Final Drug Substance Research Articles

Beyond viability: Advancing CHO cell culture process strategies to modulate host cell protein levels

Chinese Hamster Ovary (CHO) cells are widely utilized in bioprocessing industry for monoclonal antibody (mAb) production. In many instances, challenges persist in achieving sufficient clearance of Host Cell Proteins (HCPs) in the final drug substance. While purification strategies usually offer substantial HCP clearance, certain "problematic" HCPs, particularly lipases, continue to pose significant challenges. This study investigates the accumulation of various "problematic" HCPs in CHO cell culture using transcriptomics, revealing correations between cell culture parameters and HCP level. Contrary to conventional assumptions, viability alone does not reliably predict HCP levels, with factors such as clone selection, host cell line choice, media and feed compositions significantly influencing HCP accumulation. Leveraging transcriptomics-based approaches, we demonstrate the potential of upstream process control strategies to mitigate HCP presence and improve biologic product quality. Our findings underscore the importance of considering diverse cell culture parameters in bioprocess optimization to ensure product stability and quality. While promising, further research is needed to elucidate the mechanisms underlying HCP release and propagation through downstream processing stages, emphasizing the necessity of a comprehensive integrated approach to HCP control in biologics production.

Synthesis and Characterization of Chiral Impurities of Rivaroxaban, Used as an Anticoagulant Drug

The mechanism of action of the anticoagulant medication rivaroxaban is the direct and specific suppression of activated coagulation factor X (FXa). With just (S)-Rivaroxaban exhibiting pharmacological activity and (R)-Rivaroxaban being an impurity that must be controlled, this molecule only has one chiral centre. The present work describes the synthesis of chiral impurities of each intermediate, including KSM and its drug substance, and their characterization by spectral data (IR, MS, 1H-NMR, and 13C-NMR). The chiral impurity of drug substance is challenging during the process development of Rivaroxaban, and it is challenging for all pharmaceutical industries as well. The opposite isomeric impurity is introduced form its key starting material and it follows the same reaction mechanism path up to final drug substance. This work will help quality control (QC) to identify and control these impurities in the Rivaroxban drug substance as per ICH guideline.

Factors affecting product association as a mechanism of host-cell protein persistence in bioprocessing.

Product association of host-cell proteins (HCPs) to monoclonal antibodies (mAbs) is widely regarded as a mechanism that can enable HCP persistence through multiple purification steps and even into the final drug substance. Discussion of this mechanism often implies that the existence or extent of persistence is directly related to the strength of binding but actual measurements of the binding affinity of such interactions remain sparse. Two separate avenues of investigation of HCP-mAb binding are reported here. One is the measurement of the affinity of binding of individual, commonly persistent Chinese hamster ovary (CHO) HCPs to each of a set of mAbs, and the other uses quantitative proteomic measurements to assess binding of HCPs in a null CHO harvested cell culture fluid (HCCF) to mAbs produced in the same cell line. The individual HCP measurements show that the binding affinities of individual HCPs to different mAbs can vary appreciably but are rarely very high, with only weak pH dependence. The measurements on the null HCCF allow estimation of individual HCP-mAb affinities; these are typically weaker than those seen in affinity measurements on isolated HCPs. Instead, the extent of binding appears correlated with the initial abundance of individual HCPs in the HCCF and the forms of the HCPs in the solution, i.e., whether HCPs are present as free molecules or as parts of large aggregates. Separate protein A chromatography experiments performed by feeding different fractions of a mAb-containing HCCF obtained by size-exclusion chromatography (SEC) showed clear differences in the number and identity of HCPs found in the protein A eluate. These results indicate a significant role for HCP-mAb association in determining HCP persistence through protein A chromatography, presumably through binding of HCP-mAb complexes to the resin. Overall, the results illustrate the importance of considering more fully the biophysical context of HCP-product association in assessing the factors that may affect the phenomenon and determine its implications. Knowledge of the abundances and the forms of individual or aggregated HCPs in HCCF are particularly significant, emphasizing the integration of upstream and downstream bioprocessing and the importance of understanding the collective properties of HCPs in addition to just the biophysical properties of individual HCPs.

Chemical and Genetic Modulation of Complex I of the Electron Transport Chain Enhances the Biotherapeutic Protein Production Capacity of CHO Cells.

Chinese hamster ovary (CHO) cells are the cell line of choice for producing recombinant therapeutic proteins. Despite improvements in production processes, reducing manufacturing costs remains a key driver in the search for more productive clones. To identify media additives capable of increasing protein production, CHOZN® GS-/- cell lines were screened with 1280 small molecules, and two were identified, forskolin and BrdU, which increased productivity by ≥40%. While it is possible to incorporate these small molecules into a commercial-scale process, doing so may not be financially feasible or could raise regulatory concerns related to the purity of the final drug substance. To circumvent these issues, RNA-Seq was performed to identify transcripts which were up- or downregulated upon BrdU treatment. Subsequent Reactome pathway analysis identified the electron transport chain as an affected pathway. CRISPR/Cas9 was utilized to create missense mutations in two independent components of the electron transport chain and the resultant clones partially recapitulated the phenotypes observed upon BrdU treatment, including the productivity of recombinant therapeutic proteins. Together, this work suggests that BrdU can enhance the productivity of CHO cells by modulating cellular energetics and provides a blueprint for translating data from small molecule chemical screens into genetic engineering targets to improve the performance of CHO cells. This could ultimately lead to more productive host cell lines and a more cost-effective method of supplying medication to patients.

Biocatalytic cascade process of islatravir: Analytical and regulatory control strategy of minor enantiomer

Commercial process of islatravir (MK-8591, EFdA) utilizes biocatalytic cascade reactions to construct the ribose moiety of the molecule which bears three chiral centers. However, this biocatalytic process also brought analytical challenges where all stereoisomers and process related compounds are controlled in one isolated intermediate, the final drug substance. A chiral LC method was developed to resolve all those compounds from islatravir and its minor enantiomer by thorough column screening and careful optimization. Detail of designing key method validation components such as method linearity, precision and robustness is discussed, and their results were presented. The method was successfully validated to fulfill various expectation from each individual health authority including FDA, EMA, PMDA, and ANVISA.

Characterisation of the E. coli HMS174 and BLR host cell proteome to guide purification process development.

Mass-spectrometry-based proteomics is increasingly employed to monitor purification processes or to detect critical host cell proteins in the final drug substance. This approach is inherently unbiased and can be used to identify individual host cell proteins without prior knowledge. In process development for the purification of new biopharmaceuticals, such as protein subunit vaccines, a broader knowledge of the host cell proteome could promote a more rational process design. Proteomics can establish qualitative and quantitative information on the complete host cell proteome before purification (i.e., protein abundances and physicochemical properties). Such information allows for a more rational design of the purification strategy and accelerates purification process development. In this study, we present an extensive proteomic characterisation of two E.coli host cell strains widely employed in academia and industry to produce therapeutic proteins, BLR and HMS174. The established database contains the observed abundance of each identified protein, information relating to their hydrophobicity, the isoelectric point, molecular weight, and toxicity. These physicochemical properties were plotted on proteome property maps to showcase the selection of suitable purification strategies. Furthermore, sequence alignment allowed integration of subunit information and occurrences of post-translational modifications from the well-studied E.coli K12 strain.

Use of ultrafiltration/diafiltration for the processing of antisense oligonucleotides.

Ultrafiltration/diafiltration (UF/DF) has been the hallmark for concentrating and buffer exchange of protein and peptide-based therapeutics for years. Here we examine the capabilities and limitations of UF/DF membranes to process oligonucleotides using antisense oligonucleotides (ASOs) as a model. Using a 3 kDa UF/DF membrane, oligonucleotides as small as 6 kDa are shown to have low sieving coefficients (<0.008) and thus can be concentrated to high concentrations (≤200 mg/mL) with high yield (≥95%) and low viscosity (<15 centipoise), provided the oligonucleotide is designed not to undergo self-hybridization. In general, the oligonucleotide should be at least twice the reported membrane molecular weight cutoff for robust retention. Regarding diafiltration, results show that a small amount of salt is necessary to maintain adequate flux at concentrations exceeding about 40 mg/mL. Removal of salts along with residual solvents and small molecule process-related impurities can be robust provided they are not positively charged as the interaction with the oligonucleotide can prevent passage through the membrane, even for common divalent cations such as calcium or magnesium. Overall, UF/DF is a valuable tool to utilize in oligonucleotide processing, especially as a final drug substance formulation step that enables a liquid active pharmaceutical ingredient.

Predicting Formulation Conditions During Ultrafiltration and Dilution to Drug Substance Using a Donnan Model with Homology-Model Based Protein Charge

In the manufacturing of therapeutic monoclonal antibodies (mAbs), the final steps of the purification process are typically ultrafiltration/diafiltration (UF/DF), dilution, and conditioning. These steps are developed such that the final drug substance (DS) is formulated to the desired mAb, buffer, and excipient concentrations. To develop these processes, process and formulation development scientists often perform experiments to account for the Gibbs-Donnan and volume-exclusion effects during UF/DF, which affect the output pH and buffer concentration of the UF/DF process. This work describes the development of an in silico model for predicting the DS pH and buffer concentration after accounting for the Gibbs-Donnan and volume-exclusion effects during the UF/DF operation and the subsequent dilution and conditioning steps. The model was validated using statistical analysis to compare model predictions against experimental results for nine molecules of varying protein concentrations and formulations. In addition, our results showed that the structure-based in silico approach used to calculate the protein charge was more accurate than a sequence-based approach. Finally, we used the model to gain fundamental insights about the Gibbs-Donnan effect by highlighting the role of the protein charge concentration (the protein concentration multiplied with protein charge at the formulation pH) on the Gibbs-Donnan effect. Overall, this work demonstrates that the Gibbs-Donnan and volume-exclusions effects can be predicted using an in silico model, potentially alleviating the need for experiments.

Host cell protein identification in monoclonal antibody high molecular weight species

High molecular weight (HMW) species are product-related variants that may impact therapeutic product safety and efficacy. Therefore, HMW species and aggregates are considered critical quality attributes and their levels should be closely monitored and controlled during drug development, commercial manufacturing, and shelf-life storage period for therapeutic monoclonal antibody drug products. Various biophysical and analytical methods have been developed to characterize the HMW species to understand their mechanisms of formation and assess potential product risk. However, host cell protein (HCP) analysis has seldom been conducted to characterize the impurities in aggregates. In this work, HCP analysis of enriched HMW species and drug substance (DS) from five different monoclonal antibodies (mAbs) was performed. More HCPs are identified in the enriched HMW than in the DS, thus demonstrating a potential interaction between HCPs and HMW. Certain HCPs, including commonly detected HCPs and problematic HCPs, were enriched in HMW fractions. Especially, the most abundant HCP from mAb1, CC motif chemokine, was 46 times more abundant in enriched HMW than DS. The enriched HMW was further fractionated into enriched dimers and enriched very HMW (vHMW) fractions. The CC motif chemokine was found to interact mainly with mAb1 dimer species rather than vHMW fraction. Removing the HMW species from mAb1 significantly decreased the CC motif chemokine level in the final mAb1 DS. Our findings demonstrate that some HCPs are more preferentially bound to HMW species and this finding may provide a new opportunity for removing HCPs in downstream purification steps.

Immunogenicity Risk Assessment of Spontaneously Occurring Therapeutic Monoclonal Antibody Aggregates

Aggregates of therapeutic proteins have been associated with increased immunogenicity in pre-clinical models as well as in human patients. Recent studies to understand aggregates and their immunogenicity risks use artificial stress methods to induce high levels of aggregation. These methods may be less biologically relevant in terms of their quantity than those that occur spontaneously during processing and storage. Here we describe the immunogenicity risk due to spontaneously occurring therapeutic antibody aggregates using peripheral blood mononuclear cells (PBMC) and a cell line with a reporter gene for immune activation: THP-1 BLUE NFκB. The spontaneously occurring therapeutic protein aggregates were obtained from process intermediates and final formulated drug substance from stability retains. Spontaneously occurring aggregates elicited innate immune responses for several donors in a PBMC assay with cytokine and chemokine production as a readout for immune activation. Meanwhile, no significant adaptive phase responses to spontaneously occurring aggregate samples were detected. While the THP-1 BLUE NFκB cell line and PBMC assays both responded to high stress induced aggregates, only the PBMC from a limited subset of donors responded to processing-induced aggregates. In this case study, levels of antibody aggregation occurring at process relevant levels are lower than those induced by stirring and may pose lower risk in vivo. Our methodologies can further inform additional immunogenicity risk assessments using a pre-clinical in vitro risk assessment approach utilizing human derived immune cells.

Improved and ‘Nitrosamines Free’ Process for the Preparation of an α4β2 Neuronal Nicotinic Acetylcholine Receptor Agonist-Varenicline Tartrate

An improvised and efficient approach for synthesis of α4β2 nicotinic acetylcholine receptor subtype agonist, Varenicline tartrate (1) free from the ‘N-nitrosamines’ has been described. The approach involves an improved process for a key intermediate (7,8-dinitro-4,5-dihydro-1H-1,5-methanobenzo[d] azepin-3(2H)-yl)-2,2,2-trifluoro ethanone (7) free from potential genotoxic impurities. Compound 7 is converted into Varenicline base 10 in a single pot process with improved overall yield and quality. Further, Varenicline base 10 is converted into Varenicline tartrate (1) by acid addition salt which provides in quantitative yield. Our improved process consists of technical innovations/improvements which eliminate the probability for the formation of critical impurities such as dinitro nitroso impurity 2, diamino nitroso impurity 3 and varenicline nitroso impurity 4 and other genotoxic impurities such as mono nitro impurity 11 and meta dinitro impurity 12 in the final drug substance and provides ‘Nitrosamines free’ varenicline tartrate (1) with good quality and yield.

Synthesis and characterization of impurities of Apixaban drug substance—An anticoagulant

AbstractApixaban is an anticoagulant used to treat venous thromboembolic disorders. The present work describes the origin and synthesis of related substances of Apixaban PG ester‐1 and PG ester‐2. These impurities arise in the process of Apixaban drug substance, later these impurities are transformed into the final Apixaban drug substance. These novel synthesized impurities were characterized by 1H NMR, 13C NMR, HPLC, and HRMS analysis.

Modeling the Gibbs–Donnan effect during ultrafiltration and diafiltration processes using the Poisson–Boltzmann theory in combination with a basic Stern model

The Gibbs–Donnan effect is a well-known phenomenon causing ions to be distributed unevenly across semi-permeable membranes that are permeable to ions but not to larger macromolecules such as proteins. In protein ultrafiltration and diafiltration (UF/DF) processes, this effect often leads to discrepancies between the pH and excipient concentrations in the final drug substance and in the DF buffer. In this work, a model describing the retentate and permeate composition throughout combined UF/DF processes is introduced. The model accounts for volume exclusion effects and electrostatic interactions between ions and the protein based on the Poisson–Boltzmann theory in combination with a basic Stern model. Advantages and limitations of the proposed model were analyzed using UF/DF experiments with multiple diafiltration buffers and proteins. A comparison between simulated and experimental permeate data showed good agreement for low to moderate Donnan potentials but model limitations for high Donnan potentials at protein concentration larger 100 gL -1 . In contrast, simulated retentate data showed good agreement for both low and high Donnan potentials and for protein concentrations up to 190 gL -1 . It was demonstrated that in this high protein concentration regime, the applied basic Stern model provides more accurate predictions compared to previous theories based on the Poisson–Boltzmann theory alone. This makes the model a valuable tool to describe discrepancies between pH and excipient concentrations in the final drug substance and DF buffer for highly concentrated protein formulations. As model predictions are based solely on structural information on the protein and the composition of the DF buffer, the model is particularly beneficial at an early stage in process development to streamline process development and improve process understanding. • Introduction of a model to describe the Gibbs–Donnan effect in UF/DF processes. • Advanced implementation of the cell model approximation. • Calibration solely based on structural information on the protein. • Validation using various proteins (up to 190 g/L) and buffer systems.

Post-Column Denaturation-Assisted Native Size-Exclusion Chromatography-Mass Spectrometry for Rapid and In-Depth Characterization of High Molecular Weight Variants in Therapeutic Monoclonal Antibodies.

The high molecular weight (HMW) size variants present in therapeutic monoclonal antibody (mAb) samples need to be closely monitored and characterized due to their impact on product safety and efficacy. Because of the complexity and often low abundances in final drug substance (DS) samples, characterization of such HMW species is challenging and traditionally requires offline enrichment of the HMW species followed by analysis using various analytical tools. Here, we report the development of a postcolumn denaturation-assisted native SEC-MS method that allows rapid and in-depth characterization of mAb HMW species directly from unfractionated DS samples. This method not only provides high-confidence identification of HMW complexes based on accurate mass measurement of both the intact assembly and the constituent subunits but also allows in-depth analysis of the interaction nature and location. In addition, using the extracted ion chromatograms, derived from high-quality, native-like mass spectra, the elution profiles of each noncovalent and/or nondissociable complex can be readily reconstructed, facilitating the comprehension of a complex HMW profile. The utility of this novel method was demonstrated in different applications, ranging from enriched HMW characterization at late stage development, comparability assessment due to process changes, and forced degradation study of coformulated mAbs. As this method does not require prior enrichment, it is thus desirable for providing both rapid and in-depth characterization of HMW species during the development of therapeutic mAbs.

Nitrosamine impurities: from raw materials to final drug product.

Nitrosamine impurities: from raw materials to final drug product.

Toward unbiased identification and comparative quantification of host cell protein impurities by automated iterative LC–MS/MS (HCP-AIMS) for therapeutic protein development

Host cell proteins (HCPs) are process-related impurities expressed by the host cells used for production of therapeutic proteins. Although an extensive purification process removes most of the HCPs, residual HCPs are commonly present in protein therapeutics. If not well-controlled, certain HCPs may be present in the product, impacting drug stability, and potentially affecting product safety leading to safety risks for patients. Therefore, as a critical quality attribute, the levels of HCPs must be closely monitored during drug development and determined in the final drug substance at release. Liquid chromatography-mass spectrometry (LC–MS) as an orthogonal approach to traditional ELISA for HCP analysis has shown tremendous value in drug process development and analytical characterization by providing additional critical information, such as protein identity and relative abundance of individual HCPs. To meet the challenges in HCP analysis during drug development, especially downstream process development, which entails fast turnaround time and robustness while identifying high level of HCPs and their clearance trend for further purification development, we have developed HCP-automated iterative MS (HCP-AIMS): it is a simple, automated and robust HCP analysis workflow with deep and unbiased identification and relative quantification capability. This HCP-AIMS approach only requires easy direct digestion of the samples without enrichment or pre- treatment. With the fully automated precursor ion exclusion in MS/MS mode, low abundance HCP peptides could be selected for MS/MS analysis in iterative replicates, and therefore, the identification of HCPs at low abundance can be achieved. Using an in-house mAb with various levels of spiked-in HCPs as well as the NIST mAb, we were able to achieve unbiased identification and quantitation of HCPs as low as 10 ppm level. Furthermore, robustness of the HCP-AIMS approach was also confirmed for the feasibility of large-scale and high-throughput analysis.

Recombinant protein vaccines against SARS-CoV-2

Recombinant protein vaccines against SARS-CoV-2

Identification and characterization of a residual host cell protein hexosaminidase B associated with N-glycan degradation during the stability study of a therapeutic recombinant monoclonal antibody product.

Host cell proteins (HCPs) are process‐related impurities derived from host organisms, which need to be controlled to ensure adequate product quality and safety. In this study, product quality attributes were tracked for several monoclonal antibodies (mAbs) under the intended storage and accelerated stability conditions. One product quality attribute not expected to be stability indicating is the N‐glycan heterogeneity profile. However, significant N‐glycan degradation was observed for one mAb under accelerated and stressed stability conditions. The root cause for this instability was attributed to hexosaminidase B (HEXB), an enzyme known to remove terminal N‐acetylglucosamine (GlcNAc). HEXB was identified by liquid chromatography–mass spectrometry (LC–MS)‐based proteomics approach to be enriched in the impacted stability batches from mAb‐1. Subsequently, enzymatic and targeted multiple reaction monitoring (MRM) MS assays were developed to support process and product characterization. A potential interaction between HEXB and mAb‐1 was initially observed from the analysis of process intermediates by proteomics among several mAbs and later supported by computational modeling. An improved bioprocess was developed to significantly reduce HEXB levels in the final drug substance. A risk assessment was conducted by evaluating the in silico immunogenicity risk and the impact on product quality. To the best of our knowledge, HEXB is the first residual HCP reported to have impact on the glycan profile of a formulated drug product. The combination of different analytical tools, mass spectrometry, and computational modeling provides a general strategy on how to study residual HCP for biotherapeutics development.

Introduction and clearance of beta-glucan in the downstream processing of monoclonal antibodies.

β‐Glucan process‐related impurities can be introduced into biopharmaceutical products via upstream or downstream processing or via excipients. This study obtained a comprehensive process‐mapping dataset for five monoclonal antibodies to assess β‐glucan introduction and clearance during development and production runs at various scales. Overall, 198 data points were available for analysis. The greatest β‐glucan concentrations were found in the depth‐filtration filtrate (37–2,745 pg/ml). Load volume correlated with β‐glucan concentration in the filtrate, whereas flush volume was of secondary importance. Cation‐exchange chromatography significantly cleared β‐glucans. Furthermore, β‐glucan leaching from the Planova 20N virus removal filter was reduced by increasing the flush volume (1 vs. 10 L/m2). β‐glucan concentrations after filter flush with 10 L/m2 were consistently <10 pg/ml. No or only limited β‐glucan clearance was attained via ultrafiltration/diafiltration (UF/DF). However, during the first run with monoclonal antibody (mAb) 4, β‐glucan concentration in the UF/DF retentate was 10.8 pg/mg, potentially due to β‐glucan leaching from the first run with a regenerated cellulose membrane. Overall, β‐glucan levels in the final mAb drug substance were 1–12 pg/mg. Assuming high doses of 1,000–5,000 mg, a β‐glucan contamination at 20 pg/mg would translate to 20–100 ng/dose, which is below the previously suggested threshold for product safety (≤500 ng/dose).

Measuring Lipolytic Activity to Support Process Improvements to Manage Lipase-Mediated Polysorbate Degradation.

Polysorbates are critical stabilizers in biopharmaceutical protein formulations. However, they may degrade in drug substance (DS) or drug product (DP) during storage. Degradation catalyzed by lipases present in host cell proteins (HCPs) is one suspected root cause. The purpose of this study was to develop an assay to detect lipolytic activity in biopharmaceutical DS and DP formulations. The assay is based on the hydrolysis of the lipase substrate 4-methylumbelliferyl oleate to yield the fluorescent product 4-methylumbelliferone. First, the assay components and their concentrations (buffer salts and pH, solvent and inhibitor Orlistat) were established and optimized using a model lipase (Porcine pancreatic lipase) and cell culture harvest fluid that exhibited lipolytic activity. The assay was then successfully applied and thereby qualified in protein formulations and at lipase concentrations possibly encountered in actual biopharmaceutical DS and DP formulations. The lipase assay can be used to detect lipolytic activity in intermediate and final DS, for example during process optimization in downstream purification, to better and specifically reduce the level, or deplete, lipases from HCPs. The assay is also suitable to be applied during root cause investigations related to polysorbate degradation in biopharmaceutical DP.