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A thorough knowledge of both the upstream and downstream processes is required to ensure effective removal of residual impurities in biopharmaceuticals.
Detection and removal of residual impurities is a critical and mandatory part of downstream processing (DSP). To find out more about the importance of detecting residual impurities in DSP, the challenges facing scientists when dealing with impurities in DSP, and the potential solutions available to help overcome the difficulties associated with residual impurities, BioPharm International spoke with Olaf Stamm, PhD, technical business development director, Charles River Laboratories, and Luc-Alain Savoy, global head of Biologics, SGS Health and Nutrition.
BioPharm: Why is it important to detect and remove residual impurities in DSP?
Stamm (Charles River Laboratories): The principle of ‘evidence-based medicine’ requires that the API is purified using best practices and scientifically sound technologies. The demonstration of the successful removal of process-/product-related impurities is mandatory. As such, impurities are considered a critical quality attribute, and any impure product would call into question the quality, safety, and efficacy of the compound.
Savoy (SGS Health and Nutrition): Some of these residual impurities are [extremely] toxic; it is therefore essential to remove them to guarantee the safety, the quality, and the efficiency of the drug.
BioPharm: Could you highlight the challenges faced by scientists in relation to residual impurities in DSP?
Savoy (SGS Health and Nutrition): Residual impurities or process-related impurities are compounds that are usually present at [significantly] low concentrations in complex biomanufactured products and can vary from large biological entities to small chemicals. Therefore, their detection and evaluation require extensive knowledge of analytical methodology to cover a large range of products to be monitored.
The objective of DSP is to purify from the bulk [product] made in the upstream process (USP), the active ingredient. Hence, good knowledge of the USP is required to propose a list of impurities to follow, which may simply be
ingredients added in the bulk, but which also may be generated in the bioreactor during the incubation. A good knowledge of the DSP is also essential as residual impurities, such as leachable or solvents, may come from these purification steps.
In summary, the monitoring of residual impurities needs expertise and knowledge of all steps of bioproduction process (upstream and downstream), and in analytical testing.
Stamm (Charles River Laboratories): Host cell proteins (HCP) are an inevitable impurity of biopharmaceuticals, regardless of whether they are produced by recombinant fermentation or extracted from natural sources. Even after multiple sophisticated purification steps, HCPs remain or copurify.
HCPs represent a heterogeneous variety of different proteins that need to be quantified in the drug substance and in intermediates from the downstream purification process. The risk for adverse effects, such as immunogenic reaction, does not necessarily correlate with the amount of certain host cell proteins. That is to say, even small traces of certain ‘high-risk’ HCPs can be highly immunogenic.
BioPharm: Do these challenges differ depending on the therapeutic being developed?
Stamm (Charles River Laboratories): In contrast to products from recombinant protein expression, the majority of advanced therapeutics are facing more challenges with respect to HCP impurities. Gene therapy products consist of multiple viral proteins, which significantly increases the complexity of the therapeutic compared to most biologic drugs that are typically expressed as single proteins in host cells. The gene therapy manufacturing process is associated with additional process related impurities (e.g., serum, bovine albumin, Benzonase, etc.), which are needed to check for contaminating proteins from several very different species.
Additionally, gene therapy products are often manufactured on human cell lines—the proteome of which is far more complex than that of CHO [Chinese hamster ovary] cells. The increased proteomic complexity adds challenges to the development of suitable immunoassays for HCP control for several reasons described below.
First, full-cell lysate needs to be used as an antigen rather than the much less complex cell culture supernatant, because many virus products lyse the host cell or require active lysis of the host cell during the harvest. Second, human HCPs are less immunogenic in standard species, such as rabbits and goats. Third, there is currently no single commercial ELISA [enzyme-linked immunosorbent assay] capable of detecting such a heterogeneous mix. As such, there is much more emphasis on having project-specific assays even at an earlier stage in development. Fourth, gene therapies are mostly fast-tracked by regulators due to the fact that they are typically aimed at treating the most serious illnesses. This adds additional pressure to the early process development activities. If a gene therapy is delayed getting regulatory approval because of inadequate HCP detection, clearance etc., the setback could hold up drug development long enough for a competitor to bring their gene therapy to market first.
Savoy (SGS Health and Nutrition): Yes indeed. The bioproduction of peptides, proteins, oligonucleotides, cell lines, or viruses requires very different USP and DSP; therefore, challenges differ. The monitoring of residual impurities in new therapeutic modalities may be more challenging because of the limited experience we have on their production mode. On the contrary, the manufacturing of a mAb [monoclonal antibody], for example, is now [highly] standardized, and the residual impurities needing to be followed [are] well identified.
BioPharm: What solutions are available for developers to help them overcome the challenges of residual impurities in DSP?
Savoy (SGS Health and Nutrition): The development of online LC–MS [liquid chromatography–mass spectrometry] method to monitor in real time critical quality attributes, such as residual impurities, will allow a better understanding of the manufacturing operation. Moreover, when coupled to a semi-continuous downstream process this will result in consistently shorter processes, an improved control strategy, and a reduction in the number of QC tests needed for batch release.
Stamm (Charles River Laboratories): In the past, the HCP mixture has been a ‘black box’, so to speak. The overall content was measured by a set of polyclonal antibodies, which are raised against an antigen that has been derived from a mock fermentation. The knowledge about the coverage/specificity of this antibody cocktail has historically been very limited and based on the spot pattern comparison of 2D-SDS-PAGE [two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis] and corresponding Western blot.
The 2D gel approach is quite illustrative, but the pattern only provides an approximation of the relative linear coverage as the method is, by definition, focused on linear epitopes due to the use of detergent. The protein spots typically remain unidentified and quantitated.
However, despite the limited intelligence gained on the reagents of the assay, such HCP immunoassays are powerful tools and essential in drug development due to their extreme sensitivity, agnosticism for different matrices, and ease of use and validation in a GMP [good manufacturing practice] environment.
Over the past decade, proteomic tools such as mass spectrometry have become extremely powerful, much easier to use, and far more affordable than ever before. Meanwhile the analysis of several hundred or even several thousand proteins present in one sample has become routine in the hands of experienced CROs [contract research organizations].
Today, mass spectrometry can identify and quantitate each individual HCP present in samples from crude harvest to the highly purified drug substance. This new level of granularity better enables a ‘quality by design’ approach during DSP development with respect to impurities. Knowing identity and concentration of each individual HCP at every step of the purification process enables manufactures to identify the so called ‘hitchhiker’ HCPs. These HCPs co-purify with the drug protein, and it is imperative to determine whether any of them belong to a group of ‘high-risk’ HCPs that are known to cause adverse effects.
By using an immunoaffinity chromatography approach with a mass spectrometry readout, the ‘conformational’ coverage of the antibodies used in a corresponding HCP immunoassay can be determined—this is another way to lighten the ‘black box’.
In contrast to the 2D gel/blot approach, this experiment mimics the physiological conditions of the immunoassay to check the antibody cocktail for potential detection gaps. A detection gap would mean that a high-risk HCP co-purifying with the drug protein would be inadequately recognized by the antibodies present in the immunoassay.
Once such gaps are identified, the immunoassay can be tailored by a targeted approach using peptide immunization to achieve complete coverage for the final-release testing.
BioPharm: Are there any gaps/areas that are lacking perhaps where novel approaches/techniques to help overcome the challenges of residual impurities are required?
Stamm (Charles River Laboratories): As alluded to in the previous question, the novel approaches are available. The tools (mass spec applications) have become standard in the hands of experienced CROs—so much so, in fact, that these projects are typically executed within a few weeks. However, 10 years ago, such work would have filled several PhD thesis projects.
The main gap is in the mind of the manufacturers who are not aware or have not yet fully adopted the advantages of mass spectrometry and improved immunoassays. Leveraging such tools during the early stages of drug development significantly improves process/product quality while reducing the risk of running into unwanted surprises in later stages.
Savoy (SGS Health and Nutrition): The emerging therapies such as cell therapy or gene therapy products are dynamic, heterogeneous, and, hence, very complex entities making them impossible to be fully characterized. Therefore, distinguishing them from an impurity which may be closely related can be very challenging and will require the use of innovative analytical techniques. These techniques will include PCR [polymerase chain reaction]-related assays, NGS [next-generation sequencing], and biological assays. Developing methods to identify and quantify impurities that are distinguishable from the desired product only by their genetic signature is certainly a serious challenge requiring novel approach/techniques.
Felicity Thomas is the European editor for BioPharm International.
Vol. 35, No. 11
Pages: 22–23, 26
When referring to this article, please cite it as F. Thomas, “Removing Residual Impurities,” BioPharm International 35 (11) (2022).