A Practical Look At Modern Downstream Processing For Biologics

By Smiriti Gupta, NIRAS

In part 1, we built the upstream foundation — from cell bank revival and inoculum expansion to staged seed trains and tightly controlled fermentation. Those controls ensure that, at the point of harvest, the broth contains the target biologic at the highest achievable yield and quality window. We now continue with the downstream steps that recover and purify that product into compliant drug substance and, ultimately, drug product.

Harvest And Downstream Processing: From Broth To Highly Purified Drug Substance¹

Once fermentation is complete, the process transitions from a biologically driven environment to a mechanical, physicochemical, and separation‑focused domain. The objective of downstream processing is not only to recover the product but to progressively remove impurities while preserving molecular integrity and biological activity.

Harvest And Primary Separation

At the end of production fermentation, the bioreactor contains a complex broth of host cells, cell debris, media components, metabolites, and the target product (mostly protein). This broth is typically discharged into a harvest or broth tank, from which downstream operations begin.

A key decision at this stage depends on product localization:

• Extracellular products secreted into the media proceed directly to separation.
• Intracellular products require homogenization to rupture cell walls and release the product.

Homogenization is a mechanical process designed to disrupt cells without chemical agents, preserving product integrity while enabling efficient recovery.

Primary separation is most commonly achieved using a disk‑stack centrifuge, depth filter, or a tangential flow filter.

Centrifugation

1. The centrifuge separates liquid and solid phases based on density.
2. The clarified liquid phase is discarded if the product is intracellular. It is forwarded for the next step if the product is extracellular.
3. The solid phase (cell mass) remains highly hydrated after disc‑stack centrifugation, exhibiting both liquid‑like and solid‑like characteristics, as described in rheological studies of biomass solids.

The collecting vessel may be equipped with agitation to allow simultaneous addition of buffers or stabilizers, preparing the material for subsequent purification steps.

Depth filtration

Depth filtration is a clarification method where the product stream passes through a porous matrix that traps cells, debris, and particulates throughout the filter’s depth — not just on the surface — making it ideal for high‑load harvest streams.

It is commonly used before chromatography or ultrafiltration to reduce particulate burden, protect downstream equipment, and improve overall process robustness. It also helps to remove liquid from the API sludge, thereby increasing capacity later in the downstream process.

In some processes, depth filtration or microfiltration is introduced either before or after centrifugation to improve clarification and reduce particulate load.

Concentration and diafiltration: the role of TFF

Following harvest and clarification, the product stream is typically too dilute and could be in an unsuitable buffer. This is addressed through tangential flow filtration (TFF).

TFF is preferred over dead‑end filtration because it:

• handles large process volumes efficiently,
• minimizes membrane fouling, and
• maintains product integrity under controlled shear conditions.

TFF works by driving the feed stream tangentially along a semi‑permeable membrane while applying a transmembrane pressure (TMP) to push solvent and small solutes through as permeate, retaining the target product in the retentate.

Because the bulk flow sweeps parallel to the membrane surface, it continuously reduces cake buildup and membrane fouling compared with dead‑end filtration, enabling efficient concentration and buffer exchange (diafiltration) at larger volumes. It is generally favored for large volumes, high‑solids feeds, or viscous and aggregation‑prone proteins because its crossflow design minimizes membrane fouling and maintains stable flux. Dead‑end filtration is better suited for low‑solids or shear‑sensitive applications-such as sterile filtration of drug product, where simplicity and gentle handling are critical. Choosing between the two ultimately depends on product characteristics and the nature of the feed stream.

Two distinct but related operations occur in TFF systems:

Concentration

• Solvent and small impurities pass through the membrane (permeate).
• The desired protein is retained and concentrated (retentate).

Diafiltration

• This involves exchanging one buffer system for another.
• Fresh buffer is added while permeate is removed.
• It prepares the product for downstream purification, particularly chromatography.

This step is critical in conditioning the product to the correct pH, conductivity, and ionic strength required for effective binding and separation in chromatography columns.

Chromatography and polishing filtration

Chromatography is the core purification technology in biopharmaceutical manufacturing. It separates the target molecule from impurities based on physicochemical properties.

In chromatography:

• The product is transported in a mobile phase (buffer).
• Separation occurs on a stationary phase (resin) packed in a column.

Common chromatography modes include:

• affinity chromatography – highly selective capture of the target product
• ion‑exchange chromatography – separation based on surface charge
• size‑exclusion chromatography – separation based on molecular size.

Because no single step achieves full purity, multiple chromatography stages are typically combined in a defined sequence. Between and after these steps, microfiltration and ultrafiltration are applied to:

• remove particulates and aggregates,
• support viral clearance strategies, and
• protect final product quality and patient safety.

At the end of downstream processing, the product meets predefined specifications for purity, potency, and safety, qualifying it as drug substance (DS).

Viral Inactivation In Between Chromatography Steps

Viral inactivation is a dedicated downstream unit operation that intentionally destroys or denatures viral structures — typically using low pH, solvents, or heat — to prevent any infectious virus from reaching the final product. It is most critical in cell culture–based bioprocessing, because mammalian cell lines (e.g., CHO) inherently carry higher viral‑contamination risk from raw materials, animal‑derived components, and endogenous retroviral-like particles.

In contrast, microbial fermentation (E. coli, yeast) carries far lower risk of adventitious viral contamination since these hosts do not support replication of human or animal viruses, making viral inactivation less stringent but still part of the overall viral clearance strategy.

Regulators therefore emphasize robust viral inactivation and removal steps specifically for biologics produced from mammalian cell culture, where virus-related safety risks are inherently greater.

Drug Substance Holding And Release

The purified drug substance is either:

• frozen and stored under controlled conditions for future use or
• the drug substance is either transferred directly to the drug product area for formulation, or — in cases where the product or process requires a solid form — it may undergo a drying step at this stage. The specific drying methods appropriate for such products are described in the Drug Product section of this article.

Before release, DS undergoes extensive quality testing to confirm compliance with regulatory and process requirements.

Drug Product Manufacturing: From Drug Substance To Patient‑Ready Medicine

Drug product manufacturing transforms the purified drug substance into a safe, stable, and clinically deliverable dosage form. This stage is characterized by the highest sterility requirements in the entire life cycle.

Thawing and formulation logic

When frozen drug substance is used, controlled thawing is the first critical step. Thawing rates are carefully managed to prevent:

• product denaturation
• aggregation
• loss of biological activity.

Formulation serves two primary purposes:

• Dilution – highly concentrated drug substance is adjusted to a human‑consumable dosage.
• Stabilization – excipients are added to protect the molecule against degradation during storage, transport, and administration.

These stabilizers play a vital role in maintaining product efficacy throughout its shelf life.

Sterile filtration and aseptic filling

Following formulation, the product is passed through sterile microfilters and transferred to aseptic filling lines. This stage is conducted under strictly controlled cleanroom conditions:

• Class A zones for critical operations such as filling and stopper placement
• Class B environments as the surrounding background

This cleanroom hierarchy ensures that microbial and particulate contamination risks are minimized. Aseptic processing is non‑negotiable - any breach can compromise patient safety and result in batch rejection.

Drying

For products requiring enhanced stability, the product may need drying. This can be achieved by various ways:

Lyophilization (freeze drying)

Lyophilization removes water by first freezing the product and then sublimating the ice under deep vacuum, avoiding the liquid phase entirely. This makes it ideal for highly sensitive biologics, preserving structure, activity, and long‑term stability. The process produces a porous, easily reconstituted cake but is time‑ and cost‑intensive.

Spray drying

Liquid feed is atomized into droplets → hot air removes moisture → particles collected as powder.

Vacuum drying

Vacuum drying removes moisture through low‑temperature evaporation under reduced pressure without freezing the product. It is suitable for more stable biomolecules that can tolerate mild heat and retain quality during liquid‑phase drying. Compared with lyophilization, it is faster, simpler, and more economical but offers lower protection for fragile proteins.

Contamination Control: CIP And SIP As Invisible Guardians

A biopharmaceutical facility is defined by its cleanliness. Contamination is the industry’s greatest risk, and two systems form the backbone of hygienic design.

Cleaning‑in‑place

CIP cleans equipment without dismantling, using automated cycles that typically include:

1. pre‑rinsing (to remove the soil)
2. alkaline cleaning
3. intermittent flush (can be performed with lowest category of water available as clean utility)
4. acid cleaning(generally embedded as part of recipe for maintenance cycles, not often used in routine)
5. intermittent flush (can be performed with lowest category of water available as clean utility)
6. final rinsing with water quality used in product manufacturing (purified water (PW), potable water or hot water for injection (WFI).

CIP prevents cross‑contamination and ensures batch‑to‑batch integrity.

Sterilization‑in‑place

Sterilization-in-place (SIP) is a validated moist-heat sterilization step performed on closed, fixed equipment (tanks, lines, filters) using saturated/clean steam after CIP to achieve a defined sterility assurance rather than a provable “zero bioburden.”

Because microbial kill follows log-linear kinetics, sterility is expressed as a sterility assurance level (SAL) — commonly 10⁻⁶, meaning a one-in-a-million probability of a non-sterile unit— rather than an absolute claim that no microorganism can exist.^2,3

Typical SIP regimes hold the coldest point at around 121–134 degrees C for a validated exposure time (often ≥20 to 30 minutes at ~121 degrees C, depending on design and qualification). ^4,5

After the hold phase, systems are cooled and often kept under sterile air or nitrogen overpressure to maintain sterile boundaries and prevent recontamination until use.

Together, CIP and SIP ensure compliance with GMP requirements and protect both product and patient.

Biowaste Deactivation: Protecting People And The Environment

Biopharmaceutical manufacturing generates waste containing live or genetically modified organisms. Direct discharge would pose serious risks.

Biokill and decontamination systems provide:

• thermal sterilization at 121.1 degrees C for 30 to 40 minutes ^2,4
• periodic chemical inactivation (e.g., formaldehyde).

After deactivation, waste can be treated as standard chemical effluent in effluent treatment plants (ETPs). This step is essential for environmental safety, worker protection, and regulatory compliance.

Conclusion: Engineering The Future Of Medicine

Biopharmaceutical manufacturing is more than a sequence of unit operations — it is a symphony of biology, engineering, and quality science. As the industry advances toward biosimilars, gene and cell therapies, and personalized medicine, the demand for robust, flexible, and compliant manufacturing ecosystems will only intensify.

References:

1. ISPE Guide: Biopharmaceutical Process Development & Manufacturing — All DSP unit operations under cGMP
2. ISO 17665:2024 – Sterilization of health care products — Moist heat — Requirements for development, validation, and routine control of a sterilization process.
3. CDC, “Guideline for Disinfection and Sterilization in Healthcare Facilities” (Steam Sterilization section).
4. FDA – Sterilization Process Controls Guidance.
5. ISPE-Sterilization – Validation, Qualification Requirements

About The Author:

Smiriti Gupta is a senior consultant with NIRAS International Consulting in Denmark where she focuses on process automation and optimization; commissioning, qualification, and validation; and life cycle management. Previously she worked at WuXi Biologics as a computerized system validation engineer, at Siemens as a technical support specialist, and for DD Enterprises as a senior bioprocess and automation manager. Her career began as an operator on the biopharma manufacturing floor, giving her first‑hand, shop‑floor perspective that directly informs the insights shared in this article. She has a Bachelor of Engineering degree from the Madhav Institute of Technology and Science.