There are several distinctive features that must be considered in the ATMP development:

1. ATMPs are mainly for the unmet medical needs = severe, rare, or chronic diseases with no adequate conventional treatments.
2. Risk-based approach = Dominant strategy of the ATMPs development.
3. ATMP = strong scientific data + consistent/robust/aseptic manufacturing process.
4. Early start with the potency assays development (mode of action/the intended effect).
5. Quality = starting materials, raw materials, supply chain, etc.
6. Preclinical safety assessment (immunogenicity, biocompatibility, viral or bacterial contamination, ectopic tissue formation, tumorigenicity, mutagenesis, tropism and tissue distribution, stability, biodistribution, shedding, etc.).
7. Difficulties with the reproducibility and high variability (donors, patients, batches, etc.).
8. Bidirectional Traceability system.
9. Designing In Process Control Tests (comparability tests) and Product Release Tests (identity, concentration, viability, recovery, impurities, etc.), including method/tool/equipment validations behind the tests.
10. Formulation, packaging, storage/cryopreservation, and transportation conditions do have a strong impact on the intended effect.

Have you experienced additional difficulties in your ATMP development? We would love to learn more about your development program and explore how we can impact your work.

Let us facilitate your ATMP advancement in the minimum of time and with the most efficient use of resources, while ensuring the high quality. It would be great to establish a cooperation between Venn Life Sciences and your company.

Discover how we can support and accelerate the ATMP development:

• Technical, scientific, and regulatory expertise.
• Provide CMC input into development strategies for your ATMP.
• Assessment of the regulatory requirements for your ATMP development.
• Clinical trial execution under GCP with specific protocol design and considerations for your ATMP.
• Advise on the specific ATMP logistic from the production site to the clinical site.
• QA and audits.
• Biostatistics and Medical Writing.
• Guide, coach, and train professionals in your company.

Blog written by Ion Tcacencu

Ion Tcacencu (MD, PhD) is an ATMP expert with over 17 years of experience as scientist within regenerative medicine and immunology, being responsible for projects developing new ATMPs.

Appropriate scale-up/down is key for further larger production. Important is to focus to the right parameters that can help us to set the design for all experiments at lab scale.
The common approach within the up-stream processing (USP) part in biologics is to increase the scale 10 times. It means from e.g. 25L lab scale to max. 250L pilot scale and later on from pilot scale to max 2500L production scale. Key parameters are vessel geometry, agitation, aeration, back-pressure and feeding profiles (in case of fed-batch fermentation). Next diagram shows typical construction of a common fermenter with key parameters:

Geometric similarity of fermenter geometry is a pre-requisite for applying established scale-up relationship. It´s expressed as follows: D_T2/D_T1 = (V_T2/V_T1)^1/3, where D_Ti is fermenter diameter and V_Ti total fermenter volume. Geometric similarity also assumes reasonably constant impeller geometry such as impeller diameter (D_I) and number of impellers (N). Based on target total a/o working volumes obtained from geometric similarity, the desired working volume in the fermenter may be altered during experimentation. The ratio of the impeller to fermenter diameter (D_I/D_T) in standard fermenters is between 0.3 to 0.45. Fermenters with a standard geometry are beneficial within scale-up correlations assuming constant geometry. Common pilot scale-up fermenters have H_T/D_T ratios of 3:1, but they can also decrease to 1:1.

The first approach within agitation is to check the design of impellers, number of impellers, diameter and location. Common impellers used within microbial fermentations are Rushton turbines or Hollow-blade (U-shape), but could also be Hydrofoil, Maxflo, etc. Stirrer tip speed (STS) is the simplest approach normally used in case of same design of impellers between two scales. It is formulated as STS = πN_ID_I, where π is a constant, N_I and D_I are the impeller speed (s^-1) and impeller diameter (m) in fermenter respectively. Typical STS ranges from 3.8 to 7.6 m/s.

More complex approach within agitation is Constant (gassed) power input per liquid volume (P_G/V_L) which characterizes energy generated by impeller to liquid volume used in the fermenter. It is normally used in a case of scale-up for various design of impellers between two scales. P_G/V_L is expressed as P_G/V_L = P₀/V_L*0.5 = ((N_PN_I³D_I⁵ρ)/V_L)*0.5, where N_P is the power number, which means proportionality factor based on impeller design (N_P for Rushton is 5 and for Hollow-blade  is 1.5). N_I and D_I are the impeller speed (s-1) and impeller diameter (m), ρ is specific broth density (kg/m³) and V_L is volume of fermentation broth (L). General values of P_G/V_L in large scale (fermenter with a total volume more than 1500L) are between 1 to 3 W/L. It´s difficult to have high power per unit volume at the large scale due to practical limitation of the motor size.

Additional scale-up parameters are focused to oxygen transfer. They are Oxygen Uptake Rate (OUR) and Mass transfer coefficient (K_La). Scale-up based on OUR assumes that the OUR is equal to Oxygen transfer rate (OTR). This equation is expressed as OTR = K_La(c_sat – c_L) = OUR = μX/Y_X/O2 , where c_sat is the broth dissolved oxygen (DO) at saturation, c_L is the measured broth DO concentration, μ is the specific growth rate, X is the measured cell density and Y_X/O2 is the cell yield calculated per amount of consumed oxygen. There are several correlations for the determination of K_La using Gassed power per liquid volume (P_G/V_L) described previously and Superficial gas velocity V_S, where the formula looks is K_La = f₂(P_G/V_L)^aV_S^b , where f₂ is a proportionality constant. In general, the “a” value within this equation decreases with increasing working volume. The “a” value is  0.95, 0.67 and 0.5 for fermenters from lab scale (e.g. 10L scale), pilot scale (300L) and production scale (more than 20,000L) respectively. Same holds for the “b” value: 0.67 for lab and pilot scale and 0.50 for production scale. The formula for Superficial gas velocity (V_S) is V_S = φ_G/(π/4)*D_I²), where φ_G is Gass flow rate (m³/s)  and D_I is impeller diameter (m). Finally Gass flow rate can be calculated using φ_G = (Q * (t_act/t₀)*(P_atm/P_absolute))/3600, where Q is airflow rate (Nm³/s), t_act and t₀ are actual temperature and temperature of absolute ZERO in Kelvin and finally P_atm and P_absolute are the pressures. Production fermenters up to 100,000L scale have K_La value between 400 to 800 h^-1. Scale-up based on K_La is complicated approach by the fact that it is process specific and it changes over the fermentation, making it difficult to reliably quantify. Alternatively, measurement of broth DO concentration (C_L) can be used as an adequate scale-up parameter. The minimum acceptance value of C_L is well known from lab scale experiments. Cascade control of DO by agitation, aeration and back-pressure can be effective in maintaining of DO above critical value.

The Table 1 shows an example of most important parameters within scale-down model from 300L pilot fermenter to 75L lab fermenter. Appropriate set of all required parameters in small scale can be predictive for consecutive production in large scale. Finally, all set process parameters need to be confirmed in larger scale via pilot or ENG/Validation batches within further cGMP production.

CMC experts from Venn Life Sciences have strong expertise within theoretical and practical application of these models. Do you need more insight or support with your up-scaling process? Do not hesitate to contact us via our website: www.vennlifesciences.com.

Table 1. Practical application of scale-down model from pilot fermenter (300L) to laboratory fermenter (50L).

300L pilot fermenter	Fermenter parameters used for Scale down	75L lab. fermenter
3.32	Ratio HT/DT	3:1
200L	Working volume Vmax	50L
Rushton, 3 times	Impeller type and No.	Rushton, 3 times
0.23	Impeller diameter DI	0.12
4 kW	Engine power PE	1.1 kW
22 kW/m3	Engine input per working volume PE/VL = PE/(Vmax/1000)	20 kW/m3
420 rpm = 7 rps	Agitation Nmax	800 rpm = 13.33 rps
5.06	Stirrer tip speed STSmax = πNmaxDI	5.03
16.8 Nm3/h = 280 L/min	Airflow rate Qmax	6 Nm3/h = 100 L/min
1.40 vvm (1/min)	Volumetric airflow rate per volume Qmax/Vmax	2 vvm (1/min) Scale down recalculation to 1.4 vvm corresponds to airflow 70 L/min
5.15 W/L (for Nmax)	Constant (gassed) power input per liquid volume P0/VL*0.5 = ((NPNI3DI5ρ)/VL)*0.5	5.50 W/L (for Nmax) Scale-down recalculation to 5.15 W/L corresponds to agitation 782 rpm

Note: For successful scale down model is necessary to set appropriate parameters in lab. scale such as agitation, aeration and back-pressure, that mimic parameters in pilot scale.

Date: 24.10.2022

Author: Juraj Boylo

CMC evolution during gene therapy development

Gene therapy offers significant potential to treat diseases with high unmet medical need. However, the unique nature of these therapies poses challenges in product development, namely: safety concerns, efficacy issues, or obstacles related to Chemistry, Manufacturing and Controls (CMC). CMC is an integral aspect of gene therapy development. Done right, CMC development of complex manufacturing processes and analytical testing panels can accelerate clinical development and bring these important medicines to patients faster. For gene therapy products, CMC development often needs to be done in a highly compressed timeline, and there are important CMC challenges that need to be addressed to bring these products into the clinic and beyond, amongst which:

Setting the scene

Over the past century, vaccines have made a large impact on public health, as recently demonstrated by the treatment of the worldwide Covid-pandemic. Since, in general, vaccines are administered to healthy individuals, including infants and children, it is important to demonstrate the safety of vaccines with proper in vitro testing and in vivo animal testing prior to starting clinical studies with the vaccine candidate. Therefore, over the past decade, there has been an increased focus on nonclinical safety assessment of vaccines, including testing for potential toxicity.

Non-clinical guidelines for vaccines

Over time, the extent of nonclinical safety testing has been greatly increased and current regulatory guidelines (see inset) require a comprehensive package of safety studies to be performed. These guidelines are aligned with overall principles of toxicological evaluation (i.e., detection of potential for systemic and local toxicity) but allow for appropriate flexibility in study designs according to the type of the vaccine candidate, the specific disease for which the vaccine will be used, the human population to be treated and the dosing regimen to be applied in the clinical use (see inset).

Strategy for nonclinical safety/toxicity testing

Prior to starting clinical studies with vaccines, adequate information about the pharmacology and toxicological effects of the vaccines should be available. The program of safety studies to be performed varies depending on the type of vaccine, disease to be treated and intended use in humans (e.g., number of injections). It is important to have an optimized strategy and planning of the proper non-clinical studies to best and quickest transition into clinical studies. Nonclinical testing of vaccines is different from testing of small molecules and the appropriate (combination of) studies has to be deducted for each specific vaccine candidate based on applicable guidelines and experience.

Regulatory toxicology studies for testing the safety of vaccines are performed in vitro and in animal studies and need to be performed in compliance with Good Laboratory Practice (GLP). Venn experts can help you in selecting CRO’s, in monitoring the progress of your studies, reviewing reports and in drafting regulatory documentation.

Next step - clinical (challenge) studies

A clinical development approach that has become more commonplace in recent years, is to test different dosing regimens in a Human Challenge Study. In example, different adjuvants and dosing approaches (with or without booster) are tested in these challenge trials, to obtain the best possible dosing regimen for phase 2 / 3 field trials. In the design of your nonclinical safety/toxicity testing program, you also have to take into account these options to allow you to take the most out of the challenge trials and optimalise your development program.

Venn experts can help you to select the appropriate studies and define a Drug Development Plan for your vaccine candidate.

Why Venn’s Non-Clinical team

The IND filing and first clinical studies for your lead vaccine can be a lengthy and costly process. Developing the right strategy and study designs is the first step in the direction toward an optimized nonclinical program. The combination of specific scientific non-clinical expertise combined with detailed knowledge of regulatory requirements for vaccine development, commitment to your projects and the constant pursuit of quality excellence make Venn a reliable source for Non-Clinical Consultancy Service for your vaccine projects.

Each journey begins at a starting point, and we believe that few journeys are as exciting as developing a drug. In order to succeed and reach your destination, you need a solid roadmap, the drug development plan (DDP), this is the outline of your drug development strategy. 

A comprehensive drug development strategy outlines each step for advancing a new compound from the lab through each stage of development, ultimately arriving at the envisioned marketed drug product. 

This requires a multi-disciplinary project team of experienced experts in strategic planning, which outlines the chemistry, manufacturing, and controls (CMC) and formulation activities, key non-clinical studies, and Phase I-III clinical trials, regulatory submissions, and health authority interactions, market launch activities, and life-cycle management. A drug development strategy is a dynamic process that moves forward in defined stages. A carefully planned set of steps and action plans, progressing from non-clinical to the first-in-human Phase I to Phase II “proof of concept” and pivotal Phase III trials for registration.

The drug development process can be complex and involves various strategic steps that can reduce timelines and costs

A proper development strategy should answer the following:

1. What is the intended target population and indication?
2. What are the desired product attributes: indication or usage & dosage, administration & pharmacology, adverse reactions & toxicology, clinical data, clinical safety, and efficacy. 
3. Identifying the most feasible country for clinical trials. global data acceptance, developmental infrastructure, clinical developmental costs.
4. Identifying the most efficient and effective ways for clinical developments
5. What are the potential markets and what are your marketing strategies?

Identifying these steps early is an essential part of a successful development. Our Venn experts can guide through your journey.

At Venn Life Sciences, our team of experienced consultants have the expertise to help with the various steps in drug development to ensure that you receive the support and direction required to formalise your drug development. We have integrated drug development partners offering a unique combination of drug development consultancy, clinical trial design and execution.

Learn more about our drug development services here.

The first‐in‐human (FIH) clinical trial is an important milestone for each development program. For small (bio)pharma companies the FIH trial requires a significant investment, and every sponsor wants to make sure that all is well set for starting the trial.  It is indisputable that successful execution of the FIH trials does require sponsors to coordinate the task with thorough consideration and planning across many disciplines. Especially, selection of the starting dose requires a cross‐functional collaboration, where preclinical knowledge needs to be transformed into clinical applications. This step could become a large hurdle which could even retard the progress of the program, if planned and strategic alignments are not reached within the project team as well as adequate actions are not taken well in advance of the FIH trial.

Over the past years, Venn Life Sciences as a consultant company, has been exposed to a significant number of FIH trials both in healthy volunteers as well as oncology patients. In this blog, we like to highlight several considerations, based on these experiences, that will need to be addressed for defining the starting dose and beyond in the FIH trial. 

MABEL or NOAEL?

The EMA’s FIH guideline says, “Depending on the level of uncertainty regarding the human relevance of findings observed in nonclinical studies and the knowledge of the intended target, the starting dose should either be related to the MABEL (Minimum Anticipated Biological Effect Level), PAD (Pharmacologically Active Dose) or NOAEL (No Observed Adverse Effect Level).” Thus, the choice of MABEL- or NOAEL-based starting dose selection must be driven by overall assessments of available data and risk-based considerations. Typical factors for the assessments include but are not limited to:

• predicted safety risk in human based on nonclinical studies and its monitorability in a clinic trial setting
• prior knowledge on safety in a clinical setting with marketed or competitor products with the same or similar mode of action
• whether the product has agonistic or antagonistic characteristics
• immune stimulators, or another mode of action which involves a biological cascade
• therapeutic indications (healthy volunteers or patients, taking into account the severity of the disease under investigation)

It is highly recommended to make a rationalized choice of the MABEL- or NOAEL-based approach (e.g. using decision tree) with adequate documentation in investigator’s brochure, as this is a critical point of review by the competent authority/ethics committee.

Do you have sufficient/adequate data set for MABEL calculations?

To employ the MABEL-based starting dose establishment approach, it is essential that relevant data sets have been generated by the sponsor. Unlike toxicology (NOAEL)-based approach for FIH dose selection, which is rather straightforward, the type of data required for MABEL calculation can differ case by case (e.g. in vitro and/or in vivo data and in recent years increasingly involve modelling and simulation). Since generation of relevant data may take several months, any absence of relevant data for the MABEL calculation could cause a significant delay in the program. Therefore, it is pivotal that the project team plans this exercise way in advance of the FIH preparations.

How to define “minimum” effect?

One of the most frequently asked questions in terms of MABEL calculation is what degree of a biological response is considered “minimum”. Unfortunately, there is no standard answer for the question, and this is nowhere well-defined in the guidelines, as there are multiple unique factors that may need to be considered for each investigational product. For example, since receptor occupancy required for efficacy are expected to be different between antagonists and agonists even if the same receptor is targeted, different considerations are likely required for defining the MABELs for such cases. Ultimately, the sponsor is responsible to scientifically justify underlying rationale for defining the MABEL.

Starting dose calculation

While mechanistic state-of-the-art modelling (e.g. PK/PD and PBPK) is increasingly used in the calculation of the starting dose based on NOAEL, PAD and MABEL, it should be emphasized that the EMA guideline also refers to the application of allometric factors. Unless the drug product is associated with significant safety risk in human based on preclinical finding and/or a certain drug class (e.g. immune stimulator) and thus requires more sophisticated mathematical approach, empirical allometric scaling approach in combination with a simulation of exposure-pharmacological response relationship in human might also be sufficient for the starting dose selection for the FIH trial in healthy volunteers. Irrespective of approaches chosen, careful deliberation on the planning of the starting dose assessment is required, as unnecessary assessments during this process may also cause a delay in the program and a needless financial burden.

Applying safety factors

After the calculation of the MABEL, PAD, or NOAEL, additional safety factors are applied to account for several potential risks, such as the relevance of animal models, any uncertainties in the estimation of the MABEL, PAD, or NOAEL, or other unique safety or pharmacodynamic characteristics of the investigational product. Inadequate assessment of the safety factors may result in unnecessary risk for volunteers, or unnecessarily low doses in the first cohorts and as such additional dose escalation cohorts may be needed.

Be clever in trial design/protocol

Despite of all exceptional efforts and fancy mathematical MABEL calculations, pharmacokinetic properties might behave differently in human in the FIH trial, especially at the low starting dose, where PK tends to be non-linear. The clinical trial protocol, therefore, should clearly define the dose escalation rules to allow for the dose escalation to proceed in a scientifically, ethically as well as financially acceptable way. For example, the clinical protocol should include adequate language to allow for more aggressive dose increments, in case the observed exposure at the starting dose is significantly lower than predicted. Otherwise, volunteers will continue to be administered at dose levels even lower than the MABEL, which may result in the need of additional cohorts, or in the case of patients, treatment at an unnecessarily low and ineffective dose. We highly recommend authoring an adaptive FIH protocol, which will allow adjustments in the dosing schedule, circumventing delays in the trial execution.

Concluding remarks:

• Thorough consideration and cross-disciplinary collaboration toward selection of the starting dose well in advance to the FIH trial is pivotal for the success of the program (at an early stage as possible and at latest initiate this when GLP tox study is being planned).
• There is no gold-standard approach to selecting the starting dose in the FIH trial, as each new drug entity is unique in many aspects and data set required can largely differ case by case.
• Venn Life Science is happy to think with the sponsors and apply our experiences gained in these matters for the best possible approach tailored towards the client’s goals and means.

If you are interested to learn more about how to define a safe starting dose for your FIH trial, please contact Venn life Sciences at BD@vennlifesciences.com. We would welcome the opportunity to discuss efficient set up of your FIH trial and other related aspects.

Did you ever ask yourself one of the following questions?:

• “…a significant difference (p=0.002) was observed between the two groups…” What does this mean? What exactly is a p-value and what does it tell me?
• What is the difference between an independent 2-samples t-test and a paired t-test?
• Can I trust this publication of a clinical trial I’ve just read? How can I evaluate its quality?
• Estimand? What exactly is an estimand? How does the ICH E9 (R1) addendum on estimands and sensitivity analysis in clinical trials to the guideline on statistical principles for clinical trials impact the clinical trial I am planning?
• What is an adaptive trial? Should I consider one in the Clinical Development Plan of my product?
• The vaccine efficacy is 95%, does that mean that 95% of vaccinated subjects will not develop the disease?
• Intention-To-Treat. Why does it matter?

Statistics play a fundamental role at each step of a clinical trial (trial design, protocol development, the statistical analysis planification and execution, interpretation of the results). With the evolution of statistical science (e.g., progresses in the understanding of mechanisms generating missing data and methods to handle missing data) and the development of new clinical trial methodology concepts (e.g. adaptive designs, master protocols…) new challenges have emerged.

As a non-statistician clinical trial professional from the pharmaceutical industry, biotech industry or health research, you may feel uncomfortable with some statistical concepts or methods and in your interactions with statisticians.

At Venn Life Sciences Biometry Services, our expert methodology and biometry consultants provide training to fulfill this gap.

Several topics can be covered, and a training program specifically tailored to your own needs can be prepared:

Example of recent training activities

The Food and Drug Administration (FDA) is an agency within the U.S. Department of Health and Human Services. It is the US Agency responsible for the regulation of drugs and biologics, including, vaccines for humans, blood and blood products and cellular and gene therapy products.

As the United States remains the largest pharmaceutical market in the world, any drug development program will require a close interaction with the FDA to ensure your development rationale is sound and that the design of your studies is fit-for-purpose.

Although a first contact with the FDA might sometimes feel intimidating, an early interaction will prove very valuable. Your first interaction with the FDA will likely be a pre-IND meeting. Sensu-stricto a pre-IND meeting is not required, it is however highly recommended to engage with the FDA in a pre-IND meeting for the following reasons:
Early Feedback from the FDA - The most valuable benefits of the pre-IND meeting are to receive early feedback directly from the FDA on your development program and to gain an understanding as to what the FDA’s expectations are for your drug.

Fine-Tuning of your Development Strategy – Taking the feedback in the FDA into account, offers you the opportunity to fine-tune your program development strategy, potentially saving time and money.
Relationship Building – The pre-IND meeting is also the opportunity to start building a working relationship with the FDA and individuals within the Agency that will be your contact persons.

In preparation of your pre-IND meeting, you will need to prepare a briefing package. In this briefing package you will provide background information on your compound and prepare the list of questions. When preparing these questions, there a few things you need to keep in mind:

• FDA meetings are most effective when they are focused on specific scientific or regulatory issues, such as clinical trial design, pharmacology studies, toxicology studies, acceptability of novel formulations, dosing limitations, data requirements for an IND application, etc.

• Speculative and open-ended questions are difficult to address; meetings are typically most productive when questions are focused and specific. Questions for the FDA should be posed in such a way that the agency can either agree or disagree with the question.

As the briefing package forms the basis of a successful meeting, it is important that it is well worked out. Taking advantage of the valuable input that can be gained from pre-IND meetings is essential for the further development of the drug and may reduce the drug’s time to market and ensure that the proposed studies are designed to provide useful information. Sponsors who are forthcoming with potential issues of concern during the drug development process will benefit from input provided by the regulatory agency.

If you are interested to learn more about how our Regulatory Affairs department can support your development activities, please contact Venn life Sciences at getintouch@vennlife.com.