What Are Critical Process Parameters (CPPs)?

Critical Process Parameters (CPPs) are the process variables that have a direct and significant impact on the Critical Quality Attributes (CQAs) of a therapeutic product. In other words, CPPs are the parameters that must be controlled within predefined limits to ensure that the final product consistently meets its intended quality, safety, and efficacy specifications.

What is the difference between CPP and CQA?

CQAs are the what — they describe the final product attributes that must meet predefined specifications to ensure the product’s safety, quality, and efficacy. Examples include:

• Cell viability ≥ 80%
• Correct cell phenotype (e.g., CD4/CD8 ratio, CAR expression ≥ 20%)
• Potency (e.g., ability to kill target cells in vitro)
• Purity (absence of residual beads or vector)

CPPs are the how — they are process variables that, when not properly controlled, directly affect CQAs. They are the knobs and dials of your manufacturing process. Typical CPPs in cell therapy manufacturing may include:

• Temperature, pH, and dissolved oxygen during cell expansion
• Transduction parameters such as multiplicity of infection (MOI) and exposure time
• Centrifugation speed and time during cell washing or harvesting
• Cell seeding density and media exchange frequency
• Cryopreservation conditions, including cooling rate and DMSO concentration

The two are similar because they are tightly connected — you control CPPs to achieve the desired CQAs. However, they differ because CQAs describe what the product must be, while CPPs describe how the process must be run to get there.

For example, in MSC or CAR-T manufacturing:

• The multiplicity of infection (MOI) during viral transduction is a CPP because it directly affects a key CQA: the level of CAR expression on the final T cells.
• Keeping dissolved oxygen (CPP) within tight limits reduces oxidative stress, preserving cell viability and stemness (CQAs).
• Maintaining culture parameters such as pH, temperature, and nutrient levels (CPPs) within optimal ranges supports predictable cell growth, phenotype, and potency (CQAs).

For companies developing scale-up solutions for cell therapy, understanding CPPs is especially crucial, as each step involves fine-tuned adjustments to process variables.

Typical CPPs in cell therapy manufacturing may include:

• Temperature, pH, and dissolved oxygen during cell expansion
• Transduction parameters such as multiplicity of infection (MOI) and exposure time
• Centrifugation speed and time during cell washing or harvesting
• Cell seeding density and media exchange frequency
• Cryopreservation conditions, including cooling rate and DMSO concentration

Startups leveraging hassle-free bioprocessing tools and curated lab instruments and services gain an advantage by accessing pre-validated systems that help maintain control over these CPPs, reducing trial-and-error at early stages.

Why CPPs Matter in Cell Therapy: Living Products with High Variability

Unlike small molecules or biologics, cell therapies are living drugs—dynamic, heterogeneous, and highly sensitive to their environment. Even minor changes in culture conditions or raw material quality can significantly affect the phenotype, potency, or safety profile of the final cell product.
To succeed, startups need cell culture scale-up solutions that not only handle larger batch sizes but also ensure precise control over CPPs, from lab-scale runs to manufacturing.

This inherent variability makes it critical to identify and tightly control CPPs throughout the manufacturing process. A small deviation in a CPP may lead to:

• Loss of cell viability or function
• Inconsistent expression of transgenes or markers
• Altered cytokine profiles or immunogenicity
• Batch failure or out-of-specification results
• Ensuring quality, safety, and consistency

Identifying and controlling CPPs is central to building a robust, reproducible, and scalable manufacturing process. In clinical development, this helps demonstrate product comparability and lot-to-lot consistency. In commercial manufacturing, well-defined CPPs reduce batch failures, support regulatory inspections, and enable continuous improvement through process control strategies.

Companies offering scale-up solutions for cell therapy play a key role here, providing startups with the tools and systems they need to translate small-batch processes into robust, reproducible manufacturing pipelines.

Quality by Design (QbD) is a systematic approach that emphasizes understanding and controlling manufacturing processes to ensure product quality by design, not just by end testing.

In cell therapy, QbD applies by:

For example, in MSC or CAR-T manufacturing:

• Keeping dissolved oxygen within tight limits reduces oxidative stress, preserving cell viability and stemness (CQAs).
• Tightly controlling the MOI and duration of transduction ensures consistent gene delivery efficiency while minimizing the risk of insertional mutagenesis.
• Maintaining culture parameters within optimal ranges supports predictable cell growth, phenotype, and potency.

By using QbD, you move from empirical, trial-and-error processes to science-based, risk-managed manufacturing, which improves reproducibility, reduces batch failures, and supports regulatory confidence.

Regulatory Expectations for CPP Identification, Control, and Justification

Global regulatory agencies place strong emphasis on CPP identification and control as part of the Chemistry, Manufacturing, and Controls (CMC) section of clinical trial and marketing applications.

FDA (USA)

Expects a risk-based approach in identifying CPPs through process development and validation. CPPs must be linked to CQAs through data-driven analysis (e.g., Design of Experiments, multivariate analysis).

EMA (Europe)

Requires a comprehensive control strategy that includes CPPs, in line with the principles outlined in ICH Q8–Q11.

HSA (Singapore)

Aligns with ICH and WHO guidance, emphasizing the importance of CPPs in ensuring product consistency and patient safety.

Regulators may request:

T cell therapy example: 

During CAR-T cell manufacturing, the multiplicity of infection (MOI) used during viral transduction is a CPP. 

Developers must generate data showing that transduction at MOIs between 3 and 5 achieves a stable CAR expression level (e.g. >20%) without causing excessive cell stress or reducing viability. 

Design of Experiments (DoE) studies can help determine the optimal range and demonstrate that small fluctuations within this range do not compromise the product’s potency or phenotype.

MSC therapy example:

In mesenchymal stem cell (MSC) expansion, dissolved oxygen levels are a CPP because MSCs are highly sensitive to oxidative stress, which can compromise their therapeutic potential. 

Culturing MSCs under physiological (low) oxygen tensions (~3–5%) compared to atmospheric oxygen (~20%) results in significant advantages: lower oxygen enhances MSC proliferation, better preserves key stemness surface markers (CD73, CD90, CD105), and reduces the accumulation of senescence-associated markers such as β-galactosidase. The study further shows that low oxygen conditions improve genetic stability and reduce oxidative DNA damage, supporting long-term MSC expansion for clinical use.

As such, clinical manufacturing processes often adopt 5% ± 1% O₂ as the target control range, recognizing that deviations outside this range can increase oxidative stress, induce premature senescence, or impair the differentiation potential of MSCs. Maintaining this CPP ensures consistent product quality, potency, and safety for MSC-based therapies.

T cell therapy example:

The rotation speed of a rocker platform used during CAR-T cell expansion in G-Rex flasks may be found to have no significant impact on viability, cytokine profile, or CAR expression in a sensitivity analysis. The rotation speed serves mainly to prevent cell clumping or ensure even distribution but has minimal direct effect on key product attributes when kept within a validated range.

As such, developers can reasonably justify treating the rotation speed as a non-critical process parameter (non-CPP) — meaning it should be monitored and recorded but does not require tight control or real-time adjustment. Instead, it is sufficient to operate within a predefined acceptable range (e.g., 10–20 rpm) established through process characterization. 

T cell therapy example: 

A deviation occurs when transduction was performed for 18 hours instead of the validated 16 hours due to operator error. Investigations must document whether this affected CAR expression, transduction efficiency, or T cell viability. If the final product meets all CQAs (e.g., identity, potency, purity), a deviation report with root cause analysis and impact assessment must be submitted to the regulatory authority.

MSC therapy example:

A batch of MSCs experienced a short-term drop in incubator CO₂ levels during passage (from 5% to 2% for 2 hours). The deviation report should assess whether this affected cell doubling time, surface marker expression, or immunomodulatory activity. Results from in-process and final product testing (e.g., IDO activity, flow cytometry) would support the quality impact assessment.

T cell therapy example:

If switching from open transduction in tissue culture plates to closed-system transduction in bags, the MOI and duration may need to be adjusted due to different surface area-to-volume ratios. A comparability study must assess whether the new process results in equivalent CAR expression, T cell phenotype (CD4/CD8 ratio), and potency (e.g., tumor killing in vitro).

MSC therapy example:

Changing the culture platform from T-flasks to a stirred-tank bioreactor can alter shear stress and oxygenation. A comparability study is needed to evaluate whether MSCs expanded in the new system maintain key characteristics: surface markers (CD73, CD90, CD105), trilineage differentiation potential, and immunomodulatory function (e.g., T cell suppression assay).

CPPs in Startups: Why Focus on Them From Day One?

Limited Resources Require a Risk-Based Focus

Start-ups often operate with limited resources — small teams, tight budgets, and minimal time. This makes it impossible to monitor and control every single process variable with equal rigor. Instead, a focused, risk-based approach is essential, and CPPs provide a clear way to prioritize effort.

By identifying which parameters directly impact product quality, safety, and potency, start-ups can determine what absolutely must be controlled versus what can be more flexible without compromising outcomes. This prioritization guides where to allocate Quality Control (QC) efforts, testing resources, and process monitoring, ensuring that limited capacity is focused on the areas that matter most.

It also helps companies make smart trade-offs between regulatory compliance, operational cost, and development speed — avoiding overengineering non-critical steps while maintaining the controls needed for regulatory approval.

Startups Need to Build Scalable Processes from the Ground Up

Unlike big pharma with established platforms, startups are often developing processes from scratch. Identifying CPPs early ensures they don’t bake instability or variability into their pipeline. This affects:

• Process scalability (from lab to GMP)
• Batch consistency (needed even for early clinical trials)
• Cost-efficiency (avoiding repeated batch failures)

By working with providers of hassle-free bioprocessing tools for startups, they can focus efforts on the CPPs that matter most, streamlining quality control and reducing unnecessary complexity. Accessing curated lab instruments and services allows small companies to scale up confidently, knowing they’re using fit-for-purpose tools optimized for their needs.

Early Regulatory Submissions Require Process Understanding

Even at the Investigational New Drug (IND) stage, when a company is seeking FDA approval to begin clinical trials in humans, regulators expect sponsors to demonstrate a basic but meaningful understanding of their manufacturing process. 

The IND application includes Chemistry, Manufacturing, and Controls (CMC) sections, where companies must describe how the product is made, what materials and equipment are used, how quality is monitored, and what process parameters might influence product safety, identity, strength, purity, and potency.

For start-ups, this means that having a clear CPP strategy is not optional — it is essential. Regulators will review whether the company has identified and controlled key parameters that could affect the safety and consistency of the product administered to patients. Without this, reviewers may raise concerns about process robustness, batch-to-batch variability, or the risk of unexpected failures, which could delay or block clinical trial approval.

By showing thoughtful, science-driven control over CPPs, start-ups strengthen their regulatory positioning. It signals to the FDA (or other authorities) that even as a small or early-stage company, they understand their product and process well enough to ensure patient safety, comply with GMP expectations, and reliably deliver material for clinical trials.

Avoid Expensive Rework in Tech Transfer or Scale-up

Without clearly defined CPPs, scaling up manufacturing is risky and costly. Start-ups often begin with manual or small-scale protocols, but shifting to semi-automated or fully automated systems can introduce small differences that cause major issues — like unexpected changes in cell growth, viability, or product quality.

If critical process parameters aren’t well understood, teams may find too late that the process isn’t reproducible at larger scales or in new systems, leading to failed batches, regulatory delays, or costly rework.

Early investment in cell culture scale-up solutions helps minimize these risks by offering pre-engineered systems that scale smoothly from lab to production. Defined CPPs are key for successful tech transfer, ensuring both the original developers and the receiving site know what must be controlled, what ranges are acceptable, and how to maintain product quality across equipment, teams, and locations.

By identifying and controlling CPPs early, start-ups save time, money, and frustration on the path from lab bench to commercial-scale production.

Conclusion

For cell therapy start-ups, mastering CPPs isn’t just about compliance — it’s the gateway to reliable scale-up, faster clinical progress, and commercial viability. Early focus on CPPs reduces costly surprises by pinpointing which variables truly impact product quality, avoiding wasted effort on noncritical details.

Strategic use of tailored cell culture scale-up solutions and process analytics minimizes trial-and-error, enabling smarter, data-driven decisions. This not only boosts manufacturing robustness but also strengthens regulatory positioning, accelerating time to market.

In short, a sharp CPP strategy is not overhead — it’s a competitive advantage!

References

1. Estrada JC et al. (2012). Culture of human mesenchymal stem cells at low oxygen tension improves growth and genetic stability by activating glycolysis. Cell Death & Differentiation, 19, 743–755.
2. Timmins et al. (2021) Selecting a Cell Engineering Methodology During Cell Therapy Product Development. Cell Transplant. 30:9636897211003022.