimizing damage to the protein product. There were reviews of past research on freezing and thawing cycles, downstream processing cycles, and impact on protein products. Synthesis and analysis of information provide insights on technologies and techniques that are helpful to reduce damage to protein products during the freezing and thawing cycles and downstream processing cycles. Analysis of physicochemical characteristics and changes after freezing-thawing and downstream processing cycle indicate choosing techniques that are less likely to damage proteins helps maintain protein stability. Various studies highlighted multiple freezing and thawing and downstream processing cycles. Optimizing bioprocessing to reduce protein product degradation and improve their stability requires understanding how different processes affect protein stability. Chromatographic processes are more widely used for initial product capture and bioseparation operations than non-chromatographic processes, but advances in technology have made the latter viable for high-value products. Advances and research in continuous downstream processing have led to better cost-efficiency in processing biopharmaceuticals than conventional processing. Prioritizing improving efficiency in freezing and thawing cycle and downstream processing have improved product quality. Technological advances have enhanced continuous bioprocessing with less variability in protein product quality.
Key Words: Freezing and thawing cycle, downstream processing cycles, protein quality, bioprocessing, biopharmaceuticals, protein-based therapeutics, continuous processes chromatography
Introduction  
Optimizing freezing and thawing events and downstream processing cycles of protein products is necessary to limit and prevent damage to protein products. In bioprocessing production, upstream processing is the production, while purification is downstream processing, and the latter includes protein purification. The freezing and thawing processes are commonly used in biopharmaceutical processing, but the processes damage the quality of proteins compared to room temperatures (Hauptmann et al., 2018, p. 1). This research aims to evaluate the impact of freezing and thawing cycles on protein products during the downstream processing cycle. The research also identifies technologies available to minimize damage to protein products from the freezing and thawing cycles and downstream processing cycles.
When biological materials such as cells, proteins, nucleic acids, tissue culture fluid,  and plant tissues are subjected to the freezing and thawing cycles, there may be changes in the cellular, protein, or DNA structure. The freezing and thawing cycles are used to maintain the stability of biopharmaceutical products and storing process intermediates. Still, potential changes in the chemical and physical properties are one way that influences protein to denature in freezing and thawing. Repeated freezing and thawing can affect optimal activity. Freezing and thawing help ensure manufacturing flexibility and the long-term storage of cells or tissue at low temperatures. Care must be exercised so that freezing does not cause protein destabilization, loss of bioactivity, and damage to the protein molecules (Bhatnagar et al., 2007, p.511).  Figure 1 represents the freeze‐concentrated proteins of different protein sizes and concentrations with -30°C at the level of medium freeze concentration. Figure 2 indicates the variations in ATP-related compounds in frozen T. murphyi samples.The K-value is the ratio of the sum between the ATP-related compound (Hx and HxR) to the sum of ATP-degrading compounds denotes the K-value.
Figure1: Protein size‐dependent freeze concentration based on capillary electrophoresis
Source: Weber, Sitting and Hubbuch, 2021, p. 3919
Figure 2: The K-value of the samples in varying freeze–thaw (F–T) cycles.
Source: Hu and Xie, 2021, p.6
Downstream processing (DSP) is a multistep process leading to purified and homogeneous target products, including proteins. Removing or lowering contaminants to acceptable levels is necessary for recovering and purifying biosynthetic products. Downstream processing is essential in biomanufacturing, biopharmaceutical and biotechnology products can often help minimize costs and maximize product recovery. Freezing and thawing are one of the options for disrupting cells. Integrating innovative downstream processing technology and dealing with potential bottlenecks is helpful to get the desired target products, such as recovered and purified proteins (Singh and Herzer, 2017, p.119). Figure 3 represents a continuous downstream bioprocess beginning with media in a perfusion bioreactor, and impurities are removed to get the desired formulation.
Figure 3: Generic continuous downstream bioprocess
Source Andrew L. Zydney, 2015, p. 466
Critical Review
Protein-based therapeutics
Manufacturing and storage are some of the stresses affecting the manufacturing development of protein-based therapeutics. As such, understanding what affects protein aggregation is helpful, and protein should be stored at optimum temperature for the optimum expression of such proteins. One of the priorities to make protein-based therapeutics safer is minimizing aggregation and helping maintain stability (Jain, Salamat-Miller, and Taylor, 2021, p.2). Optimum conditions for protein-based therapeutics and purified proteins ensure they do not get compromised as the loss of structural integrity affects their therapeutic action. Still, researchers are looking into ways to maintain the efficiency and stability of protein-based therapeutics to expand their use and get the desired therapeutics to release rate. The process of protein stabilisation (or destabilisation) is complex, and mammalian cell systems are the most commonly used in biopharmaceutical proteins. Understanding the mechanisms of recombinant protein expression is potentially helpful to improve target product stability. Proteins get exposed to freezing-thawing events during bioprocessing, but downstream processing and freezing cause complex changes in recombinant therapeutics cells (Tripathi and Shrivastava, 2019, p.2). Recombinant proteins increase the solubility of products, but it is still challenging to purify and optimize them as part of the commercial biopharmaceutical protein processes.
The freezing and thawing cycles even affect the structural changes in frozen foods, such as the myofibril protein secondary structure (Hu and Xie, 2021, p.2). Purification helps improve the efficiency of the downstream process and liming damage to protein products (Brämer et al., 2019, p.7). Research on improving the product quality is essential to enhancing the production process of complex biological products. . It is essential to overcome protein aggregation and denaturation in the production process to maintain protein quality (Zydney, 2016, 465). Figure 4 shows the thermal stability is lower with freezing and thawing, and the reduced protein thermal stability may be because of the intermolecular hydrogen bond breakdown.
Figure 4: Differential Scanning Calorimetry Measurement (DSC) the sample with different freezing–thawing (F–T) cycles.
Source: Hu and Xie, 2021, p. 9
Method
The proposed methodology is reviewing past research, identifying relevant research information, and appraise what is relevant to the research topic. Synthesizing and appraising research will also highlight updated information. Analysing the existing literature is also helpful in collecting useful data and information on the topic and expanding knowledge while contextualizing conclusions. There is better clarification of the problem when there is a synthesis of information. The research choice is based on the research objective and the methodologies' weaknesses and strengths, and weaknesses. Researchers have used different methods and materials to study the impact of freeze-thaw processes and downstream processing. Some of these materials and methods are the preparation of the freeze-thaw treatment, preparation of protein, cell separation, the purification process, stability experiments, protein structure analysis, and analytical methods (Brämer et al., 2019, p. 5; Hu and Xie, 2021, p. 2). Controlling the freezing and thawing process is helpful to evaluate how the FT process affects stability at different concentration levels (Weber, Sittig, and Hubbuch, 2021, p. 3916).
Downstream...