CREATE Bio Examples: Milestones

What are milestones?

A milestone is a finding or set of findings that signal the achievement of a specific aim in your research plan.   Applicants must propose one or more milestones for each Specific Aim. For each milestone, provide sufficient details on methods, assumptions, experimental designs, data analysis plan (if the results are quantitatively measured), and specify the quantitative criteria for measuring success and related rationale. Quantitative criteria should be robust and consistent with the state-of-the-art in the field. Most of the time, the quantitative criteria for success in the milestones will also be used for making go/no-go decisions and this should be specified. Specify the timeline for each milestone. There should be at least one milestone each year.  

How are they going to be used?

Because therapy development is an inherently high-risk process, it is anticipated that there may be significant attrition as projects move through the therapy development process. Achievement of milestones, then, can be utilized in decisions to continue funding throughout the lifecycle of the proposed project.

Prior to funding an application, NINDS program staff will contact the applicant to discuss the proposed milestones and any changes suggested by the NINDS review panel or NINDS program staff.  A final set of approved milestones will be specified in the Notice of Award.

Progress towards achievement of the finalized set of milestones will be evaluated by NINDS program staff.  NINDS program staff may consult as necessary with independent consultants with relevant expertise. If justified, future year milestones may be revised based on data and information obtained during the previous year. If, based on the progress report, a funded project does not meet the milestones, funding for the project may be discontinued.  In addition to milestones, the decision regarding continued funding will also be based on the overall robustness of the data package that adequately allows an interpretation of the results (regardless if they have been captured in the milestones), overall progress, NINDS portfolio balance and program priorities, competitive landscape, and availability of funds.

NINDS emphasizes the importance of the reliability and internal consistency of experimental results.  In some cases, conducting additional critical experiments will be important for NINDS to have confidence in making a funding decision.  Therefore, NINDS program staff, in consultation with the PI, may add experiments that need to be conducted prior to or during the award as an additional milestone(s). In most cases, these studies will be supported by additional funds from NINDS.

What are good practices for developing milestones?

  • Milestones should be described in the Research Strategy Section C: Detailed Plans for Research Strategy (Including Milestones and Timelines).
  • Provide sufficient details on methods, assumptions, experimental designs, data analysis plan (if the results are quantitatively measured), and specify the quantitative criteria for measuring success and related rationale.
  • Quantitative criteria should be robust and be consistent with the state-of-the-art in the field. Most of the time, the quantitative criteria for success in the milestones will also be used for making go/no-go decisions and this should be specified. If a criterion is not to be used for making go/no-go decisions, it should be clearly noted and justified.
  • Specify the timeline for each milestone.
  • Be aggressive yet realistic
  • Define at least one milestone for each year of funding
  • Include FDA interactions in the milestones (if appropriate?)

Disclaimer:  The examples shown below are for illustrative purposes ONLY. NINDS is not endorsing particular development plans or models, parameters, or cut-off values for any disease, modality, or stage of development. Therefore, NINDS expects Investigators to establish milestones appropriate for each development plan, but aligned with the recommendations indicated above.

Example #1 (Discovery Track)

Specific Aim 3:  Demonstrate in vivo efficacy of PB-19 in the Y-maze test for its effect on working memory.  

Methods:  The spontaneous alternation behavior in a Y-maze is a measure of spatial working memory, exploratory behavior and responsiveness to novelty, and can be used to evaluate therapies that can potentially improve working memories. We will test mice in a Y-maze with 34 x 8 x 14 cm arms. Each mouse will receive one 5 min trial during which arm choices and total numbers of arm entries are recorded. Spontaneous alternation, expressed as percentage, refers to the ratio of arm choices differing from the previous two choices, to the total number of arm entries.

Experimental design and data analysis plan: Mice (n=12/group) will be treated subcutaneously once-daily for 1 month with vehicle or PB-19 (25 mg/kg). Transgenic (Tg) or non-transgenic littermates (non-Tg) will be dosed with each treatment arm (positive control and PB-19). Mice will be assessed in the Y-maze with the experimenter blinded to genotype and treatment, according to a randomization schedule generated by a statistician. Data will be collected on the total arm choices and arm choice as compared to the previous choice. Data will be expressed as % spontaneous alteration (Mean +/-SEM), which is a measure of the ratio of arm choices differing from the previous two choices to the total number of arm entries. Statistical analysis will be performed using a 2-way ANOVA with genotype and treatment as the factors. Primary endpoint is prevention of cognitive decline (p<0.05) with PB-19 treated Tg mice versus vehicle-treated Tg animals compared to non-Tg controls.

Assumptions for power analysis: In our laboratory, mice have the opportunity to do repeated entries into a single arm, resulting in a chance performance level of 22% (2/9) for spontaneous alternations. Healthy young mice make 60-70% spontaneous alternations. In the transgenic mice we observed them to make a 26% reduction in % spontaneous alternations relative to age and litter-matched wild type mice. Typical within-group variance is < 10%. This is consistent with the literature using the same test at the same age.   

Power analysis: To detect a 50% prevention of cognitive decline in Tg mice compared  to non-Tg mice at alpha level of < 0.05, within-group variance < 10%, with a group of 12 mice, the power is >80% in detecting treatment effect.

Milestone associated with specific aim 3: Demonstrate prevention (Tg) from cognitive decline with treatment in a mouse behavioral model (Tg) in Y-maze test as measured by spontaneous alternations. (Complete end of year 1).

Cognitive endpoints will be the Y-maze measured as number of arm entries (as a measure of exploratory/activity levels) and the percent spontaneous alternations as the working memory measure. Data will be presented as spontaneous alternation, expressed as percentage. Spontaneous alternation, expressed as percentage, refers to the ratio of arm choices differing from the previous two choices, to the total number of arm entries. All treatment groups (Vehicle, PB-19, in both Tg and non-Tg mice) will be represented in one data set.

• Criteria for success and go/no-go:

1. >35% statistically significant (p<0.05) difference between non-Tg (Veh) and Tg (Veh) littermates so there is large enough window for detecting treatment effect.

2. Demonstrate a >50% statistically significant prevention of cognitive decline (p<0.05) with PB-19 treated Tg mice versus vehicle-treated Tg animals (i.e., >50% of the difference between vehicle treated Tg mice and non-Tg controls).

Example #2 (Discovery Track)

Specific Aim 3: Develop and establish process controls and specifications to facilitate the reproducible manufacturing of [humanized antibody] in support of future nonclinical studies. 

Experimental Design: A panel of biochemical and release assays will be developed to assess the purity and quality of [humanized antibody] produced from an existing stable clone. A production lot of [humanized antibody] will be extensively analyzed and then designated as a reference reagent (stored at -80°C) for all future comparison purposes. This will be followed by process development and scale-up of [humanized antibody] optimizing the up-stream (cell culture) and down-stream (purification) processes in order to maximize the amount of [humanized antibody] produced for continuation of nonclinical studies, and to prepare for future technology transfer to a Contract Manufacturing Organization (CMO) for large scale clinical manufacturing.

Assumptions:

The development of biochemical, potency, and endotoxin assays that meet the criteria stated below as project milestones will facilitate future scale-up production and product characterization studies needed to complete the nonclinical development of [humanized antibody]. Completion of this aim will also position us for initiating the PK/PD and toxicology experiments planned for further nonclinical development of [humanized antibody].

Milestones:

Milestone 3a: Develop and optimize biochemical and release assays for [humanized antibody] with SOPs for each to be used in future downstream process development and manufacturing. The table below summarizes the SOPs to be developed, attribute measured, and the proposed specification for each test method. (Completed by end of year 1)

 

Test SOP

Attribute

Specification

SDS-PAGE or SEC-HPLC Identity, Mass ~150 kDa
SDS-PAGE or HPLC-SEC Identity, Purity > 90% monomer
HPLC-SEC Identity, monomer < 4% aggregate
ELISA Potency Equivalent to reference standard
A280 Concentration > 0.4 mg/ml
LAL Endotoxin Endotoxin < 0.5 EU/ml

 

a. Biochemical assays: The [humanized antibody] will be characterized by A280 nm absorbance to determine concentration, SDS-PAGE and analytical HPLC-SEC methods to assess molecular weight, structure, and purity.

b. Potency assay: Initially, a quantitative ELISA based binding assay detecting the binding of [humanized antibody] to [target] will be used to assess antibody potency. As we enter clinical development, we intend to develop a biological potency assay based on the functional activity of [humanized antibody].

c. Endotoxin assay: We will use an endotoxin detection test to determine the [humanized antibody] endotoxin level in EU/ml.

Milestone 3b: Develop an optimized manufacturing process for [humanized antibody] with documentation (SOPs) to ensure reproducibility and to facilitate transfer of manufacturing process. (Complete in year 2)

a. Cell culture process development: The optimal production conditions for the CHO-S cells will be determined using pilot production runs using a bioreactor system. The cell culture optimization SOP will describe parameters such as seeding density, viability and growth rate monitoring, harvest time, and media components.

b. Purification process development: We expect that purification will consist of Protein A affinity purification, cation-exchange, and a final hydroxyapatite chromatography step. Removal of host cell DNA and steps for viral reduction will also be included. All these methods will be scaled and optimized during process development to improve yield and purity of the [humanized antibody] and to inform large scale manufacturing. SOPs will be developed describing each step of the production and purification processes including acceptance criteria for each step.

Milestone 3c: Produce a single > 1 gram lot of [humanized antibody] and successfully test and release for future PK/PD and toxicology studies. (Completed by end of year 2)

Produce lot for PD/PK and toxicology studies: A single large scale (> 1 gram) production run of [humanized antibody] will be conducted following the SOPs developed in milestone 2a. The purified [humanized antibody] will be vialed, tested, and qualified using the optimized test methods described in the SOPs developed in milestone 2b and compared to a designated reference standard.

Milestone 3d: Demonstrate that the [humanized antibody] is stable for up to a year in solution at 4°C using biochemical and potency tests criteria established above and as compared against the designated reference standard retaining  >90% bioactivities. (Ongoing activity, but 1 year stability data available by end of project)

We will determine if [humanized antibody] maintains its biological activity up to 1 year in solution at 4°C. The [humanized antibody] will be produced and stored in individual sample containers at room temperature, 4°C, -20°C, and -80°C. At 3, 6, 9 and 12 months of storage, samples will be analyzed using the same tests and release criteria when it was produced a year earlier and compared to designated reference standard.

Example #3 (Development Track)

Specific Aim 3: Complete IND-enabling CMC studies for Ab305.

Experimental Approach: Based on input from previous meetings with FDA and our progress to date with the nonclinical development of Ab305, we will conduct final product characterization and formulation studies and then prepare and file an IND package to FDA.

Assumption: We anticipate that correspondence with FDA will be required to assess the adequacy of our data or answer specific questions prior to filing the IND package.

Milestones:

Milestone 3a. Finalize the description, qualification, and characterization of Ab305 master and working cell banks (Complete in year 1).

We will establish procedures to describe the characterization and testing of the master and working cell bank to confirm the identity, purity, and suitability of these cell banks for manufacturing use:

 

Element

Test

MCB

WCB

Specification

Pass/Fail

Identity Gene: DNA Sequencing

X

N/A

Confirm correct DNA sequence

 
  Vector: Plasmid retention in colonies

X

X

Confirmed by mini-prep extraction and DNA electrophoresis

 
  Vector: Restriction endonuclease mapping

X

X

Confirm EcoR1 map compared to standard

 
  Vector: Copy number determination

X

X

> 1

 
  Host: morphology on media

X

 

Homogenous

 
  Identity of secreted protein

X

X

Western-blot positive 

 
  Peptide mapping of purified protein

X

N/A

Confirm correct amino acid sequence compared to standard

 
Purity Culture purity: Colony morphology on media

X

X

Homogenous

 
  Absence of bacteriophages

X

X

Not detected

 
Suitability Cell viability

X

X

> 80%

 

* MCB: Master Cell Bank, WCB: Working Cell Bank

X: Applicable

N/A: Not applicable

Milestone 3b. Finalize the optimization of critical process parameters for Ab305 purification process (Complete in year 1).

We will establish procedures describing all successive steps with a description of critical process parameters to purify Ab305 starting with the clarified bulk to the purified API.

 

Purification Step

Critical Parameter

Specification(s)

Pass/Fail

Protein A Chromatography
  1. Protein Load
  2. Elution Buffer pH

1 g – 2 g

4.5 - 5.0

 
Low pH Incubation
  1. pH
  2. Time
  3. Temperature
  4. Protein Load

5.0

1 – 1.5 hrs

4 – 8 °C

> 1 g

 
Cation Exchange Chromatography
  1. Load Conductivity
  2. Elution pH

> 80 mS/cm

4.5

 
Anion Exchange Chromatography
  1. Load pH
  2. Wash Conductivity

4.0 – 4.5

< 10 mS/cm

 
Ultrafiltration/Diafiltration
  1. Operating Pressure
  2. Filtration Volume

1.0  - 1. 2 bars

200 – 300 ml

 

 

Milestone 3c.  Establish control parameters of the final product manufacture (Complete in year 1).

We will establish procedures describing all successive steps with a description of critical process parameters to assemble the final product starting with purified API to final biologic product:  

 

Manufacturing Step

Critical Parameter

Specification(s)

Pass/fail

Formulation

Excipients Used

 

Temperature

Mixing Times

Rotating Speed

25 mg/ml sucrose and 0.01% (v/v) polysorbate 80

18 – 24 °C

30 – 60 min

80 -100 rpm

 
Sterile Filtration Filter integrity test Pass filter integrity test pre and post-use  
Filling/Stoppering/Capping Dosing accuracy < 2% deviation from label  
Assembly and Labeling Absence of protein aggregation  Bioburden

Clear solution

 

NMT 10 CFU/100 ml

 

* NMT: Not more than

Milestone 3d: Finalize the qualification of the potency assay for Ab305 (Complete in year 1).

We will qualify an ELISA to measure binding of Ab305 to its ligand. The following assay performance characteristics will be evaluated and test specifications set:

Performance Characteristic

Measured

Specification

Pass/Fail

Linearity R squared > 0.9  
Range Upper to lower Upper and lower standards  
Specificity % Interference *NMT 1%  
Limit of detection Low end of linear range *NMT lowest range limit  
Precision %CV *NMT 5%  
Accuracy % Bias *NMT 5%  

* NMT: Not more than