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.