Introduction
Host cell proteins (HCPs) are a significant class of process-related impurities that need to be monitored and adequately removed during bioprocess development (Hogwood, Bracewell, & Smales; X. Wang, Hunter, & Mozier, 2009). Residual HCP impurity in biological drug substance or drug product are generally considered as a critical quality attribute (CQA) due to its potential impact on product quality, safety, and efficacy (FDA, 1997; Guideline, 1999). The impacts can be classified into four main categories: 1) immunogenicity; 2) adjuvant effects; 3) biological activities; and 4) enzymatic activities (Jones et al., 2021; Vanderlaan et al., 2018; X. Wang et al., 2009). Since most of biologics are produced using non-human cell lines, immunogenicity is one of the major concerns caused by residual HCP impurities in biological products if not adequately removed. Immunogenicity can lead to various degrees of adverse effects in patients ranging from more severe cytokine storm, chronic inflammation, hypersensitivity to mild injection site reactions (Reijers et al., 2019). Although the majority of commercialized biologics have proven to be safe with the presence of only trace amount of residual HCPs, there are a few cases where the presence of high level of immunogenic HCPs delayed product development and approval (Vanderlaan et al., 2018). IB1001, a recombinant coagulation factor IX, caused anti-CHO HCP immune response in 30% patients receiving treatment at the time of its clinical development, which resulted in the suspension of two clinical trials of IB1001 by FDA in 2012. Despite the lack of clear correlation to adverse events, the manufacturing process was modified with the addition of hydrophobic interaction chromatography (HIC) step to reduce the level of residual HCPs in IB1001 to < 26 ng/mg from 58500 ng/mg as detected in former process, and the product was later approved by FDA in 2015 with the trade name IXINITY® (Cheung et al., 2016). Lebrikizumab, a humanized IgG4 monoclonal antibody targeting IL-13, was found to contain 242-328 ng/mg CHO phospholipase B-Like 2 (PLBL2) in the clinical material used for a pivotal phase III Lute and Verse clinical trials. Anti-PLBL2 immune response was observed in 76-90% of subjects enrolled in a Pivotal phase III Lute and Verse clinical trials. The trials were later converted to phase IIb studies although the observed immune response has not shown a direct link with clinical adverse effect or an impact to the efficacy of lebrikizumab. Phase III clinical trials done with substantially reduced levels of PLBL2 (0.2-0.4 ng/mg) showed significantly less and dose-dependent frequency of immune response to PLBL2 (Fischer et al.; Hanania et al., 2015). Other than eliciting immune response against themselves, the presence of residual HCPs in biological product can also act as adjuvant. One example is Somatropin Sandoz powder for injection (Covance), a biosimilar recombinant human growth hormone(hGH) of Pfizer’s Genotropin® derived from E. coli . During its development, ~60% of patients enrolled developed anti-hGH antibodies during clinical trials, potentially due to the presence of high levels (~1400 ppm) of ribose phosphate isomerase (RPI) (Vanderlaan et al., 2018). The manufacturing process was later changed to further remove E. coli HCPs and the drug was later approved in 2006 and marketed as Omnitrope® (Romer et al., 2007; Saenger, 2009; Vanderlaan et al., 2018).
Apart from immunogenicity concerns, residual HCPs can also cause serious adverse effects due to their biological activity. For example, the presence of monocyte chemoattractant protein-1 (MCP-1) in a CTLA4-IgG1 fusion protein led to a clinical hold due to serious adverse effects related to histamine release in patients (Vanderlaan et al., 2018). Similarly, presence of an E. coli protein Flagellin in a biological product caused acute toxicity mediated by Toll-like receptor 5 (TRL-5) and resulted in a clinical hold of the product during development (Vanderlaan et al., 2018). In contrast to the relatively rare immunogenicity and biological activity problems related to residual HCPs, residual enzymatic activity caused by insufficient removal of HCPs appears to be quite common. Many HCPs with protease activity can cause product degradation, some of the examples including Adam 19 and Furin (Clarke et al., 2019), Cathepsin D (Bee et al.), and Cathepsin L(Luo et al., 2019). Other than degrading the drug product, certain enzymes could also degrade excipients used in the formulation of biological drugs. Some notable residual enzymes in this category include PLBL2, group XV lysosomal phospholipase A2 (LPLA2), and lipoprotein lipase (LPL) (Chiu et al.; Dixit, Salamat-Miller, Salinas, Taylor, & Basu; Hall, Sandefur, Frye, Tuley, & Huang).
As a CQA, the level of residual HCPs present in final drug substance and drug product often needs to be tested for batch release. It is both an industry-wide common understanding and a regulatory requirement to remove HCPs to acceptable low levels that will not affect product quality, safety, and efficacy (FDA, 1997; Hogwood et al.).
Unlike biologics, small molecule drugs are usually chemically synthesized, and the manufacturing process is typically free of protein residuals. However, with the increased use of biocatalysis in organic synthesis, recombinant enzymes used to catalyze chemical reactions can be introduced into small molecule chemical drugs as a new class of impurities, along with host cell components including HCPs, especially when whole-cell lysate is used as a catalyst (Reetz, 2013). Biocatalysis contributes to a greener pharmaceutical process by 1) use of highly selective enzymes so that protection and deprotection steps associated with chemical synthesis can be reduced or eliminated, hence, reducing the number of process steps and associated E-factors (Kg of waste produced per Kg of product) when compared to chemical synthesis; 2) use of mild conditions and aqueous solutions that reduce the use of hazardous reagents and organic solvents in the reactions; 3) use of enzymes with high selectivity and activity to achieve an excellent stereochemical purity and high conversion rate (Patel, 2006; Reetz, 2013; Woodley, 2008). Recent successful examples include the use of enzyme cascades to produce opioids in yeast utilizing 21-23 enzymes from plants (de Maria & Hollmann, 2015; Galanie, Thodey, Trenchard, Interrante, & Smolke, 2015) and the use of biocatalytic cascade for the manufacture of islatravir, an investigational HIV treatment (Huffman et al., 2019). Although isolated or immobilized enzymes are the ideal candidates for biocatalysis, at times, whole-cell lysate or partially purified enzymes have also been used to maintain enzymatic activity and reduce operational cost.
The analytical control strategy for enzyme and total residual proteins in biocatalytic synthesis of APIs for oral delivery is well summarized in the two papers published by Wells et al. (Wells, Finch, Michels, & Wong, 2012; Wells et al., 2016). However, when the whole-cell lysate is charged in the reaction and the API is used for parenteral dosing, there is an increased risk of immunogenicity if the enzyme and/or HCPs were not adequately removed during the isolation process. One recent example is the use of evolved cyclic GMP-AMP synthase (cGAS) to convert ATP and GTP derivatives to a cyclic dinucleotide API, MK-1454 (John A. McIntosh1*, 2022; Novotna et al., 2019), which binds to Stimulator of Interferon Genes (STING) to initiate a downstream transcription cascade and type I interferon signaling to stimulate the immune surveillance against tumor cells(Lama et al., 2019; Zhou et al., 2018). Since cGAS needs the presence of DNA and other cofactors to be active, enzyme purification or immobilization yields very limited activity. Therefore, whole-cell lysate overexpressing cGAS is used for biocatalysis, which creates additional challenges in process development to remove cell debris, endotoxin, DNA (genomic and plasmid), and host cell proteins. Particularly, traditional chemical approaches using liquid-liquid extraction, solid phase extraction, crystallization, and recrystallization for API isolation and purification are not effective in removing residual HCPs especially when a bulk volume of cell lysate was added to the reaction mixture. As mentioned previously, the presence of residual proteins in small molecule chemical drugs can have a detrimental impact on product quality, safety, and/or efficacy, with a major concern being the immunogenicity of proteins of microbial origin. This is challenging not only in process development but also in analytical development since the methods used for measuring trace amount of residual proteins are not typically used for oral dosage form drugs synthesized by biocatalytic routes. In this article, we will use the residual protein measurement and control in MK-1454 as an example to illustrate the analytical challenges and control strategies for residual HCP control in parenteral dosage form API synthesized using a biocatalytic route.