Biologics Manufacturing Processing Steps: From Cells to Life-Changing Medicines

In the world of pharmaceuticals, biologics have emerged as a groundbreaking class of drugs, offering innovative therapies for a range of diseases, from cancer to autoimmune disorders. Behind these life-changing treatments lies an intricate manufacturing process. In this blog post, we will take you on a journey through the key steps of a typical biologics manufacturing process.

Upstream Manufacturing

Upstream manufacturing encompasses all the steps involved in cultivating and expanding the cell culture to produce the desired therapeutic protein. This stage focuses on the growth and multiplication of cells that have been genetically modified to produce the target protein.

Here are the key unit operations involved in upstream manufacturing:

Vial Thaw: Bringing Cells Back to Life

The journey begins with a vial of frozen cells. These cells are the foundation of the biologic drug and were meticulously engineered to produce the desired therapeutic protein. The first step is to thaw the cells, reawakening them from their frozen slumber. This process requires precision and care to ensure the cells regain their viability.

Shake Flask Expansion: Cultivating the Cells

Once the cells are thawed, they are transferred to shake flasks filled with a nutrient-rich growth medium. In this environment, the cells begin to multiply, adapting to their new surroundings. This stage is crucial for building up a healthy population of cells before moving to larger bioreactors.

Bioreactor Expansion Train: Scaling Up the Production

From the shake flask, the culture is transferred to progressively larger bioreactors. These bioreactors provide controlled conditions of temperature, oxygen levels, and nutrient supply to support optimal cell growth and protein production. The cells continue to multiply exponentially, increasing the yield of the therapeutic protein.

Bioreactors that compose the expansion train (or "seed train") are identified by the nomenclature "N-X", where X is the step preceding the "N stage" production vessel.  So, for instance "N-1 stage," is the bioreactor that inoculates the production vessel and "N-2 stage" is the bioreactor that inoculates the bioreactor that inoculates the production vessel.  

• Production Vessel = N Stage
• Bioreactor that inoculates the production vessel = N-1 stage
• Bioreactor that inoculates the N-1 Stage = N-2 stage

N Stage Production: Enhancing Protein Output

The N stage, also known as the production stage or the main production bioreactor, is where the majority of cell culture expansion and protein production occur. Cells from the N-1 stage are transferred to larger bioreactors and provided with the ideal conditions for growth and protein expression. The cells are allowed to multiply and produce the therapeutic protein at significant levels.

The N stage is where the highest density of cells is achieved, resulting in a substantial yield of the desired protein.

Harvest: Collecting the Protein

Once the cells have reached their peak productivity, it's time to harvest the culture. This involves separating the cells from the culture medium to obtain the protein of interest. The harvested cell culture is clarified, typically by centrifugation or depth filtration to remove large cell debris and aggregates. 

Many processes will also utilize a viral inactivation step during the harvest as a contamination control mechanism.  Viral inactivation is typically the transition step in the process between upstream manufacturing and downstream manufacturing. 

Downstream Manufacturing:

Downstream manufacturing, also known as downstream processing or purification, involves the purification and formulation of the harvested material to isolate the target protein and remove impurities. This stage is all about refining the clarified, harvested material into a pure and stable form that can be safely administered to patients.

Key components of downstream manufacturing include:

Column Chromatography: Purifying the Protein

In biologics manufacturing, column chromatography is a vital technique used for the purification of proteins and other biomolecules. It employs a stationary phase (usually a resin or gel) and a mobile phase (liquid buffer) to separate components based on their interaction with the stationary phase.

The harvested material contains not only the desired protein but also various impurities. Column chromatography is a sophisticated technique used to separate and purify the target protein from these impurities. There are several types of column chromatography techniques used in biologics manufacturing to purify and isolate target proteins.

Here are some of the most common ones:

Affinity Chromatography:

Affinity chromatography exploits the specific binding between a target protein and a ligand immobilized on the resin. This ligand can be an antibody, enzyme, receptor, or other molecule that binds to the protein of interest. Affinity chromatography is highly selective and can be used to isolate proteins with very high purity.

Ion Exchange Chromatography:

Ion exchange chromatography separates proteins based on their net charge. Positively charged proteins are attracted to negatively charged ion exchange resins (anion exchange), while negatively charged proteins are attracted to positively charged resins (cation exchange). This technique is effective for separating proteins with different isoelectric points or charge characteristics.

Size Exclusion Chromatography (SEC) or Gel Filtration:

SEC separates proteins based on their size. Smaller molecules enter the pores of the gel and take longer to travel through the column, while larger molecules pass through more quickly. It's useful for separating proteins from aggregates, fragments, and other impurities based on their molecular size.

Hydrophobic Interaction Chromatography (HIC):

HIC utilizes the hydrophobic regions of proteins to facilitate separation. A hydrophobic resin is used, and proteins interact with the resin based on their hydrophobicity. The more hydrophobic a protein is, the stronger its interaction with the resin. This technique is particularly useful for purifying proteins with similar charges but different hydrophobic properties.

Multi-Modal Chromatography:

Multi-modal chromatography (also known as mixed-mode chromatography) uses resins with multiple types of interactions. These interactions could be a combination of ion exchange, hydrophobic, and/or affinity interactions. This approach allows for enhanced selectivity in separating complex protein mixtures.

Reversed-Phase Chromatography:

Although less common in biologics manufacturing, reversed-phase chromatography uses a hydrophobic stationary phase and a polar mobile phase. It's often used for separating peptides and small hydrophobic proteins.

HILIC (Hydrophilic Interaction Chromatography):

HILIC is another less common method in biologics manufacturing. It's used to separate polar compounds based on their hydrophilicity and polarity.

The choice of which chromatography methods to use depends on the specific characteristics of the protein being purified and the desired level of purity.  Each of these chromatography techniques has its advantages and limitations. Biologics manufacturers often employ a combination of these methods in a purification process known as chromatography "resin cascades" or "chromatography trains." These cascades involve multiple chromatography steps to gradually isolate the target protein from impurities, resulting in a highly pure and concentrated product ready for further processing and formulation.

Viral Filtration

Viral filtration is a critical purification step in biologics manufacturing that employs specialized filters to remove potential viral contaminants from the final drug product. As the product passes through the filter under controlled pressure, viruses are trapped on the filter's surface, preventing their presence in the finished drug. This crucial step ensures the safety of biologic products by significantly reducing the risk of viral contamination.

Formulation Using Tangential Flow Filtration: Preparing for the Next Phase

After purification, the protein is concentrated and formulated to ensure stability and compatibility with the body. Tangential flow filtration is a key process used for this purpose. 

Tangential Flow Filtration (TFF), also known as Cross Flow Filtration, is a widely used technique in biopharmaceutical manufacturing for concentrating and purifying biomolecules, including proteins, enzymes, and other macromolecules. TFF operates on the principle of pressure-driven filtration, where the liquid is passed across a semi-permeable membrane, allowing small molecules to pass through while retaining larger molecules.  Through this process, the target protein is concentrated and excess salts, buffer components, and smaller impurities are removed while retaining the therapeutic protein in its active form.

Bulk Drug Substance and Final Filling

Bulk drug substance filling, also known as "bulk fill" or "BDS Fill", is the step where the purified and concentrated biologic drug substance is filled into containers or vessels that are suitable for storage and transportation.  Testing is performed, production records are reviewed and upon confirmation that the target protein meets Safety, Quality, Identity, Purity and Potency (SQUIPP) requirements, the bulk drug substance can proceed into the final drug product filling step.

Final drug product filling is the last stage in the biologics manufacturing process before the product is distributed for use by patients. This stage involves taking the bulk drug substance and transferring it into the final product format that will be administered to patients.

The journey from a vial of frozen cells to a life-changing biologic drug involves a series of meticulously orchestrated steps. Each stage of the manufacturing process plays a crucial role in ensuring the production of a safe, effective, and high-quality therapeutic protein. The collaboration between scientists, engineers, and specialists in various fields is essential to bring these complex treatments from the lab to the patients who need them most.