Regulatory Success Tips for Bringing Protein Purification to Regulatory Agencies: Speed process development and path to clinic

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Protein purification is a crucial part of biopharmaceutical manufacturing process development, especially in the development of gene and cell therapies (CGT). Ultimately, these process development details will end up facing regulatory agencies as part of the manufacturing quality, or "Chemistry, Manufacturing and Controls (CMC)" (also known internationally as "Module 3") write-up that enable drug developers to start clinical trials and gain drug approval.

What is Protein Purification in Biopharmaceutical Process Development?

Protein purification involves separating and isolating the therapeutic protein from a complex mixture of cellular components, ensuring that the final product is pure and free from contaminants.

Accuracy in protein purification is vital for several reasons:

1. Safety: Purifying the therapeutic protein to its highest degree of purity is essential to eliminate any impurities or contaminants that could potentially cause adverse effects in patients.
2. Efficacy: Impurities and contaminants can reduce the therapeutic effectiveness of the protein, impacting its intended pharmacological activity or triggering undesirable immune responses in the patient.
3. Regulatory Compliance: Stringent regulatory requirements demand that therapeutic proteins are of high purity and quality, with stringent specifications to meet safety and efficacy standards.

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Importance of Protein Purification in Process Development

?At the discovery stage, selecting the right equipment to achieve optimal purification is important, and it may be quite easy to iterate, optimize, and change the purification process. As companies get closer to clinical trials, more control and process "lock" will occur that enables a repeatable and compliant manufacturing process for the drug material used in critical animal toxicology studies (or "Good Laboratory Practice (GLP)" studies) and in humans, under current Good Manufacturing Practices (cGMP).

In process development, protein purification is important in:

Process Optimization: The purity of the therapeutic protein influences downstream processes such as formulation, stability, and storage. A well-established purification process contributes to the overall efficiency of the manufacturing process.

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1. Product Quality: The purity of the therapeutic protein directly impacts its quality attributes, including potency, stability, and bioavailability. Consistent and optimal purification ensures a reliable product with predictable performance.
2. ?Cost Consideration: Efficient purification processes impact the overall cost of manufacturing, making it essential to develop cost-effective and scalable purification methods for commercial production.

Protein Purification and its?Impact on Investigational New Drug (IND) Development and Clinical Trials

Delays in protein purification optimization can significantly impact the progress of investigational new drug programs and clinical trials, in particular by delaying the readiness of cGMP drug product for investigational testing. Some key concerns are:

1. Inadequate Purity: The presence of impurities can lead to safety concerns, requiring further process optimization and validation before initiating clinical trials.
2. Yield Variability: Inconsistent purification processes can lead to unpredictable protein yields, affecting the availability of sufficient material for preclinical and clinical studies.
3. Long Process Development-- and ultimately manufacturing delays: Iterative optimization of purification processes can prolong the development timeline, delaying IND submissions and subsequent clinical trial initiation.

There are several ways we can optimize our proteins purification strategy to avoid these setbacks by focusing on

1. Robustness: The purification process should be robust, reliable, and scalable to ensure consistency in yield and purity across multiple batches.
2. Selectivity: The strategy should effectively remove impurities and contaminants while preserving the stability and biological activity of the therapeutic protein.
3. Process Integration: Seamless integration with upstream and downstream processes, such as cell culture and formulation, is essential for process efficiency and product quality.
4. Validation and Compliance: The purification process should be validated to meet regulatory requirements, with stringent documentation of purification steps, critical parameters, and quality control measures.
5. Cost-Effectiveness: Balancing purification costs with process efficiency is critical to ensure that the strategy aligns with commercial production requirements.

Each of these areas also plays into downstream complexity of regulatory filings, and the complexities added to programs (and regulatory interactions) when there is a need to make changes during critical manufacturing activities for key toxicology studies in animals or in product for use in human clinical trials.

Examples of Delay Due to Protein Purification

Example 1: Cell Therapy

A cell therapy candidate required meticulous purification of a specific membrane glycoprotein to ensure safety and efficacy. Challenges in achieving high purity caused delays in process development and subsequent IND filing and clinical trial first-patient in -- which can mean months of delays to major milestones and millions of burn during small biopharma development programs.

In this case for a novel cell therapy candidate targeting a membrane glycoprotein as its critical therapeutic target, the membrane glycoprotein, when isolated and purified, demonstrated potential for targeted therapeutic efficacy in specific disease indications. However, due to the complex nature of membrane proteins, achieving high purity and maintaining biological activity posed significant challenges.

As scientific background, membrane proteins are known for their hydrophobic properties and complex tertiary structures, making their isolation and purification technically demanding. Techniques such as affinity chromatography, ultrafiltration, and diafiltration are commonly employed, but the delicate balance between purity and preservation of protein structure and function requires meticulous optimization.

In this case, early optimization was essential to the regulatory filings and clinical trial start date, and later changes can not only delay manufacturing but also regulatory clearance to proceed with the trial.

Case details from: Butler TJ, Smith SM. Strategies for the Purification of Membrane Proteins. Methods Mol Biol. 2023;2699:477-491. doi: 10.1007/978-1-0716-3362-5_20. PMID: 37647009.

Example 2: Viral Vector Gene Therapy

Gene therapy products with viral vector proteins necessitate stringent purification to eliminate host cell DNA and residual viral components. Optimization of purification strategies was critical to avoid potential safety risks and regulatory hurdles. The development of gene therapy products involved the use of viral vectors, and the purification of viral vector proteins is a critical aspect of the manufacturing process. Ensuring the elimination of host cell DNA, residual viral particles, and other contaminants is essential to mitigate safety risks and regulatory concerns.

As scientific background, the purification of viral vector proteins involves specialized techniques to remove impurities while preserving viral vector integrity and functionality. Purification methods may include ultracentrifugation, chromatography, and filtration, with a focus on selective separation and removal of contaminants.

For gene therapies, it is critical to have the correct and optimized protein purification steps in an IND to start a clinical trial. And downstream changes later on can cause delays with clinical trials.

Case details from: Kearney AM. Chromatographic Purification of Viral Vectors for Gene Therapy Applications. Methods Mol Biol. 2023;2699:51-60. doi: 10.1007/978-1-0716-3362-5_4. PMID: 37646993.

These examples illustrate the intricate challenges involved in purifying specific types of proteins for advanced therapeutic applications, emphasizing the critical role of robust protein purification strategies in advancing cell and gene therapy development for clinical use. For further scientific literature and detailed research references, I recommend consulting established academic databases or scholarly journals specific to protein purification and biopharmaceutical manufacturing.

Common purification methods for viral vectors in gene therapy

Common purification methods for viral vectors in gene therapy manufacturing include:

1. Chromatography: Chromatographic techniques, such as affinity chromatography, ion exchange chromatography, and size-exclusion chromatography, are frequently employed for the purification of viral vectors. These methods allow for the separation and purification of viral vector particles based on size, charge, or specific interactions with ligands.
2. Ultracentrifugation: Ultracentrifugation is utilized for the concentration and purification of viral vectors by pelleting viral particles at high centrifugal forces. This method can help to separate viral vector particles from impurities and cell debris based on their density.
3. Filtration: Tangential flow filtration (TFF) and size-based filtration methods are employed for the removal of impurities and the concentration of viral vectors. TFF allows for continuous filtration and concentration of viral vectors, while size-based filtration can effectively eliminate larger impurities and contaminants.
4. Precipitation: Viral vector purification may involve the use of precipitation techniques, such as polyethylene glycol (PEG) precipitation, to concentrate and purify viral particles by inducing their precipitation while leaving impurities in the supernatant.
5. Aqueous Two-Phase Systems (ATPS): ATPS can be utilized for the partitioning and purification of viral vectors based on their interactions with polymer–polymer or polymer–salt systems, allowing for the separation of viral vectors from host cell components.

In the context of gene therapy and viral vector manufacturing, the selection of purification methods is influenced by factors such as the specific characteristics of the viral vector, product yield requirements, scalability, purity targets, and the need to ensure the safety and efficacy of the final gene therapy product.

Regulatory CMC Aspects of Protein Purification

The Quality section of the Common Technical Document (ICH M4Q) provides a harmonized structure and format for presenting CMC (Chemistry, Manufacturing and Controls) information, including that for protein purification, to regulatory agencies as part of clinical trial applications and drug approvals. ICH M4Q was formally recognized by FDA in 2001 and is the most widely accepted international standard for presenting drug manufacturing information.

Importance of CMC Flow Charts and Visuals to Regulatory Agencies

According to ICH M4Q, a flow diagram should be provided that illustrates the purification steps (i.e., unit operations) from the crude harvests up to the step preceding filling of the drug substance. All steps and intermediates and relevant information for each stage (e.g., volumes, pH, critical processing time, holding times, temperatures and elution profiles, selection of fraction, and storage of intermediate, if applicable) should be included.

Example Flowchart and Corresponding ICH Standards

Image Source: Ho & Afssaps "Manufacturing Process of Biologics" ICH 2011

The CMC regulatory dossier is broken down into "Module 3" or "3.X" sections that are primarily about the drug product (DP in sections 3.2.P.xxx) and drug substance (DS sections in 3.2.S.xxx). You cna find the full structire of a drug filings here: eCTD structure headings. Key sections for protien puridicaiton processes include 3.2.S.2.2 to 3.2.S.2.6:

• 3.2.S.2.6 Process development (same principles as Q8)
• 3.2.S.2.2 Description of Manufacturing / Process Controls
• 3.2.S.2.3 Control of Materials
• 3.2.S.2.4 Controls of Critical Steps and Intermediates
• 3.2.S.2.5 Process Validation and/or Evaluation

According to ICH M4Q, critical steps for which specifications have been established are mentioned in 3.2.S.2.4, and a description of each process step (matching the visual or flow diagram) should be included in the regulatory filing CMC sections. Descriptions should include information on, for example, scale, buffers, and other reagents (this goes into section 3.2.S.2.3), major equipment (in the annex to Module 3, 3.2.A.1), and materials. For materials such as membranes and chromatography resins, information for conditions of use and reuse is also required to be in the regulatory filing (also in3.2.A.1; provide validation studies for the reuse and regeneration of columns and membranes in 3.2.S.2.5). The description should include process controls (including in-process tests and operational parameters) with acceptance criteria for process steps, equipment, and intermediates. (include in 3.2.S.2.4.) Reprocessing procedures with criteria for reprocessing of of intermediates or drug substances should also be detailed in the regulatory filing (include in 3.2.S.2.5.)

What does it take for regulatory success in protein purification?

In summary, the success of cell and gene therapy manufacturing and clinical development hinges on the optimization of manufacturing processes, including optimizing protein purification processes early and in a timely manner that minimizes the risk of delay to key toxicology and human clinical trial activities-- including filing of the IND/CTA required to start clinical trials containing the appropriate level of regulatory detail in the CMC (Module 3 Drug Substance manufacturing sections). By addressing challenges, adhering to regulatory standards, and prioritizing purity and yield, drug developers can streamline the path from process development to IND submissions and clinical trial initiation for novel therapeutics.

Angela Johnson, PhD, RAC is the head of global regulatory compliance for Cytiva and lecturer on regulatory strategy at Northeastern University. She has led two venture-backed biotech regulatory teams to IPO and held leadership roles at IQVIA, GE Healthcare, and APAC consulting firms in drug development, and is an active member of the Regulatory Affairs Professionals Society (RAPS) and regulatory board member of the American Society of Cell & Gene Therapy (ASGCT), with more than 30 publications on regulatory and drug development.

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