Two case studies are presented in which a DOE approach was used to determine the robustness of protein formulations to changes
in protein, excipient, and pH levels. In case study 1, the results indicated that the formulation was robust to wide variations
in excipient, protein, and pH levels; much wider than could occur during manufacturing of the product. In case study 2, the
results indicated that the formulation was robust to wide variations in excipient and proteins levels, but not pH. Thus, in
case study 1, the results were used to support the conclusion that variations in excipient levels had a minimal impact to
product whereas in case study 2, results indicated that the pH of the formulation needed to be tightly controlled.
Case Study 1
In case study 1, the robustness of a high protein concentration lyophilized product to protein, excipient, and pH ranges was
evaluated. The excipients consisted of a buffering agent, lyo/cryoprotectant, and surfactant. During manufacturing, excipient
levels are weighed within 1.5% of target, but some vary wider in the product because of the retention of excipients during
the UF–DF process. Excipient levels evaluated in the study were well beyond excipient concentrations expected so that a broad
design space could be evaluated. Ranges were ±25% of target concentration for the cryo/lyo-protectant, ±50% of target concentration
for the surfactant, and –50% to +100% of target concentration for the buffering agent. The buffering agent was evaluated up
to +100% to establish design space at higher buffer concentrations. To accommodate the nonlinear nature of the buffering range,
the factor levels were log-transformed, arising in a buffering range of ±0.301 log units of target. It is important to keep
the design dimensions equidistant around the center point of interest because this allows for an analysis in which each of
the design points gives equal weight to the responses.
Table 2. Screening design for case study 1
Although a full-factorial, response-surface design would have provided information on curvature in the response and all possible
interactions, this would have required the preparation of 42 different formulations excluding the center points. To reduce
the number of formulations and still be able to determine formulation components and some 2-factor interactions that significantly
affected stability, a fractional factorial screening study was used (Table 2). Samples were monitored at intended and accelerated
storage conditions by size-exclusion and ion-exchange chromatography, as well as SDS-PAGE, and tested for moisture and potency.
Degradation observed was limited to aggregation. Statistical analysis of the stability data using JMP (Cary, NC) showed that
protein and cryo/lyo-protectant concentrations were the only formulation components that significantly affected stability.
The prediction profile for the impact on stability as a function of formulation components is shown in Figure 2A. Increasing
protein concentrations and decreasing cryo/lyoprotectant concentrations negatively affected stability. In addition, a protein:cryo/lyo-protectant
interaction was observed such that the impact of the cryo/lyo-protectant on stability was more evident at the higher protein
level. Similarly, the impact of protein concentration on stability was more evident at lower cryo/lyo-protectant concentrations
(Figure 2B). The robustness of the formulation was demonstrated by the limited degradation observed at 5 °C, the intended
storage temperature for the product, even in the least stable formulations containing the highest protein:cryo/lyoprotectant
Case Study 2
In case study 2, the robustness of a high concentration liquid product to protein, excipient, and pH concentrations was evaluated.
The excipients consisted of a buffering agent, tonicifying agent, cryoprotectant, and surfactant. Excipient ranges evaluated
in the study were within 20% of cryoprotectant and tonicifying agent target concentrations and 50% of buffering agent and
surfactant target concentrations. Because of the number of formulation components, a fractional–factorial screening design
was also used in this study. Samples were monitored at intended and accelerated storage conditions by size-exclusion, ion
exchange, and reverse-phase chromatography, as well as by potency assay. Degradation was observed by size-exclusion and ion
exchange chromatography. The only factor that had a significant effect on stability was pH, resulting in increased aggregation
and deamidation (data not shown). The results from the excipient robustness study prompted well-defined pH acceptance criteria
in buffer preparation records and on the product specification.
The two case studies illustrate two outcomes that can be obtained from an excipient robustness study:
- the product is robust to wide variations in formulation components
- the product is not robust to wide variations in one or more of the formulation components and limits are put in place to control.
Irrespective of the outcomes, controls for protein, excipient, and pH levels must be established in the batch records or product
specifications. Controls in the batch record may include acceptance criteria for buffer and excipient level weigh-outs for
the formulation buffer, the pH of the formulation buffer, as well as pH and protein concentrations of the formulated BDS and
FDP. Additionally, controls in the BDS and FDP specifications may include acceptance criteria for pH, protein concentration,
and osmolality. The acceptance criteria are selected such that the quality of the drug product is not affected beyond an acceptable
level. If the robustness study indicates that the formulation is not robust to wide variations in one or more of the formulation
components, controls are put in place to ensure that acceptable levels are achieved consistently.