Design of Experiment – Applications of DOE

Cell Culture – Use of DOE in cell culture

Customers – Who benefits from our services

Contracts – A summary of how they work

Design of Experiment (DOE)

Q. What is DOE?
A. Design of Experiment is a structured analytical method for determining the relationship between factors affecting a process and the output of that process. It can also be said that DOE is a statistical method to plan experiments and analyze data so that the maximum amount of information is obtained with the fewest number of runs. DOE has broad applications in science and engineering in comparison tests, process development & optimization, formulation & product design, validation tests, and process management.

Q. Why do I need DOE?
A. The classical strategy of experimentation is the one-factor-at-a-time (OFAT) approach. This method consists of varying one factor at a time while keeping the others constant. The OFAT approach is time-consuming, does not focus on the critical factors affecting the process and does not detect factor interactions. To make matters worse cell culture development and in particular, formulation development has frequently used trial and error to find the desired settings, leading to inconsistent results. By detecting factor interactions, concentrating on relevant factors and eliminating trial and error, statistically designed experiments reduce variability, improve process performance, and reduce developmental time and cost.

Q. When do I need DOE?
A. Statistical modeling minimizes the risk of making incorrect decisions, and is particularly useful to:
Screen unknown factors and select the vital factors from the trivial ones.
Determine how critical factors interact and affect a process.
Define the best mixture combination in a formulation.
Find the right factor settings for optimum performance.
Improve process control and minimize variability.

In other words, designed experiments are useful at any stage of cell culture development from bench to production. In addition, because cell culture processes are subject to experimental errors, this statistical approach to planning experiments is critical to draw meaningful conclusions from the data obtained.

Q. How do I integrate DOE with OFAT?
A. You cannot use DOE and OFAT at the same time. However, practical information you have obtained using OFAT can be used for DOE. In fact, the best DOE results are obtained when you have good knowledge of your system. Let’s say you know which factors affect your process but want to reduce process variability. In this case, we use your previous knowledge of the factors to design an experiment that will bring your process under control.

Q. Can I use DOE to fix a problem quickly?
A. If you are looking for quick fixes, DOE is not your answer. DOE uses a sequential approach where the next design is based on the results obtained from the previous design. This is repeated until the desired response is achieved. DOE is used to improve the quality, consistency, and efficiency of a process whether at the research or production level. It is also used to determine the commercial viability of a product and/or process, by looking at efficiency versus cost.

Q. How many DOE reiterations does it take to complete a project?
A. The number of designs required to complete a project is highly dependent on the stage of the cell culture process. Screening assays for example require more reiterations than validation assays. We always try to minimize the number and size of the designs to save time and cost, but never at the expenses of valid information.

Q. What other considerations should be kept in mind when using DOE?
A. First and foremost, the quality of the data obtained from a statistically designed experiment depends on the quality of the input data, i.e. garbage in = garbage out. Do not expect reliable results when factor specifications are incorrect, historical data are used, equipment is not properly calibrated, and the experimenter has poor technical skills or limited knowledge of the system. In addition, designed experiments must follow a structured lay out (random runs, replicates, blocks, etc) which should not be modified without affecting final data analysis. This is why interactions with customers are so important during the planning stages of DOE to ensure proper execution of the experiments.

Q. Do I need a statistical background to use a DOE design?
A. No, it is not required. All you need to do is the laboratory work according to the layout of the experimental design provided to you. This design consists of a random combination of factors at specified concentrations (or settings) in a structured matrix created by our statistical software, which is very easy for you to follow. Statistical analysis of the final data is also performed by our software. We do the design, interpret the results, review them with you and provide you with easy-to-understand graphs and summary of data evaluation.

Q. How does Cell Culture Solutions design experiments?
A. Experimental design and data analysis are performed using “Design-Expert” statistical software from Stat-Ease, Inc. By combining our expertise in cell culture with DOE techniques, we are able to design experiments that provide valid and objective conclusions.

Q. Is the statistical software properly validated?
A. Stat-Ease, Inc. uses several validation procedures to test its software. Because the FDA's 21 CFR Part 11 ruling does not apply to the software, it can be used for developmental and validation studies in the biopharmaceutical industry. In fact, the DOE approach is part of a set of tools recommended by the CBER/FDA for the Process Analytical Technology (PAT) Initiative. The goal of PAT is “to design and develop processes that can consistently ensure a predefined quality at the end of the manufacturing process”. When designing experiments using statistical tools, Cell Culture Solutions will ensure that compliance issues are thoroughly reviewed with customers before issuing a DOE protocol.

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Cell Culture

Q. Is DOE applicable to all phases of cell culture?
A. DOE can be used both at the research and production levels regardless of the size of your laboratory. Whether you are using small dishes in an incubator or a production bioreactor, DOE helps you optimize and validate your cell culture process.

Q. What are the advantages of using DOE in culture development?
A. DOE has extensive application in the development of cell culture processes. Designed experiments can substantially improve formulation design, bioreactor settings and process parameters resulting in optimized culture performance, consistent product quality and reduced operational costs.

Q. Can I use DOE to improve a primary culture?
A. You certainly can. Primary cultures however have some limitations that need to be kept in mind when designing statistical experiments. First, the number of primary cells obtained from human or animal tissues is usually small, which limits the size of the cell inoculum in startup cultures. Second, primary cells have a finite lifespan, which restricts culture expansion and scale up. With these limitations in mind, statistical designs can be very helpful to optimize media formulations, remove serum or animal-derived components from the culture medium, improve culture characteristics (i.e. growth, viability, differentiation, etc), and determine the most appropriate scale up conditions for these cells.

Q. Are there limitations to the application of DOE in cell culture?
A. DOE does not substitute for experience. Without cell culture knowledge, the experimenter’s input is limited and goals are ill defined. Furthermore, poor technical skills can seriously compromise the experimenter’s ability to perform the assays. A typical example is the harsh dissociation of primary cells from donor tissues, which results in loss of viability before cultures are even initiated. Another example is to expect a low producer cell line to generate high product yields in a bioreactor. DOE can do little in these instances.

Q. Does your Company provide services in all areas of cell culture?
A. We prefer to focus on the development of chemically defined formulations and determine the correct settings for optimal culture performance.

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Customer Profile

Q. What is the general profile of your customers?
A. Most of our customers work in research projects that routinely require utilization of cell cultures. Some however, are novice to cell culture technology.

Q. Who benefits from your services?
A. Anyone who needs to establish a new cell culture but has had some difficulties, or needs to improve a cell culture process for better efficiency. For example, smaller bioscience companies may require assistance in developing cell culture assays or products, while customers from academia are often interested in starting projects with new cell types including primary cells. Others may require assistance in the development of a serum free formulation, or improvement of their current culture system.

Q. Do you assist customers who have limited experience in cell culture?
A. Of course, but we are very cautious about using DOE in these cases. We are more likely to utilize our expertise in cell culture to provide initial guidance than to give them designed experiments, which require knowledgeable input from the experimenter.

Q. How do customers handle the use of DOE?
A. Many customers are not familiar with DOE when they contact us, and prefer to continue using OFAT for culture development. However, many like the idea of working in collaboration with us to design an experiment that significantly improves their cell culture processes. Therefore, it does not take too long for our customers to realize they are better off using DOE than OFAT.

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Contracts

Q. Do we always sign a contract?
A. You do not sign a service contract while using General Consulting Services (no fee), but you may want to sign a CDA (see below). All other services require a contract, which usually includes a CDA.

Q. Can you describe these in more detail?
A. General consulting is free of charge. Most customers need less than one hour worth of information before deciding whether to proceed with a service contract. Therefore, Cell Culture Solutions does not charge for the first hour of consultation, even if it happens over a few days. We give priority to those who ask questions pertinent to their research project. On the other hand, Contract Services are required for all other in-house and on-site services, including technical advice beyond the first hour, and therefore are charged a fee. A signed contract is required between parties before work can proceed.

Q. How Do I protect proprietary information?
A. Both parties must sign a confidential disclosure agreement (CDA) to protect confidential information from unauthorized disclosure. Some customers, even after signing a CDA, prefer not to disclose all proprietary information. Proprietary information does not have to be disclosed to prepare a designed experiment. Factors can be entered as codes (+1, 0, -1, etc) in the design matrix without affecting the statistical analysis. A CDA may be signed between parties without a service contract.

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