The Role of Downstream Development in Ensuring Biologic Product Purity and Quality

• Downstream development isolates stable, homogeneous therapeutic proteins from host cell proteins, DNA fragments, and process contaminants. This guarantees reproducibility across production campaigns, which is critical for both novel biologics and biosimilars. 
• Purity is fundamental to the therapeutic value of biologic products. Even minimal contamination can compromise safety, reduce product stability, or prevent regulatory approval. 
• Downstream processing transforms raw harvest into clinical-grade medicines, ensuring safety and efficacy. This process is multi-step and relies on various techniques for purifying biologics. 
• Mabion’s downstream strategy integrates capture Protein A chromatography, virus inactivation, polishing by ion-exchange chromatography, ultrafiltration, diafiltration, nanofiltration, and sterile viral filtration. Each step is executed in controlled Grade D cleanroom environments to guarantee regulatory and scientific excellence. 

Downstream Development in Biologics

Biologics are complex molecules produced through living systems. Their development requires precise process design from early stages. Upstream processes focus on cell growth and expression. Yet, the true definition of product quality emerges during downstream development. 

Downstream processing (DSP) development involves recovering, purifying, and stabilizing the product. It transforms a raw biological mixture into a safe and effective therapeutic. Without effective downstream purification, biologics manufacturing cannot meet modern standards. 

Biologics process development integrates upstream and downstream phases into a seamless strategy. This ensures that therapeutic proteins, monoclonal antibodies, and other advanced molecules are not only produced but also refined to meet stringent regulatory expectations.¹ 

At Mabion Biologics CDMO, downstream development integrates capture, virus inactivation, polishing, ultrafiltration/diafiltration, nanofiltration, and sterile filtration into a robust sequence. Each step is carried out in Grade D cleanroom environments, under rigorous quality control frameworks. The aim is not only to isolate proteins of interest but also to ensure compliance with international regulatory standards. 

Purity Really Matters in Biologic Drug Manufacturing

The therapeutic value of biologics depends on purity. Even trace impurities can compromise safety, causing immunogenicity, or reduce therapeutic effect. Therefore, biologic drug development requires purification strategies that deliver clinical-grade consistency. 

Regulators require detailed quality control in biologics. Impurities must be identified, quantified, and removed.^2,3 Downstream purification technologies ensure that proteins are isolated from host cell proteins, DNA fragments, and process-related contaminants. A consistent purification process secures lot-to-lot reliability, critical for both innovative biologics and biosimilars. 

Key Steps in the Downstream Purification Process 

Downstream development follows a sequence of engineered steps, each designed for efficiency and precision. The process transforms a complex biological harvest into a highly defined, safe, and efficacious drug substance. Each purification stage contributes a unique function, ensuring that the final biologic meets the highest standards of purity, safety, and stability. These steps are interdependent scientific filters designed to progressively remove distinct classes of impurities and to preserve the integrity of the therapeutic protein. 

Protein A Affinity Chromatography

The capture step is the cornerstone of downstream development, particularly for monoclonal antibodies. Protein A affinity chromatography exploits the strong, specific interaction between Protein A ligands and the Fc region of antibodies.⁴ Using advanced platforms such as ÄKTA Ready, ÄKTA Process, and ÄKTA Pilot systems, Mabion achieves high-yield binding and selective recovery of target molecules. 

This step drastically reduces process-related impurities by removing the bulk of host cell proteins (HCPs), DNA, and other cellular debris. Its high selectivity allows for efficient concentration of the therapeutic protein, setting the foundation for subsequent refinement. Without this capture step, downstream purification would lack the robustness required for reproducibility and clinical-grade output.

Viral Inactivation 

Following capture, the product still carries the inherent risk of viral contamination originating from cell substrates or raw materials. To mitigate this, low pH treatment is applied in FlexAct systems equipped with pH and temperature sensors, magnetic mixers, and peristaltic pumps. The therapeutic protein is exposed to a controlled acidic environment for a defined duration, sufficient to inactivate potential enveloped viruses without compromising protein structure or function. 

Viral inactivation ensures that the final drug product is free of latent biological threats that cannot be removed by standard filtration alone. Typically, at least two orthogonal viral inactivation steps are implemented in the whole downstream processing to ensure the safety of biotherapeutic products.^5,6 These steps should utilize different mechanisms to reduce viral load. The robustness of this step, validated through viral clearance studies, is essential to building confidence in the biologic’s safety profile.⁷ 

Ion-Exchange Chromatography 

Even after capture and viral inactivation, trace impurities remain. These include aggregates, misfolded variants, residual HCPs, leached Protein A, and adventitious viral particles.  

Ion-exchange chromatography separates molecules based on their electrical charge using a stationary phase (resin) with either a positive (cation exchange) or negative (anion exchange) charge. Molecules with the opposite charge bind to the resin, while those with the same charge are washed away. The bound molecules are then eluted and released by changing the buffer conditions, usually by increasing the salt concentration.⁸ 

Mabion applies cation- exchange chromatography (CEX) and anion-exchange chromatography (AEX) in flow-through mode, which separates molecules. Both single-use and multi-use columns are used, depending on project scale. 

This step is indispensable for ensuring product homogeneity, since aggregates or charge variants can alter pharmacokinetics and immunogenicity. Each layer of polishing selectivity addresses specific, clinically relevant impurities that earlier steps cannot fully resolve.  

Ultrafiltration & Diafiltration & Nanofiltration 

While purification yields a clean protein, the drug substance must also be concentrated to therapeutic doses and formulated into the correct buffer system. As a part of downstream development, ultrafiltration/diafiltration (UF/DF) achieves this dual role by using single-use membrane pumps, UF cassettes, and tangential flow filtration (TFF) units. 

UF concentrates the protein, while DF exchanges the buffer to achieve stability under storage and administration conditions. The choice of system depends on production scale: compact TFF units are applied for volumes between 10-30 L, while FlexAct platforms with flow, pressure, and temperature sensors handle production exceeding 100 L.⁹ 

To further enhance viral safety, nanofiltration is employed, physically excluding viral particles using membranes with pore sizes in the nanometer range.¹⁰ Conducting polishing on FlexAct systems with membrane pumps and precise sensor integration guarantees that fine impurities are eliminated without eroding product yield. 

This step is critical for maintaining molecular stability. Without proper UF/DF, biologics may precipitate, degrade, or lose activity, undermining the drug’s therapeutic reliability. It is here that process development expertise directly intersects with clinical performance. 

Sterile filtration 

The last stage, sterile filtration, is the ultimate quality safeguard before storage and formulation. Using 0.2 μm-rated sterilizing-grade filters, any potential microbial contaminants are removed. This barrier is indispensable, as even undetectable microbial contamination can compromise patient safety and shelf stability. 

Sterile filtration ensures that the drug substance entering the formulation and fill-finish stages is devoid of viable microorganisms. While earlier steps target host cell- and process-related impurities, sterile filtration uniquely addresses environmental risks that could arise during processing.¹¹ 

Common Challenges in Ensuring Biologic Product Quality 

Unlike small molecules, biologics are produced in living systems, which inherently generate variability, impurities, and risks that cannot be ignored. Despite advances, DSP process development faces persistent challenges. High product titers from upstream intensify purification complexity. Host cell proteins, DNA, and aggregates can be difficult to remove completely. High throughput must be achieved without compromising yield. 

Downstream processing normally accounts for 50-70% of biologics manufacturing costs.^{12, 13} The need for multiple purification cycles, expensive chromatography resins, and validated viral clearance studies increases financial burden. The economic challenge is to optimize process steps to achieve regulatory-compliant purity without excessive cost-of-goods (COGs). CDMOs that invest in single-use technologies, continuous chromatography, and high-capacity resins can strike this balance better than those relying on traditional infrastructure.^{14, 15} 

For biosimilars, downstream development is even more demanding. Success depends on demonstrating molecular comparability to the reference product, not just in purity but also in glycosylation, charge distribution, and aggregation profiles. Minor deviations in downstream performance can jeopardize approval. This requires CDMOs to apply high-resolution purification and characterization technologies that go beyond the minimum.¹⁶ 

Agencies such as the FDA, EMA, and global authorities increasingly demand not only evidence of impurity removal, viral safety, and product stability, but also a clear demonstration that these controls are embedded within a sustainable and scientifically sound framework. 

One of the most significant shifts has been the widespread adoption of Quality by Design (QbD) principles.¹⁷ Regulators no longer view downstream purification as a fixed recipe but as a system that must be understood in depth. Developers are expected to identify critical process parameters (CPPs), define their impact on critical quality attributes (CQAs), and demonstrate robust control strategies. This means that biologics manufacturers must go far beyond simply validating a single “happy path.” Instead, they must show that their purification process can withstand variability in raw materials, scale transitions, and long-term manufacturing campaigns while consistently meeting product specifications. 

Closely tied to QbD is the increasing emphasis on Process Analytical Technology (PAT) and real-time monitoring.¹⁸ Authorities encourage companies to integrate in-line sensors and advanced analytics into downstream processing, enabling immediate detection of deviations rather than relying solely on end-point testing.¹⁹ This reflects a shift in regulatory philosophy: product quality must be built into the process itself, not inspected in afterwards. For downstream development, this requires investment in platforms capable of capturing and analyzing data in real time, ensuring that purification is both scientifically controlled and transparently documented. 

Another area of evolving expectation is lifecycle process management. Agencies expect companies to treat process development as a living system, not a one-time exercise completed before approval. Continuous Process Verification (CPV) is increasingly required, ensuring that every manufacturing run generates data demonstrating ongoing compliance with validated parameters. This creates pressure on organizations to maintain digital traceability and advanced data management systems, linking laboratory-scale development directly to commercial manufacturing.²⁰ 

In our facility all downstream processes are executed in Grade D cleanroom environments, fully aligned with EMA and FDA expectations. This provides assurance of compliance across global regulatory jurisdictions and accelerates pathways to both EU and US markets. For many CQAs, we offer capabilities that significantly exceed regulatory requirements. Mabion’s DSP development does not stop at demonstrating impurity clearance. Every purification run is treated as a scientific experiment, generating data on protein stability, aggregation propensity, viral safety margins, and buffer compatibility. This ensures that scaling up downstream processes does not dilute control, but instead strengthens product reliability. 

How Advanced Technologies Improve Downstream Efficiency?

Innovation in DSP process development has led to more efficient purification. Filtration techniques in DSP are advancing toward single-use systems. These reduce cross-contamination risks and shorten turnaround times. Integration of automated monitoring ensures real-time quality control in biologics. 

A particularly transformative advancement is the adoption of multimodal chromatography. Unlike classical ion-exchange or affinity techniques that rely on a single interaction mode, multimodal resins combine ionic, hydrophobic, and hydrogen-bonding interactions in a single ligand. This complexity allows purification of challenging molecules that do not separate well under traditional methods, such as antibodies with atypical glycosylation, fusion proteins, or antibody-drug conjugates.²¹ Multimodal platforms expand purification flexibility by enabling robust removal of host cell proteins, aggregates, and charge variants in a single step, reducing the need for multiple sequential polishing columns.²² Scientifically, this results in higher process efficiency, lower resin consumption, and improved resolution of critical quality attributes (CQAs). 

Equally important to downstream efficiency is the use of modular chromatography systems, such as Mabion’s adoption of FlexAct modular systems and ÄKTA platforms reflects this trend toward flexibility and scalability. Modular chromatography provides an adaptable framework in which purification units can be configured or reconfigured depending on the specific molecule, scale, or project stage. Unlike fixed stainless-steel systems, modular platforms can handle a broad spectrum of production volumes, from early-stage clinical batches to full-scale commercial campaigns, while maintaining identical process control logic. This scalability reduces technology transfer risks, ensures reproducibility, and shortens the path from laboratory to market. For decision-makers, the benefit lies in flexibility. Modular systems allow rapid response to shifting pipeline priorities, biosimilar comparability requirements, or evolving regulatory expectations without costly infrastructure changes.^23,24 

Today, efficiency in biologics purification depends on adopting advanced technologies that enable higher selectivity, shorter cycle times, and greater scalability. Real-time analytics support in-line monitoring of protein quality, reducing deviations. Such technologies drive consistency, lower costs, and ensure quality control in biologics. 

Best Practices for Maintaining Product Purity and Compliance

Ensuring purity and compliance requires best practices throughout biologics process development. Best practices in downstream development begin with holistic design. Processes should anticipate regulatory requirements from the earliest stages. Aligning biologics manufacturing strategies with compliance frameworks avoids costly redesigns later. 

Robust characterization is essential. Analytical technologies should monitor critical quality attributes during each step. Chromatography in DSP and filtration must be validated under real-world stress conditions.²⁵ 

Collaboration between upstream and downstream teams enhances process efficiency. Integrating scientific knowledge and experience ensures smoother scaling up downstream processes. It reduces bottlenecks and improves cost predictability in biologic drug development. 

Ultimately, biologics manufacturing is not only a technological process but also a compliance-driven discipline. Continuous improvement in downstream process optimization ensures that monoclonal antibody purification, viral safety, and sterile filtration meet both scientific and regulatory expectations.

Prepared by:

Jakub Knurek

Marketing Specialist

j.knurek@mabion.eu

Jakub Knurek

Marketing Specialist

Jakub Knurek is a marketing and business development expert specializing in CDMO operations with scientific and biopharmaceutical background. During the COVID-19 pandemic, he contributed to the development of the Novavax protein vaccine. As a side project, he runs his own pharma company.

As the author of numerous articles, he is recognized as an important voice of the younger generation of scientists. He is a member of Mabion’s representation at industry trade shows, including BIO International Convention 2025 in Boston, 2nd Global Biologics India 2025 in Hydrabad, CPHI 2024 in Milan, CPHI 2023 in Barcelona, Life Sciences Baltics 2023 in Vilnius. He actively participates in academic, business and industry discussions, such as the “Building the Future Pharma Workforce” panel at CPHI Milan 2024.

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