Roundup: Removing Impurities from Biomanufacturing

Effective impurity control is critical to biomanufacturing, supporting process, cell line, and formulation stability while preserving biotherapeutic potency and shelf life. Residual impurities can drive oxidation, enzymatic degradation, protein unfolding, and aggregation. GEN asked leaders in biomanufacturing: What technologies do you use to carry out effective impurity removal at specific points throughout upstream and downstream processing to ensure product quality and patient safety?

Agilent Technologies

Effective impurity removal in biomanufacturing requires strategically integrated separation and analytics across both upstream and downstream workflows. Early-stage control focuses on removing process-related impurities, such as host cell proteins (HCPs), DNA, and media components, where affinity chromatography remains foundational for bulk clearance and process robustness

As molecules increase in complexity, orthogonal analytical approaches become critical. High-resolution multidimensional separations enable deeper interrogation of complex mixtures and reduce the risk of co-eluting impurities. Agilent Bio-inert LC platforms are developed to minimize metal interactions that can compromise protein integrity or mask low-level impurities, improving sensitivity and reproducibility for biologics and metal-sensitive analytes.

Downstream purification strategies are increasingly tailored to modality. LC solutions paired with integrated software workflows, support efficient peptide purification with yield and purity tradeoffs. For oligonucleotides, impurity removal often relies on orthogonal ion-pairing reverse phase (IP-RP) and anion exchange (AEX) chromatography to resolve sequence failures and closely related variants.

Ultimately, combining robust purification technologies with advanced analytical workflows ensures not only effective impurity clearance, but also comprehensive characterization critical to maintaining product stability, regulatory compliance, and patient safety.

Lonza

Driving effective impurity control in biomanufacturing requires orthogonal diversity coupled with capacity redundancy and holistic process stewardship.

Bioinformatics can inform liabilities before even picking up a pipette. Cell line stability, clone selection and high-throughput upstream optimization reduce host cell protein (HCP) accumulation by preventing upregulation of lipases and proteases that cause oxidation and unfolding but serve to protect cellular function under prolonged stress.

Often overlooked, effective primary harvest depth filtration combined with charged media is the first line of defense, separating impurities based on size and electrostatic, hydrophobic, and hydrogen bonding mechanisms. This focus is central as upstream processes intensify.

Downstream processing should leverage heterogeneous product characteristics: selection of specialized affinity resins, enhanced washes that disrupt “hitchhiking” HCP-target molecule-histone complexes, and reduced proteases that contribute to ligand leachate. Viral inactivation precipitates impurities to aid physical removal when followed by charged depth filtration, which should be optimized for pH and conductivity conditions. Multimodal resins combining IEX, HIC and hydrogen bonding provide powerful synergistic polishing for removing aggregates, fragments, stubborn HCPs, endotoxins, viruses, and residual affinity ligand to provide representatively pure product, essential for successful liquid formulation studies. Changes to regulatory guidance mandate future HCP characterization using LC-MS informed early process development, to ensure patient safety, product stability, and promote the maximum possible shelf life.

MilliporeSigma

Effective impurity control is enabled through a combination of advanced analytics, targeted purification technologies, and upstream material design. Upstream, MilliporeSigma minimizes impurity introduction through stringent raw material qualification, trace element (TE) control strategies, and high-sensitivity analytical platforms such as ICP-MS for multi-element profiling. These capabilities allow precise characterization and specification of low-level metal impurities that can catalyze oxidation or destabilize proteins. Engineered cell culture media further reduce variability by controlling impurity ingress at the source, supported by supplier qualification and risk-based raw material selection. In-process controls and real-time monitoring ensure consistency throughout bioreactor operations.

A recent supply chain disruption involving ferric ammonium citrate (FAC) put these capabilities to the test. When alternative sourcing introduced material with markedly different TE impurity profiles, MilliporeSigma leveraged its integrated chemical manufacturing expertise and ICP-MS analytical platforms to engineer an FAC solution aligned with historically established TE baselines. Defined impurity targets preserved process comparability and avoided customer reformulation, demonstrating how analytical comparability and change management frameworks keep impurity profiles within defined limits when supply or process adjustments are required.

In summary, MilliporeSigma integrates analytical rigor, engineered impurity control, and robust purification technologies across upstream processes, providing a scalable, supply-resilient foundation that safeguards product quality, process consistency, and patient safety while reinforcing leadership in differentiated bioprocessing solutions.

Repligen

Effective impurity control begins with a well-designed purification strategy. Across downstream processing, the objective is straightforward: remove process- and product-related impurities while preserving the quality, potency, and stability of the therapeutic.

Repligen has built a comprehensive portfolio of purification and analytical technologies designed to address impurity challenges across a wide range of biologics. Affinity chromatography remains one of the most powerful tools available, routinely removing more than 95% of process-related impurities in a single step while maintaining yields above 90%. Our portfolio includes both catalog affinity resins and industry-leading capabilities for the rapid development and commercialization of custom affinity ligands. These custom solutions can address particularly challenging separations, including product-related impurities that are difficult to resolve using conventional polishing techniques.

Filtration plays an equally important role throughout the purification workflow. Tangential flow filtration, using either flat-sheet or hollow-fiber formats, supports impurity clearance, enhances chromatography performance, and enables efficient formulation of the final drug product. Together, chromatography and filtration create a highly effective platform for delivering consistent product quality and process robustness.

What differentiates Repligen is the integration of purification technologies with advanced process analytical technologies (PAT). We believe effective impurity control requires not only removing contaminants but also measuring and understanding them in real time. Our portfolio spans at-line, on-line, and real-time analytical solutions that provide actionable process insight directly to operators and manufacturing teams.

Because no single analytical technology can address every challenge, we have deliberately built a broad PAT portfolio that enables customers to apply the right tool to the right problem. Repligen provides solutions that provide real-time UV-Vis and Raman analytics for upstream and downstream process monitoring, while platforms enable deeper characterization of media components, proteins, and critical impurities.

Ultimately, successful impurity control requires the seamless integration of purification, filtration, and analytics. By combining these capabilities into a connected bioprocessing ecosystem, Repligen helps customers accelerate development, strengthen process understanding, and consistently manufacture safe, high-quality biologics.

Sartorius Stedim Biotech

Effective impurity control is essential in biomanufacturing, with strategies tailored to the target molecule and process. Downstream processes for monoclonal antibodies (mAbs) have demonstrated a direct impact on product quality and patient safety. Key impurities—host cell proteins (HCPs), residual DNA, endotoxins, aggregates, and process-related chemicals—originate from the expression system or manufacturing steps. If not adequately removed, HCPs and DNA can trigger immune responses or toxicity in patients, aggregates may induce immunogenicity or reduce efficacy, and endotoxins can cause severe inflammation. Process chemicals, like Protein A leachates or buffer residues, further contribute to safety risks.

Industry-leading technologies are applied in downstream processing to ensure impurity removal. Protein A affinity chromatography captures mAbs and eliminates bulk HCPs and DNA, mostly followed by a prolonged hold step at low pH to facilitate virus inactivation. Polishing steps with ion exchange as well as hydrophobic interaction chromatography remove charged contaminants, viruses, and aggregates. Sartorius membrane chromatography enables a complete membrane-based workflow for efficient impurity removal and high productivity, while bioburden, sterile, and virus filters ensure reliable removal of particulates, bacteria, and viruses, maintaining the critical quality attributes before final formulation. All process steps are usually complemented by several ultrafiltration and diafiltration steps, which facilitate further impurity removal, buffer exchange, as well as concentration of the molecule of interest.

Regulatory entities rigorously assess these purification steps during drug approval, ensuring impurity removal meets stringent safety standards to protect patient health, as demonstrated in many commercial processes.

Veranova

For antibody-drug conjugates (ADCs), it is critical to tightly control reaction stoichiometry, buffer composition, temperature, and reaction time. Hold times during antibody modification and conjugation should also be assessed, as delays in processing the antibody-linker intermediate may alter reaction stoichiometry or increase product-related impurities that complicate downstream purification.

Tangential flow filtration (TFF) is widely used with minimal yield loss to remove small-molecule stabilizers from antibody storage buffers prior to conjugation, as well as for ADC concentration, buffer exchange, and clearance of process-related impurities such as free payload, linker, and organic solvents. Compared to chromatography, TFF is typically more scalable, cost-effective, and higher yielding, largely due to the significant size difference between the ADC (~150 kDa) and payload (<2 kDa).

In some cases, carbon filtration is applied after TFF as an orthogonal step to remove residual linker-payload species. Depending on antibody subtype, conjugation method, and payload, product-related impurities may include aggregates and undesired isoforms. In these situations, size exclusion chromatography or hydrophobic interaction chromatography may be necessary, although they can reduce yield and increase manufacturing cost.

As novel conjugation technology evolves using bispecific mAbs, Fc-fusion proteins, and chemical site-specific conjugation, traditional TFF systems will be sufficient for purification alone or in combination with chromatographic separation techniques to deliver homogeneous ADCs with uniform drug-to-antibody ratios.

WuXi Biologics

Residual impurities are primarily process-related, including DNA, host cell proteins (HCPs), leached Protein A, and upstream additives. Effective control of these impurities requires an integrated strategy across both upstream and downstream processes. While cell line and process development help define and minimize the initial impurity burden, downstream purification takes the main responsibility for their removal.

Among the residual impurities, lipases and metal ions can be particularly detrimental due to their roles in protein degradation and instability. In downstream processing, Protein A affinity chromatography is one of the most effective unit operations for removing such impurities, owing to its high selectivity for antibodies/Fc-fusions. However, certain HCPs and other impurities may interact with the target molecule and copurify.

To address this, it is critical to disrupt these interactions. Therefore, we extensively optimize Protein A wash conditions using additives such as amino acids, chaotropic salts, organic solvents, or detergents to enhance HCP clearance. In one case, we successfully removed cathepsin B, a CHO endogenous protease that chops the target antibody, by adding sodium caprylate into the wash buffer.¹ Similarly, EDTA is used in the wash buffer to remove metal ions. High-pH eluting Protein A resins and Protein A membranes have also demonstrated advantages for HCP clearance, reducing HCP levels to below those achieved with regular Protein A resins.^2,3

Beyond the affinity step, intermediate depth filtration following low-pH inactivation/ neutralization and subsequent polishing steps are further optimized to reduce residual impurities. Among polishing techniques, hydrophobic interaction chromatography (HIC) has shown particular effectiveness in removing lipases, and ongoing evaluations focus on resins with varying hydrophobicity to enhance performance.

References

1. Hu L, Tang J, Zhang X, Li Y. Sodium caprylate wash during Protein A chromatography as an effective means for removing protease(s) responsible for target antibody fragmentation. Protein Expr Purif. 2021, 186: 105907.

2. Li Z, Hu L, Li Y. The advantage of high pH eluting Protein A resins over their regular counterparts in aggregate and host cell protein clearance. Protein Expr Purif. 2026, 239: 106859.

3. Yang Y, Zhang X, Li Y. Deciphering the inconsistent performance of Protein A membrane on host cell protein clearance. Protein Expr Purif. 2026, 240: 106891.