Quality Controls and Quality Assurance Systems Employed in Downstream Bioprocessing

A STUDY OF THE MAIN QUALITY CONTROLS AND QUALITY ASSURANCE SYSTEMS TYPICALLY EMPLOYED IN DOWNSTREAM BIOPROCESSING TO ENSURE THE MANUFACTURE OF HIGH-QUALITY PROTEIN PRODUCTS ON A CONSISTENT BASIS
Name
I declare that this project is solely the work of the author and is submitted in partial fulfillment of the requirements of the module ‘Fermentation and Cell Culture Processing’.
Abstract
Downstream processing is applied in protein processes to separate, purify, and distillate the previously manufactured drug substance, in this case, protein products, from the intricate bulk matrix. This research paper conducted a systematic review of the main quality controls and quality assurance systems typically employed in downstream bioprocessing to consistently manufacture high-quality protein products. Secondary research entailed identifying and assessing relevant data sources on quality control checks during isolation, purification, concentration, and formulation activities, representing the transition from protein-engineered biocatalysts to therapeutic enzymes ready for human administration. After comparing and analyzing various data sources, the research paper determined that quality control methods are mainly categorized into analytical methods related to purity, identity, safety, strength, and potency of the medicinal product. The study further revealed that each of the five quality control categories entails specific measurements recommended by good manufacturing practices and regulatory bodies. Although most studies determined a strong emphasis on quality control and quality assurance issues from a manufacturing perspective, other critical facets of production, including people, equipment, and manufacturing facilities, tended to be largely ignored.
Acknowledgments
I appreciate the assistance given to me by my tutors and peers. I am also grateful for the words of support and encouragement from family and friends.
Contents
1.0 Abstract………………….……………………………………………….......……………II
2.0 Acknowledgements……………………………………………………..……..........……..III
3.0 Contents…………………………………………………………………………..………..IV
4.0 Introduction…………………………………………………………………...…....……….1
5.0 Literature Review………………………………………………………………...…………2
4.1 Quality Control and Quality Assurance Methods: Identity Testing………...………..3
4.2 Quality Control and Quality Assurance Methods: Purity Testing……………...…….4
4.3 Quality Control and Quality Assurance Methods: Potency Testing…………...……..6
4.4 Quality Control and Quality Assurance Methods: Safety Testing………………...….7
4.5 Quality Control and Quality Assurance Methods: Stability Testing………………….9
4.6 Quality Control and Quality Assurance Methods: Characterization Methods………10
6.0 Analysis……………………………………………………………………………………...11
7.0 Conclusion…………………………………………………………………………………..14
8.0 References…………………………………………………………………………………...15
9.0 Appendices…………………………………………………………………………...……...18
A Study Of The Main Quality Controls And Quality Assurance Systems Typically Employed In Downstream Bioprocessing To Ensure The Manufacture Of High-Quality Protein Products On A Consistent Basis
Introduction
Biopharmaceutical downstream processing entails recovering and purifying drug matters from natural sources, including animal and bacterial cells. It typically denotes the unit operations succeeding achievement of cell growth and expansion and product formation, including drug substance synthesis. Therapeutic enzymes are typically protein-engineered biocatalysts whose intended primary mechanism of action is to modify substrate levels in various clinical applications such as treating metabolic disorders and cancer. A broad selection of therapeutic enzymes is currently manufactured to treat various human illnesses, including enzyme deficiency diseases like Gaucher’s disease and leukemia (Gervais, 2019). The manufacture and testing of these protein-engineered biocatalysts are controlled by regulatory bodies to ensure batch-to-batch consistency. The production process begins with the cell culture phase.
After the protein-engineered biocatalyst has been expressed in a cell culture bioreactor, the target product undergoes downstream processing through vigorous and reproducible protein purification (Doblhoff-Dier and Bliem, 1999). This process is central to certifying the safe delivery of therapeutic medicines. While modern downstream processing technology has greatly improved the downstream development pathway in relation to robustness and production times, rigorous quality control testing and quality assurance systems are necessary to guarantee the medicinal product's purity, identity, safety, strength, and potency. This research paper will conduct a literature review of the quality control and quality assurance systems typically employed in downstream bioprocessing of protein-engineered biocatalysts to consistently manufacture high-quality therapeutic enzymes.
Literature Review
An extensive and systematic literature review of quality control and quality assurance systems employed in manufacturing therapeutic enzymes as protein-engineered biocatalysts revealed that the two quality checks are critical to ensuring batches are fit for human administration. In most biopharmaceutical manufacturing plants, quality control and quality assurance systems are administered by a department of highly-trained personnel. Accurate succinct recording of downstream processes is a central facet of quality control operations: the methods employed for each protein-engineered biocatalyst are pre-established and approved by regulatory bodies (Dawson and Hoare, 2019). These pre-specified downstream processes cannot be changed or modified after product licensure without comprehensive studies prior to supervisory support for the method change. Besides, stipulated specification ranges such as the required protein content range must be observed before the product is used in clinical applications. On the whole, quality control and quality assurance systems rely on reproducible and accurate procedures to ensure batch consistency.
Quality checks are also implemented under the regulations of good manufacturing practice: this means that besides the production specifications previously stated, the quality control department must be equipped with trained and qualified personnel capable of appraising downstream processing according to specified and ratified standard operating procedures. The quality control personnel are also responsible for ensuring that production methods are used as properly documented and that suitable controls exist to avert mix-ups during testing of process stages or diverse protein-engineered biocatalysts. Quality assurance in the manufacture of therapeutic enzymes also includes investigation of any deviations in manufacturing practice, over and above, improvement of production practices (Mandenius, 2021). Quality control of analytical test samples involves proper documentation of chain-of-custody and clear identification as well as traceability of the same. The unit dedicated to administering quality control and quality assurance systems functions under the all-encompassing supervision of the Quality Management System aimed at certifying that stipulated manufacturing procedures are observed and that all documentation is current and organized.
The procedures used in quality control testing of protein-based therapeutic enzymes are based on their capacity to answer the basic questions of efficacy and safety. However, quality control methods can be further categorized into analytical methods related to the medicinal product's purity, identity, safety, strength, and potency. The federal agency regulating therapeutic enzymes, the Food and Drug Administration, classifies quality control methods into several groups, including identity, potency, purity, safety, and potency. This literature review focused on these forms of quality control and quality assurance. Quality control approaches typically rely on comparing test samples against a reference standard: manufactured batches are only released into the market once they have met the stipulated acceptance criteria for each quality control method (Su et al., 2019). The reference standard is usually developed from a standard manufacturing batch of the protein-engineered biocatalyst and characterized using various analytical methods more comprehensive than those applied for regular product release.
For instance, the aggregate profile of each standard manufactured batch may be evaluated using size-exclusion chromatography (SEC). Still, the batch used as a reference standard is analyzed using SEC in addition to other orthogonal techniques like the analytical ultracentrifugation (AUC). This additional analysis ensures that the reference standard is of the highest level of quality and, therefore, suitable for comparing against batches intended for market release. Good manufacturing practice protocols require that the reference standard be kept under specific conditions and monitored for stability at specified time intervals.
Quality Control and Quality Assurance Methods: Identity Testing
Identity testing of protein-engineered biocatalysts is generally performed on manufactured batches to determine if the product constitutes a suitable therapeutic enzyme. Identity tests determine if the active ingredient in the batch is the correct enzyme using a pass or fail criteria according to an established measurement suitable for analyzing and validating the enzyme. One common method of identifying the active ingredients of therapeutic enzyme batches is the peptide mapping method, often in conjunction with mass spectrometry. Proteolysis of the product enzyme is conducted under specific conditions to map the enzyme amino acid and establish the proteolytic cut sites (Thakur and Rathore, 2021). The subsequent peptide digest is separated and quantified using a process similar to Reversed-Phase High-Pressure Liquid Chromatography (RP-HPLC). Each protein tends to give a unique peptide signature in terms of HPLC retention times, and therefore the identity of the active ingredient can be easily known. RP-HPLC, along with mass spectrometry, is usually used to identify the constituent peptides. In quality control environments, the unidentified peptides are equated to an established reference standard in order to determine peptide similarity.
A threshold is set to determine whether or not the result is positive or negative for the required ingredient. The ultraviolet (UV) absorbance peptide is particularly useful in sequencing chromatograms for protein-engineered biocatalysts (Gervais, 2019). Other diagnostic techniques such as peptide mapping using MS/MS and LC/MS, N-terminal sequence, and western blot are plausible identity test methods that also use a reference standard to confirm proof of protein integrity.
Quality Control and Quality Assurance Methods: Purity Testing
Another critical quality control method is testing for contaminating substances, whether derived from production or product-related. The existence of substantial amounts in protein-engineered biocatalysts adversely impact their efficacy in vivo or result in undesirable and possibly severe side effects. Before the therapeutic enzyme product is declared fit for clinical applications, it is essential that a purity analysis test be conducted to verify the purity of the target analyte. Product-related impurities tend to result from the degradation of the target enzyme, such as truncated or cropped forms of the therapeutic enzyme. Enzyme deviations containing post-translational alterations like glutamine deamidation, methionine oxidation, and changes to glycosylation patterns are other examples of product-related impurities. On the other hand, process-related impurities result from elements introduced during manufacturing but are required to be eliminated from the downstream processing stream to suitable levels (Bielow, Mastrobuoni and Kempa, 2015). These include host cell proteins (HCP) as well as other non-target enzymes such as antifoaming agents, antibiotics, cellular expression platforms, and leachates. Process validation is often recommended for impurity removal and constitutes routine final product testing to validate or risk-evaluating the elimination of specific elements.
The particular definition of impurities varies considerably depending on the specific therapeutic enzyme product. For instance, post-translationally altered forms of the target enzyme are regarded as product-related if certain conditions are met. One such condition is the presence of modified forms appearing in a particular ratio when compared to the unmodified product on a batch-batch basis. Some therapeutic enzyme products are satisfactory if they are of one chemical composition with a similar post-translational alteration profile. In some cases, batches with various variants are altogether regarded as acceptable. Some enzymes have been shown to have the same action and features as the wild-type equivalent, and therefore, if present, they can be thought of as part of the product. High-performance liquid chromatography (HPLC) is the most used purity testing technique owing to its transferability of equipment and methods across a broad array of column chemistries and purposes. The method is also easy to validate, thereby easing regulatory compliance. Various forms of HPLC are employed in protein synthesis, although protein analysis usually depends on charge variants (Gervais, 2019). Another purity testing method is the RP-HPLC technique used in identification analysis, where the presence and characteristics of glycosylation moieties in the enzyme are determined and analyzed. However, HPLC is more accurate when testing for impurities introduced during manufacturing, including antifoam, to reduce the same to stipulated threshold values.
Another method used in biopharmaceutical purity testing is the comparatively low-resolution method, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), to ascertain the in-process quantity of non-target protein impurities. However, gel electrophoresis has supplanted SDS-PAGE in modern quality control units. Other purity test methods that have seen an uptake in recent years are capillary isoelectric focussing (usually employed for protein charge variants) and capillary zone electrophoresis. Another critical element in impurity measurements is the valuation of protein-protein aggregates that tend to occur in pharmaceutical compositions of proteins and enzymes. While a small quantity of these protein-protein aggregates is tolerable, higher amounts of the same usually increase or amplify adverse immunological reactions in patients or even lead to the development of anti-drug antibodies that diminish the efficacy of the drug (Roch and Mandenius, 2016). One purity testing method used to determine the amount of protein-protein aggregation is the size-exclusion chromatography (SEC). The method is relatively easy to use, reproducible, and usually require similar equipment to other HPLC methods.
However, the technique requires that interaction between analyte protein and resin matrix surface be restricted since the variance in the movement of proteins through the column is driven primarily by the link between the pore arrangements of the column matrix and the dimension of each protein class. When using the SEC technique, the type of column matrix is critical since particular proteins (especially proteins with a high isoelectric charge) tend to bind to specific chromatography strains containing a negative surface charge, thereby producing artifacts in analysis. Tied to the issue of impurities in therapeutic enzymes related to quality control and quality assurance systems in the manufacture of protein-engineered biocatalysts is that of residual DNA. The federal agency, FDA, has stipulated tolerable levels of lingering DNA in biological products (Fekete et al., 2015). This regulatory oversight is founded on the theoretical dangers of specific nucleic acid classifications causing health problems to the patient receiving the therapeutic enzyme. The downstream processing of therapeutic enzymes must be capable of achieving the residual levels specified by the FDA. Manufacturing plants must also possess the analytical abilities to measure residual levels in the final product. The most applied DNA testing technique in the quality control environment is the quantitative polymerase chain reaction.
In addition to testing for residual DNA, it is recommended that residual levels of host-cell proteins not measurable by typical methods such as SD-PAGE be assessed and demonstrated to be within the levels required by FDA. The residual levels of host-cell proteins in finished pharmaceutical drugs are usually within the range of ng HCP per mg of biology-engineered biocatalysts. Higher levels than these tend to result in adverse immunological responses or even toxicity in the patient. Some profound measurement methods used in quality control for host-cell proteins detection and estimation include the ELISA or enzyme-linked immunosorbent assay. The antibodies used in the ELISA technique are crucial to the success of the quality control test. Consequently, it is essential during the evolution of HCP ELESA to show that the polyclonal antibodies employed in the assay are capable of ensuring sufficient analysis of the expression entity’s proteome (Känsäkoski et al., 2006). Regulatory expectation has increasingly preferred the application of 2D-gel electrophoresis and western blotting to evaluate antibody coverage. Mass spectrometry is the other non-ELISA technique used in host-cell proteins assessment.