Formulation Development

Transcription

1 Formulation Development Application Note NT-PR-013 Use of iformulate and nanodsf for Fast and Precise Protein Formulation Development Dennis Breitsprecher*, Rajiv Nayar #, *NanoTemper Technologies GmbH, Floessergasse 4, 1369 Munich # HTD Biosystems, 1061 Serpentine Lane, Ste E, Pleasanton, CA 94566, USA Abstract One of the challenges in protein formulation is the simultaneous evaluation of multiple key formulation variables in a rational fashion. The Design of Experiments (DOE) approach has gained significant popularity in protein formulation as well as other process development activities. HTD Biosystems has developed a robust DOE approach for protein formulations referred to as iformulate that utilizes advanced response surface quadratic modeling to evaluate the effects of four formulation variables in a multivariate fashion. The procedure allows for rapid identification of stable protein formulations, using only small amounts of material. By analyzing the thermal stability of a model protein, Lysozyme, using the Prometheus NT.4 in conjunction with the iformulate TM DOE approach, we demonstrate that ideal formulations can be identified in as little as 35 minutes with minimal sample consumption. The DOE analysis of the precise and reproducible data generated by NanoTemper Technologies Prometheus NT.4 from 25 trial formulations resulted in the identification of the formulation design space for lysozyme formulations with high melting temperatures (T ms). The formulations can be rationalized using Quality by Design principles from the DOE analysis. Thus, by using the predictions from the DOE output and high-precision thermal stability analysis by nanodsf, stable formulations of proteins can be generated and validated with unprecedented speed and precision. Introduction The new reality in biopharmaceutical drug development is that over the past 25 years, since the genome project, the pace of research has increased exponentially but the pace of development of new biopharmaceuticals has lagged behind. As a result, development groups are constantly challenged to bring an ever-growing number of biologics through the pharmaceutical development pipeline. Not only have the number of development projects increased dramatically, but the groups are under immense pressure to deliver the drug candidates under tight timelines, with limited material, and limited resources. Unfortunately, the billions of dollars spent in research have not translated into appropriate resources being dedicated to the development groups. The advent of three revolutions going on at the same time in the 21 st century such as the medical revolution, the computer revolution, and the quantum revolution has offered us the opportunity to address the challenges of the drug development pipelines. These revolutions have resulted in high throughput instrumentation that can generate an immense volume of data in a short period of time using 1

2 limited amounts of material. A perfect example is NanoTemper Technology s label free nanodsf instruments of the Prometheus series. The instruments are specifically designed to meet the requirements of modern biologics development, providing state-of-theart thermal stability quantification, maintenance-free operation, maximal ease of use and even an optional, full automation. The Prometheus instruments analyze protein unfolding transitions based on high precision detection of intrinsic fluorescence changes under label free conditions. The instrument design allows for the parallel detection of up to 4 samples with protein concentrations ranging from 10 µg/ml to more than 250 mg/ml without buffer restrictions. It does not require the addition of extrinsic fluorescent dyes like in the classical DSF technique, thereby avoiding potential detrimental interactions of the dye and protein or excipients. Furthermore, the innovative dual-uv detection method enables rapid scanning of samples, which results in a very high data point density of 20 or more data points per C, and unmatched reproducibility, which is required for a robust and precise determination of unfolding profiles. The use of DOE offers an efficient approach to experimental design. DOE effectiveness stems from the combination of a validated statistical approach and an intelligent choice of variables, so that even extremely complex problems can be addressed in an effective manner with a minimal set of experiments. This can be accomplished by utilizing Deming s wheel of quality. Deming s wheel can be divided into four parts. The first part is planning, where the variables and goals of the project are defined. The second part is doing, where the required data are recorded by appropriate experiments. The third part is analysis of the results and checking the data for validity. And the last part is acting on the conclusions generated from the data from the DOE exercise. At HTD Biosystems we call this concept bringing mindfulness to formulation development. We do this by utilizing very little material, generating data in fast time frames without compromising quality, and performing the work with optimized and thus low-cost approaches without cutting corners. We have set up a formulation systems platform using the Prometheus NT.4 that can generate high quality data with minimal material requirements to record advanced DOE data. The use of DOE also allows us to understand complex interactions between the experimental variables that are implicit but difficult to tease out. In addition, the results can be presented in a coherent fashion to a wide audience base. This leads to better information decimation and decision-making, which eventually translates into better support for the project. The iformulate TM plate is a convenient predesigned formulation plate loaded with the formulation excipients for rapid formulation of proteins. The design is based on multivariate experimental response surface design that investigates 20 unique formulations with five replicates to look at effects and interactions of ph, ionic strength, buffer, concentration, and stabilizer concentration. The advantages of using iformulate are (1) maximizing results with minimum number of trials, (2) savings in time, cost, and resources and (3) providing justification of the formulation using QbD and FDA guidelines on process validation. This application note demonstrates the use of iformulate in combination with the Prometheus NT.4 for generating stable protein formulations in rapid fashion using limited quantities of material. 2

3 Methods iformulate process using the Prometheus NT.4. iformulate is a platform technology where four of the most critical formulation variables of proteins are evaluated in a multivariate fashion using a highly advanced response-surface quadratic design. The experimental design space evaluates the effect of ph, ionic strength (NaCl concentration), stabilizer concentration (trehalose), and buffer concentration in 20 unique formulations and five replicates. Therefore, in 25 formulations, all four formulation parameters and any interaction between them can be investigated simultaneously. Since all the formulations are pre-prepared in a 96-well MTP format, protein is simply added to the formulations and then evaluated by a high throughput analytical method. Figure 1 shows the iformulate protocol. Figure 1. Protocol used in formulation of proteins using iformulate and Prometheus NT.4. 3

4 Formulations were reconstituted with MilliQ water, and a lysozyme stock solution (5 mg/ml in MilliQ water) was added to formulations to reach a final concentration of 1 mg/ml lysozyme. Thermal unfolding of lysozyme in the different formulations was analyzed using the Prometheus NT.4. Briefly, 10 µl of each of the 25 formulations were aspirated into standard-treated capillaries by capillary action and analyzed using the Prometheus NT.4 at a scan rate of 2 C/min from 20 C to 95 C. The software analyzed the thermal denaturation curve and determined the onset (T onset) and unfolding transition temperatures (T m) of each of the 25 formulations. The data were then analyzed using DOE software, and Pareto, response-surface, and interaction plots were generated to define the formulation design space. An optimum formulation with the maximum T m was identified. Results and Discussion DSF profiles of iformulate formulations. Figure 2 shows thermal unfolding curves for the 25 iformulate formulations which differ in ph, ionic strength, trehalose concentration, and buffer concentration. Depending on the formulation composition, the T m varied between 70.3 C and 7.1 C. Figure 3 shows the 1 st derivative analysis of the nanodsf profiles to better illustrate T m shifts between the formulations. The data generated by the Prometheus NT.4 are highly precise and reproducible as shown in Figure 4 and Table 1. The color-coded profiles are the five replicate formulations in the iformulate design that are placed randomly in the plate. The DOE analysis resulted in a residual S.D. of 0.22 and replicate S.D. of 0.16, indicating the high precision and reproducibility of Prometheus NT.4 nanodsf. Figure 2: Prometheus NT.4 nanodsf thermal unfolding profiles of iformulate TM formulations of lysozyme. Thermal unfolding was recorded at 2 C/min from 20 C to 95 C, A total of 1.4 mg of protein was required for analyzing 2 experimental trials in 35 minutes. The y-axis is F350/330 fluorescence ratio and x-axis is temperature ( C). 4

5 Figure 3. First derivatives of the thermal unfolding curves of the iformulate TM formulations from Figure 2 Figure 4. The thermal unfolding curves of the five color coded iformulate TM replicate formulations generated by the Prometheus NT.4 illustrating the reproducibility and precision of the nanodsf data. The upper panel shows the thermal unfolding profiles and the lower panel shows the corresponding 1 st derivative curves. 5

6 Table 1. Tm values from the five replicate iformulate formulations Replicate T m T m Δ This dataset demonstrates that the DOE approach allows for rapidly determining optimal formulation conditions with a minimal number of experiments (Figure 5D) The analysis defines an optimal design space for lysozyme formulations for high T m s of > 76 C at low ph values around ph 5 and trehalose concentrations between -10 wt %, with 50 mm NaCl and 30 mm buffer. Figure 5D shows the results of this formulation and validates the predicted T m with the actual data. DOE analysis of iformulate data. By designing a response-surface quadratic design, the linear, interaction, and quadratic terms were evaluated in a multivariate fashion (i.e. all variables simultaneously). Hence, all three interactions between the four formulation variables (ph, trehalose concentration, salt concentration, and buffer concentration) were evaluated to optimize lysozyme s T m. The output of the DOE analysis is shown in Figure 5. In the Pareto analysis, the terms are sorted in decreasing absolute effect order, so that the effect of greatest magnitude appears at the top of the graph (Figure 5A). This analysis clearly shows that ph is the most important variable in the lysozyme formulation, followed by trehalose and NaCl. The negative sign for ph indicates that ph is negatively correlated with T m (i.e. low ph conditions results in high T m values). In contrast, trehalose concentration has a positive correlation, which suggests that increasing trehalose concentration will increase the T m. This effect is illustrated by the 3-D response surface plot for ph and trehalose concentration effects on T m (Figure 5C). Note that the slope for the ph dependency is much steeper than for the trehalose dependency, validating that ph affects the T m significantly more than trehalose concentration. The effect of NaCl concentration and ph (Figure 5B) suggests that the ionic strength (concentration of NaCl) only has a minor effect on T m and is mostly independent of ph values. Conclusion In conclusion, using iformulate and the Prometheus NT.4 nanodsf technology, it is possible to formulate proteins rapidly within < 1 hour, with minimal material requirements (<< 10 mg), and with exquisitely high precision and reproducibility of experimental data. The four critical formulation variables of ph, ionic strength, stabilizer concentration, and buffer concentration can be evaluated by a multivariate approach with high statistical power. This approach can result in identification of stable protein formulations in rapid and precise fashion. We have extended such an approach to therapeutic proteins such as antibodies and other biopharmaceuticals that will be available in other application notes or publications. Figure 5. (next page) Panels showing various DOE analysis. (A) Pareto analysis showing the ranking of the variables. (B) 3-D response surface curve of ph and NaCl. (C) 3-D response surface curve of ph and trehalose. (D) is a 2-D contour surface curve of ph and trehalose showing the formulation design space that is optimized for the highest Tm (shaded area). The red cross indicates subsequently tested parameters to confirm the DEO-output. (E) Triplicate thermal unfolding curves of lysozyme in 20 mm acetate ph 5.0, 50 mm NaCl, and % trehalose. The obtained Tm value (76.7 C +/- 0.1 C) matches the prediction shown in 5D. 6

7 ECHIP Trehalose A B Tm-50C/40min BufferC = 30.0 Trehalose = ECHIP 6 ph NaCl C Tm-50C/40min ECHIP Tm-50C/40min NaCl = 50.0 BufferC = ph NaCl = BufferC = 30.0 D 4 6 Trehalose ph ph=5.00 Trehalos=10.00 Value Low Limit High Limit E 7

8 Contact NanoTemper Technologies GmbH Floessergasse Munich Germany Phone: +49 (0) Fax: +49 (0) info@nanotemper-technologies.com Information about iformulate can be obtained from HTD Biosystems and from or Information about the Prometheus NT.4 can be obtained from NanoTemper Technologies; NanoTemper, Prometheus and nanodsf are registered trademarks NanoTemper Technologies GmbH