{"id":21723,"date":"2026-10-30T11:49:19","date_gmt":"2026-10-30T11:49:19","guid":{"rendered":"https:\/\/www.sygnaturediscovery.com\/?post_type=case-study&#038;p=21723"},"modified":"2026-10-30T11:49:20","modified_gmt":"2026-10-30T11:49:20","slug":"design-of-experiments-the-experimental-method-every-scientist-should-be-aware-of","status":"publish","type":"case-study","link":"https:\/\/www.sygnaturediscovery.com\/fr\/case-study\/design-of-experiments-the-experimental-method-every-scientist-should-be-aware-of\/","title":{"rendered":"Design of Experiments &#8211; The Experimental Method Every Scientist Should Be Aware Of"},"content":{"rendered":"\n<h2 class=\"wp-block-heading has-text-xl-font-size\" style=\"margin-top:var(--wp--preset--spacing--40);margin-bottom:var(--wp--preset--spacing--40)\"><strong>What is Design of Experiments (DoE) and why use it?<\/strong><\/h2>\n\n\n\n<p class=\"wp-block-paragraph\">Design of Experiments (DoE or sometimes DoX) is an umbrella term that has been coined to cover a particular statistical design and evaluation approach to performing experiments. In essence it is a hugely powerful and very efficient way to conduct experiments where you have more than one variable that you would like to study.<br><a href=\"https:\/\/www.linkedin.com\/in\/drphilkay\/\" target=\"_blank\" rel=\"noopener\">Phil Kay<\/a>\u00a0, who describes himself as an Evangelist for Data Analysis says that\u00a0<strong>\u201cEvery scientist and engineer in the world should be able to use DoE: It is THE most important method in statistics and data science for industrial R&amp;D and manufacturing\u201d.<\/strong><\/p>\n\n\n\n<h2 class=\"wp-block-heading has-text-xl-font-size\" style=\"margin-top:var(--wp--preset--spacing--40);margin-bottom:var(--wp--preset--spacing--40)\"><strong>Would you rather do 15 experiments or 5?<\/strong><\/h2>\n\n\n\n<p class=\"wp-block-paragraph\">Perhaps the best way to illustrate this is with a relatively simple example. Say you have a system where you have 2 variable factors, temperature and pH, that you are interested in for a reaction and you want to study their impact on the final yield of your product. All the other factors; time, reagent concentrations, etc are held constant as far as is experimentally possible. You might have some historical (random trial) data for the reaction (Figure 2a) and from that you decide to perform a structured study by varying One Factor At a Time (OFAT). In your 1st OFAT set of trials, you fix reaction pH at one level (pH7.5) and then vary the temperature between 10\u00b0C and 100\u00b0C in 9 separate experiments. From this set of experiments, you discover that the optimum temperature is 40\u00b0C and it gives you a yield of 63% (Figure 1a). For your 2nd set of OFAT trials, you then fix the temperature a 40\u00b0C and run 6 experiments between pH 5.0 and 10.0 and find that at pH 6.0 you have improved the yield to 71% (Figure 1b). Hoorah\u2026\u2026.!\u2026\u2026<\/p>\n\n\n\n<figure class=\"wp-block-image size-full is-resized is-style-rounded is-style-rounded--1\" style=\"margin-top:var(--wp--preset--spacing--50);margin-bottom:var(--wp--preset--spacing--50)\"><img loading=\"lazy\" decoding=\"async\" width=\"998\" height=\"311\" src=\"https:\/\/www.sygnaturediscovery.com\/wp-content\/uploads\/2026\/10\/Screenshot-2026-10-30-111247.webp\" alt=\"Line graphs from OFAT studies evaluating temperature and pH optimisation for recombinant protein production. Maximum yield was achieved at around 40\u00b0C and pH 6.0, highlighting key process parameters affecting protein expression efficiency.\" class=\"wp-image-21724\" style=\"aspect-ratio:3.209114203888861;width:1440px;height:auto\" srcset=\"https:\/\/www.sygnaturediscovery.com\/wp-content\/uploads\/2026\/10\/Screenshot-2026-10-30-111247.webp 998w, https:\/\/www.sygnaturediscovery.com\/wp-content\/uploads\/2026\/10\/Screenshot-2026-10-30-111247-300x93.webp 300w, https:\/\/www.sygnaturediscovery.com\/wp-content\/uploads\/2026\/10\/Screenshot-2026-10-30-111247-768x239.webp 768w, https:\/\/www.sygnaturediscovery.com\/wp-content\/uploads\/2026\/10\/Screenshot-2026-10-30-111247-640x199.webp 640w\" sizes=\"(max-width: 998px) 100vw, 998px\"><figcaption class=\"wp-element-caption\"><strong>Figure 1.<\/strong> Yield data from 1st OFAT run with pH held at 7.5 (left) and 2nd OFAT run with temperature held at 40\u00b0C (right).<\/figcaption><\/figure>\n\n\n\n<p class=\"wp-block-paragraph\">\u2026.BUT\u2026\u2026 In reality it transpires that the optimum yield of 83% is obtained at 80\u00b0C and pH9.0. You have run 15 experiments (not including the initial random experiments \u2013 Figure 2) and although the yield is reasonable, you have missed the sweet spot, because you didn\u2019t experiment in that region. The red point on all of the charts in Figure 2.<\/p>\n\n\n\n<figure class=\"wp-block-image size-large is-resized is-style-rounded is-style-rounded--2\" style=\"margin-top:var(--wp--preset--spacing--50);margin-bottom:var(--wp--preset--spacing--50)\"><img loading=\"lazy\" decoding=\"async\" width=\"1024\" height=\"685\" src=\"https:\/\/www.sygnaturediscovery.com\/wp-content\/uploads\/2026\/10\/Screenshot-2026-10-30-114602-1024x685.webp\" alt=\"Comparison of experimental space studied using initial trials, one-factor-at-a-time (OFAT) methodology and Design of Experiments (DoE). The DoE approach evaluates interactions between temperature and pH across a wider design space, enabling more efficient process optimisation than traditional OFAT experiments.\" class=\"wp-image-21731\" style=\"aspect-ratio:1.494935900360418;width:1440px;height:auto\" srcset=\"https:\/\/www.sygnaturediscovery.com\/wp-content\/uploads\/2026\/10\/Screenshot-2026-10-30-114602-1024x685.webp 1024w, https:\/\/www.sygnaturediscovery.com\/wp-content\/uploads\/2026\/10\/Screenshot-2026-10-30-114602-300x201.webp 300w, https:\/\/www.sygnaturediscovery.com\/wp-content\/uploads\/2026\/10\/Screenshot-2026-10-30-114602-768x514.webp 768w, https:\/\/www.sygnaturediscovery.com\/wp-content\/uploads\/2026\/10\/Screenshot-2026-10-30-114602-538x360.webp 538w, https:\/\/www.sygnaturediscovery.com\/wp-content\/uploads\/2026\/10\/Screenshot-2026-10-30-114602-640x428.webp 640w, https:\/\/www.sygnaturediscovery.com\/wp-content\/uploads\/2026\/10\/Screenshot-2026-10-30-114602.webp 1066w\" sizes=\"(max-width: 1024px) 100vw, 1024px\"><figcaption class=\"wp-element-caption\"><strong>Figure 2<\/strong>. Experimental space studied in the initial random trials, the OFAT experiments and a full factorial DoE with a centre point. The point in red in all charts is the actual process optimum point where a yield of 83% is obtained.<\/figcaption><\/figure>\n\n\n\n<p class=\"wp-block-paragraph\">With the DoE approach, doing less, but with more focus gives you more, much more. Kind of the Tai Chi martial art of experimental design. With a DoE trial you vary both factors at the same time, with them set at the corners of the experimental range you wish to study and often include a centre point (Figure 2). In this example, you only run 5 experiments, but are much more likely to have settings that are near to the optimum condition for the system than with the OFAT approach, because you have covered more of the experimental space. Your second set of DoE experiments can then explore the region in more detail if you wish.<\/p>\n\n\n\n<p class=\"wp-block-paragraph\">The power comes if you translate this out to a study varying 3, 4, 5 or more factors at once. This is not only possible in a DoE trial, but relatively straightforward to achieve. (Figure 3 shows the design space for 3 factors set at 2 levels with a centre point), If like Doc Brown in Back to the Future you can think forth dimensionally, perhaps you can visualize the experimental ground you can cover with 4 and then even more factors studied at once.<\/p>\n\n\n\n<figure class=\"wp-block-image size-full is-style-rounded is-style-rounded--3\" style=\"margin-top:var(--wp--preset--spacing--50);margin-bottom:var(--wp--preset--spacing--50)\"><img loading=\"lazy\" decoding=\"async\" width=\"854\" height=\"552\" src=\"https:\/\/www.sygnaturediscovery.com\/wp-content\/uploads\/2026\/10\/Screenshot-2026-10-30-112438.webp\" alt=\"Three-factor Design of Experiments (DoE) model illustrating the experimental space used for process optimisation. Corner points represent combinations of low and high factor settings, while the central point represents a centre condition used to evaluate interactions, curvature and optimal process performance.\" class=\"wp-image-21726\" srcset=\"https:\/\/www.sygnaturediscovery.com\/wp-content\/uploads\/2026\/10\/Screenshot-2026-10-30-112438.webp 854w, https:\/\/www.sygnaturediscovery.com\/wp-content\/uploads\/2026\/10\/Screenshot-2026-10-30-112438-300x194.webp 300w, https:\/\/www.sygnaturediscovery.com\/wp-content\/uploads\/2026\/10\/Screenshot-2026-10-30-112438-768x496.webp 768w, https:\/\/www.sygnaturediscovery.com\/wp-content\/uploads\/2026\/10\/Screenshot-2026-10-30-112438-557x360.webp 557w, https:\/\/www.sygnaturediscovery.com\/wp-content\/uploads\/2026\/10\/Screenshot-2026-10-30-112438-640x414.webp 640w\" sizes=\"(max-width: 854px) 100vw, 854px\"><figcaption class=\"wp-element-caption\"><strong>Figure 3. <\/strong>Illustration of a factorial DoE design with 3 factors each set at 2 levels, with one centre point (red point) included. 9 experimental runs in total.<\/figcaption><\/figure>\n\n\n\n<h2 class=\"wp-block-heading has-text-xl-font-size\" style=\"margin-top:var(--wp--preset--spacing--40);margin-bottom:var(--wp--preset--spacing--40)\"><strong>How we use DoE at Sygnature Discovery<\/strong><\/h2>\n\n\n\n<p class=\"wp-block-paragraph\">Our previous managing director, Mark Abbott, was one of the early adopters of DoE for recombinant protein production. In this\u00a0<a href=\"https:\/\/doi.org\/10.1006\/prep.2001.1573\" target=\"_blank\" rel=\"noopener\">2002 paper<\/a>\u00a0he and his team used a fractional factorial design to optimize the refolding of Cathepsin S from inclusion bodies. In this\u00a0<a href=\"https:\/\/doi.org\/10.1016\/j.pep.2015.08.016\" target=\"_blank\" rel=\"noopener\">later study<\/a>, the aim was to optimize the process to transiently transfect CHO cells. In the early days you needed a card carrying statistician to help both design the experiment and then interpret the data obtained. Fortunately, nowadays, things have moved on and there are software packages that do the heavy statistical lifting for you. This now allows scientists to go a long way with DoE and the statistical interpretation. Mind you, it is still good to have a statistician available to discuss the detail with when you get out of your depth. We use the\u00a0<a href=\"http:\/\/www.jmp.com\/\" target=\"_blank\" rel=\"noopener\">JMP Software<\/a>\u00a0which allows us to design, perform and interpret a range of DoE methodologies such as Full and Fractional Factorial, Definitive Screening, Response Surface and other designs.<\/p>\n\n\n\n<p class=\"wp-block-paragraph\">In Sygnature Protein and Structure, we have team members who have significant (&gt;10 years) experience using DoE and other statistical methods for biopharmaceutical manufacturing process control and process development (both upstream fermentation and downstream purification). Currently we are aiming to leverage this knowledge and increase our use of DoE. Where appropriate, we are applying DoE to processes we are trying to optimize. This will be projects such as optimizing refolding conditions from inclusion bodies (similar to the Cathepsin S example above), how best to solubilise membrane proteins, optimisation of cell culture protocols, how to obtain the best activity of an enzyme, how to improve the yield from a protein chromatography protocol. As you can see the potential applications are wide and diverse. See our case study in one of the coming month\u2019s newsletter (Mar 2023) for an example where we have used it to optimize an\u00a0<em>E.coli<\/em>\u00a0fermentation process and the subsequent lysis and clarification steps to improve the yield of a target recombinant protein.<\/p>\n\n\n\n<h2 class=\"wp-block-heading has-text-xl-font-size\" style=\"margin-top:var(--wp--preset--spacing--40);margin-bottom:var(--wp--preset--spacing--40)\"><strong>The main considerations when starting a DoE trial<\/strong><\/h2>\n\n\n\n<p class=\"wp-block-paragraph\">There are a few key points to consider when designing a DoE trial and these influence the type of design that you select. To give you an idea, the first thing to decide is how many different factors you are going to study and balance this with how much time and resource you have. This obviously very much depends on the complexity of the experimental set up and protocol. If you have a lot of factors and want to see which are just having an impact on your process, then a screening design is a good place to start. However, often you are not able to get an indication of whether the factors interact and affect each other with these type of designs. If that is important, then factorial designs are needed, however these take more individual runs (more time and resource). Do you need to see if there are quadratic effects (curvature in the response)? Do you need to get data on the inherent experimental variation of the system? If yes, more runs are required. There are solutions to all these considerations, but as you can see time spent thinking through the overall approach and design, is time very well spent.<\/p>\n\n\n\n<h3 class=\"wp-block-heading has-text-base-font-size\" style=\"margin-top:var(--wp--preset--spacing--30);margin-bottom:var(--wp--preset--spacing--30)\"><strong>More information<\/strong><\/h3>\n\n\n\n<p class=\"wp-block-paragraph\">There are plenty of resources on DoE on the web, especially YouTube. A good place to start though is the free online course provided by JMP\u00a0<a href=\"https:\/\/www.jmp.com\/en_us\/online-statistics-course.html\" target=\"_blank\" rel=\"noopener\">\u201cStatistical Thinking for Industrial Problem Solving\u201d<\/a>\u00a0that has DoE as one of the modules.<\/p>\n","protected":false},"excerpt":{"rendered":"","protected":false},"featured_media":21726,"template":"","category":[680,702],"resource_tag":[],"class_list":["post-21723","case-study","type-case-study","status-publish","has-post-thumbnail","hentry","category-protein-and-structure","category-protein-expression-and-purification"],"acf":[],"yoast_head":"<!-- This site is optimized with the Yoast SEO plugin v28.8 - https:\/\/yoast.com\/product\/yoast-seo-wordpress\/ -->\n<title>Design of Experiments: The Method Every Scientist Should Know<\/title>\n<meta name=\"description\" content=\"Learn how Design of Experiments helps scientists optimize processes faster, reduce experimental workload, identify 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