Host cell proteins in monoclonal antibody processing: Control, detection, and removal

Abstract

Host cell proteins (HCPs) are process-related impurities in a therapeutic protein expressed using cell culture technology. This review presents biopharmaceutical industry trends in terms of both HCPs in the bioprocessing of monoclonal antibodies (mAbs) and the capabilities for HCP clearance by downstream unit operations. A comprehensive assessment of currently implemented and emerging technologies in the manufacturing processes with extensive references was performed. Meta-analyses of published downstream data were conducted to identify trends. Improved analytical methods and understanding of “high-risk” HCPs lead to more robust manufacturing processes and higher-quality therapeutics. The trend of higher cell density cultures leads to both higher mAb expression and higher HCP levels. However, HCP levels can be significantly reduced with improvements in operations, resulting in similar concentrations of approx. 10 ppm HCPs. There are no differences in the performance of HCP clearance between recent enhanced downstream operations and traditional batch processing. This review includes best practices for developing improved processes.

Introduction

Monoclonal antibodies (mAbs) have had the highest number of health authority approvals in recent years and currently constitute the most important class of recombinant protein therapeutics. The majority of mAb manufacturing processes use Chinese Hamster Ovary (CHO) cell lines to express the biotherapeutic and recover it from harvested (clarified) cell culture fluid (HCCF). Host cell protein impurities (HCPs, frequently referred to as CHOP) are secreted into the HCCF from viable cells and released through cell lysis over the course of culture and harvest.^1-3 The amount and components of HCPs in HCCF vary depending on the clone selected, mAb expressed, bioreactor types, and culture conditions.

HCPs are process-related impurities that originate from cells expressing mAbs,⁴ and can include thousands of unique proteins with widely varied concentrations, molecular weights, isoelectric points (pI), and surface charges.^5-7

HCPs are classified as a critical quality attribute (CQA) in therapeutic mAb manufacturing. Many HCPs are essential for key metabolic pathways of the cell but can be a risk to patient safety at parts per million (ppm) concentrations. They can cause an immune response in immunocompromised patients (immunogenicity) or by being biologically active. They can alter mAb stability or solubility and can enzymatically attack a mAb therapeutic to alter its potency.^8, 9 While most HCPs exhibit no enzymatic activity, some HCP components may show lipase,¹⁰ glycosidase, or protease behavior in the enzymatic breakdown of a mAb¹ and possibly promote aggregation in the mAb formulation with a consequent impact on shelf life.¹¹

Health authorities (e.g., the U.S. Food and Drug Administration [FDA] and European Medical Agency [EMA]) do not provide written guidance on specific acceptable levels of aggregate HCPs in the final drug product to be injected into patients. However, there is an informal empirical target level of HCPs as <100 ppm, or <100 ng/mg product.^12, 13 From the recent development in HCP analysis technologies and immunogenicity assays, the limit of 100 ppm is inadequate to ensure drug stability and patient safety.¹⁴ The accepted level of HCPs in a final product is determined by risk assessment and depends on multiple factors, including the concentration, therapeutic dose, frequency of drug administration, type of drug, and severity of the disease.^7, 14 Accordingly, ensuring that the product is safe and efficacious requires a manufacturing process capable of robust HCP control and removal such that the final product has a level of HCPs that neither influences product quality nor compromises patient safety. This may require monitoring and restricting a subset of “high-risk” HCPs.¹⁵

Many biopharmaceutical manufacturers are exploring new unit operations, and process intensification with high cell density cultures linked with downstream unit operations in series to operate as a continuous process. Although these are often discussed in terms of the advantages of higher productivity and lower cost of goods, managing HCPs is a key quality attribute for patient safety. Upstream developments to increase mAb titers by prolonging production time results in changed cell density and viability, which may lead to a higher HCP level and modified composition. This poses a higher removal challenge to downstream purification¹ and HCP detection.

This review covers the current biopharmaceutical industry trends relating to HCPs as a CQA, as well as innovative technologies for analytical assays and manufacturing processes. Data on in-process HCP levels and clearance were analyzed by way of a comprehensive assessment of the literature on upstream generation, control, and downstream removal of HCPs.

Control of HCPs in Downstream Bioprocessing

Protein A chromatography and HCP clearance

Protein A chromatography is widely used as a capture step in bind-and-elute mode to purify mAbs by removing both process- and product-related impurities.⁹¹ Due to its high degree of specificity for the IgG Fc region, Protein A chromatography provides high HCP removal in a downstream processing step, as most HCPs are removed in the flow-through fraction.⁹² However, high HCP levels (ranging from 2000 to >10,000 ppm, Figure 3) may occur in the mAb elution fraction due to non-specific binding and/or mAb-HCP interactions.^{67, 104, 105} The QbD case study working group characterized HCPs in the elution pool as ranging from 5575 to 9893 ppm (~0.9 LRV), depending on critical parameters of mAb load (10–50 mg/mL) and elution pH (3.2–3.9).¹⁷

Protein A carryover HCPs include those HCPs bound to the resin or the adsorbed mAb which are released along with the mAb during the elution step. The pIs of HCPs in harvest material are widely distributed, with the majority being acidic. The HCPs identified in approved mAb products also reflect a similar broad range of pIs. However, the most frequently observed HCPs across mAb products were basic pIs.¹⁷ Many possible causes can result in co-elution. The presence of chromatin in the harvest strongly affects this HCP co-elution. Prefiltration by anion exchange media for chromatin removal reduces the level of HCPs in the Protein A elute.^106, 107 As regards non-specific associations with mAbs, a small subset of mAbs form transient clusters with high charge–charge interactions. This significantly greater number of net positive charges is proposed as a reason for the co-elution of negatively charged HCPs.¹⁰⁸ A wash step can be introduced after the binding step with excipients to disrupt HCP binding and control a pH >4.5 to maintain adsorbed mAb.¹⁰⁹ Wash solutions with a pH of 8–10 would be above the pI of both most mAbs and HCPs causing them both to be positively charged and repel each other while leaving the mAbs intact.⁹³

Wash buffer excipients (Table 3) can reduce bound HCPs by disrupting attractive forces between HCPs and mAbs or the substrate.^{22, 92, 93, 110, 112} Salts (e.g., sodium chloride) disrupt electrostatic binding. And detergents (e.g., Triton X-100), and solvents (e.g., propylene glycol) disrupt hydrophobic binding.^92, 93 Combining high pH with salt and caprylate improves HCP clearance.⁹⁴ Arginine is commonly added to relax electrostatic, hydrophobic, and hydrogen bonds while stabilizing folded proteins to reduce aggregation.^111, 113 PEG 4000 addition led to sharper elution peaks, reducing HCP levels and elution pools.¹¹² Enhanced washes including additives or excipients show improvements in HCP removal (over 5 LRV) compared to standard washes (2 LRV), as shown in Figure 3. Interestingly, almost the same level of HCP clearance was reported in intensified processing, where multiple Protein A columns are used and cycled at high binding capacity. However, additional chromatographic polishing steps are used to provide a robust purification platform for HCP removal.

Polishing chromatography and adsorbent for HCP removal

Following the Protein A step, mAbs are held at low pH for retrovirus inactivation and then neutralized for subsequent processing. The neutralization step is frequently associated with HCP precipitation and requires subsequent depth filtration for clarification and HCP removal.^67, 114 The HCP removal mechanism is the same as that for the harvest, and the clearance performance is similar to that of chromatography even at low HCP concentrations.

Polishing chromatography steps are then used to remove the remaining HCPs using a variety of chromatographic matrices (resin, membranes) operated in different modes (bind-and-elute, flow-through, weak partitioning) and using a variety of binding ligands. These have been used in various sequences (Table 4), with the most common sequence of steps for commercial mAb production being as follows: Depth–Protein A–virus inactivation–cation exchange chromatography–anion exchange chromatography–virus filtration–ultra filtration/diafiltration.¹⁷ The adsorption steps employ different mechanisms to remove HCPs.

For positively charged anion-exchange (AEX) column binding in flow-through mode, operation at a pH below that of the mAb pI (typically >7.5) leaves the mAb positively charged and unbound, while HCPs with lower pIs will be negatively charged and bound.¹²⁹ Conductivity levels also need to be kept low to keep the electrostatic binding strength high and avoid charge passivation by salts. The low HCP load in the polishing step feed does not require high binding capacities for the AEX resin or membrane (Figure 4). The wide range of HCP feed concentrations also reflects different positions in the purification train. For a final step, the QbD case study working group characterized HCPs in the flow-through as ranging from 7 to 26 ppm (~0.5 LRV), depending on critical parameters of load conductivity (1.6–5.6 mS/cm) and pH (7.2–7.8).¹⁷ In the final step, HCPs also behave more like a single species, remaining as a single peak without separating into different peaks traveling through the adsorption bed at different speeds.¹¹⁴ AEX results in an HCP clearance of 2–3 LRV at an HCP feed concentration of around 10³ ppm; below 10² ppm, less than 1 LRV is obtained (Figure 4).¹³⁰

AEX membrane chromatography is particularly well-suited to this application requiring short residence times. This leads to significantly reduced sizing and facilitates single-use operation.¹³¹ A variety of membranes with a high density of binding sites and rapid mass transfer can reduce HCPs by 85–93% with membrane loads of 2–14 kg mAb/L and a residence time of only 2–6 seconds.¹¹⁹ New formats such as monoliths and packed fibers have not been widely applied for mAb polishing.

Cation-exchange (CEX) chromatography is commonly used to remove aggregates, charge variants, and HCPs from mAbs in a bind-and-elute mode. Loading at a pH of 6.0 allows high pI HCPs to have a negative charge and remain unbound in the flow-through fraction.¹³² Usually, the pH, salt concentration, and gradient length of the CEX elution step are optimized for aggregate removal, but this also influences HCP removal.

At feed HCP levels of 10⁴ to 10,⁵ CEX clears about 2–3 LRV with HCPs mainly in the flow-through fraction (Figure 4). When CEX was used as the first step after Protein A capture, the QbD case study working group characterized HCPs in the elution as ranging from 16 to 91 ppm (~2.2 LRV), depending on the critical parameters of loading (10–30 mg mAb/mL) and elution conductivity (3–7 mS/cm).¹⁷ This is comparable to HCP clearance from Protein A without a wash step. CEX clearance at feed concentrations below 10² is higher than the value reported for AEX. Resin and membrane chromatography show no significant difference in HCP LRV performance.

Hydrophobic interaction chromatography (HIC) significantly reduces HCP levels, as does mixed-mode chromatography,^133, 134 which employs both hydrophobic and ion-exchange interactions. In cases of bind-and-elute polishing chromatography, implementing an intermediate wash step with additives (e.g., arginine, urea, ethylene glycol, hexylene glycol, sodium caprylate, etc.) has been shown to improve HCP clearance, similar to Protein A capture chromatography.^{74, 135, 136} The conditions of the mobile phase for HIC can be set for non-binding of the target protein, allowing for flow-through separation while removing bound HCPs. Mixed-mode or multimode chromatography (MMC) employs novel ligands (e.g., sulfhydryl, primary amine, hydroxyl) or combinations of charged (positive or negative) and hydrophobic (e.g., alkane, phenyl) groups in the same ligand for multipoint interactions (e.g., hydroxyapatite). These ligands can improve binding selectivity (purity vs yield trade-offs) and binding solution salt tolerance. Some have even used MMC to replace Protein A. However, multipoint interactions require extensive screening using DoE with high-throughput screening (HTS) methodology to find a “sweet spot” of loading, binding, elution buffer pH, and conductivity for operation. The higher cost and lengthier process development tend to make MMC a secondary option, employed for problematic therapeutics where conventional resins are unable to meet performance targets.¹³⁷

Strong binding impurities are preferentially retained, displacing weak binding mAbs and impurities and significantly improving resin utilization. Enhanced displacement among mAb products and low-prevalence HCPs on CEX resins allows for the selective separation of HCPs.¹³⁸ Frontal chromatography takes advantage of this displacement of impurities to allow flow-through separation in CEX, thereby contributing to process integration and continuous processing of downstream purification. This mainly targets aggregate removal but also contributes to the removal of HCPs.^{102, 115, 139}

Packed columns of activated carbon (AC) utilize a porous structure with a high surface area for adsorptive binding of HCPs through non-covalent interactions.¹⁴⁰ Low molecular weight proteinaceous impurities are effectively removed using AC.¹⁴¹ However, it is difficult to make definitive conclusions on the adsorption mechanism of activated carbon, since this will also depend on the charge, shape, and hydrophobicity of the protein. AC has maximum protein binding capacity when the pH is at the protein’s pI, where protein charge and electrostatic forces are minimized, and hydrophobic interactions dominate.¹⁴¹ A reduction of 3 LRV was observed for a recombinant enzyme with sensitivity to loading, salinity, and pH.¹¹⁶ AC treatment performance has also been reported to improve with low conductivity.⁷⁵ Given the diverse properties of HCPs, a DoE approach based on pH and conductivity can be applied.¹¹⁵

HCP LRV clearance by AC is comparable to AEX (Figure 4). AC has been evaluated post-clarification without Protein A and post-Protein A prior to CEX or AEX. For a purification train with acid precipitation, diafiltration to reduce conductivity and AC, robust HCP removal was superior to that of Protein A.⁷⁵ HCP removal by flow-through AC is orthogonal to AEX, that is, removes different HCP components.¹⁴² This AC step intensifies the subsequent anion exchange column to reduce the impurity burden in the post-Protein A polishing step.^72, 115

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Ito T, Lutz H, Tan L, et al. Host cell proteins in monoclonal antibody processing: Control, detection, and removal. Biotechnol. Prog. 2024;40(4):e3448. doi:10.1002/btpr.3448

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