Formulation of biotechnological drugs

Aggressive approach

SEC-HPLC, CEX-HPLC, SDS-PAGE, DLS. Aggressive approach. More used for biotech drugs. The accelerated stability conditions used in such studies may range from 40-55°C to increase the rates of degradation and enhance the likelihood of observing significant differences among the candidate formulations over the short duration of the study. It's important to understand thermal properties (like Tm's of API) to set the stress temperature. Then a long term stability study may be performed on the selected formulation and ultimately on the actual clinical trial material prepared in the final selected formulation, in accordance with the ICH guidelines. (ICH = the International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use).

Advantages:

• Acceleration of development timelines.
• More efficient use of resources (people, lab, and API).

Disadvantages:

• Challenge of assigning predictive value to accelerated

Stability data for protein therapeutics. Degradation processes that occur at accelerated temperatures may be irrelevant to shelf life.

The translation of accelerated stability data into a real-time storage shelf life is problematic as protein degradation processes are quite complex and often do not follow Arrhenius kinetics. Cannot be used to directly predict real-time shelf life.

Case study: aggressive approach

Monoclonal Antibody for IV administration (≈25mg/mL). Goal: initiate Phase I clinical trials as rapidly as possible and develop IV formulation within 6 months. Utilized aggressive approach to identify lead candidate formulation based on statistical DOE, forced degradation and accelerated stability.

1. Initial Linear Ranging Studies.

   1. Solubility (e.g. test 5 buffers at two pH values each, to select to conditions for further tests).
   2. Biophysical characterization.

2. Forced Degradation Studies.

   1. Freeze/Thaw and agitation.
   2. Evaluate excipients in each buffer system.

Utilized Biophysical and Analytical techniques.

1. Pre-formulation DOE.
   1. Two 2-level factorial designs.
   2. 2 weeks accelerated stability, 5°C (non-stressed) and 40°C/75%RH (stressed), designed to optimize pH, buffer concentration, and excipient concentration for each candidate buffer/excipient system.
   3. Select Formulation.
2. 6 Month Non-GMP Stability Study on selected formulation.
3. Stability on cGMP Drug Substance & Drug Product.

Tool Box Methodology

The study is as good as the methods used that must be:

• Biophysical, Analytical, Biological: investigate thermal, conformational, chemical, and biological stability.
• Orthogonal Techniques (SEC-HPLC and IEX-HPLC; DSC and DLS): conditions optimal for reducing formation of one impurity may be sub-optimal for others.
• Designed to detect/quantify product-related impurities of interest: HMW species, deamidation, oxidation, other charge variants, clips/truncated species.
• Robust with acceptable accuracy and precision.

and understood variability: differentiate analytical variability from changes to product and suitable for use in statistical DOE (Design Of Experiments: software working in reducing the number of samples to test).

Comparison

Aggressive Pros:

• Delivered formulation composition in approximately 4 months.
• Facilitated the completion of process development and scale-up activities.
• Completed 6 months of real-time non-GMP stability within 11 months of project initiation.
• Plenty of real-time stability data to support IND.
• The goal is to obtain a formulation with excellent 5°C stability.
• Similar profile for cGMP drug product lot after 6 months storage.

Aggressive Cons:

• No 5°C real-time stability data beyond 6 months.
• Virtually no real-time stability data on "back-up" formulations in the event of precipitous drop in purity after 6 months.

Conservative Pros:

• Resulting formulations showed excellent 5°C stability.
• ≤0.5% increase in HMW

species via SEC-HPLC after 12 months for all three formulations.

≤4% decrease in MP purity via CEX-HPLC after 12 months for all three formulations.

• High level of confidence in ultimate shelf life of clinical supplies.
• Strong "fall back" position of alternative formulations with extensive real-time stability to support their use should problems arise during manufacturing or early clinical development.
• Plenty of real-time stability to support IND.

Conservative Cons:

• Value relative to resources expended and time spent.
• Little/no evaluation of different buffer/pH systems.
• Platform approach, did not screen a broad range of buffers/pH values.

Useful information for protein pharmaceuticals

Summary on chemical degradation:

• Tips for storage-packaging
• Smooth glass walls best to reduce adsorption or precipitation.
• Avoid polystyrene or containers with silanyl or plasticizer coatings.
• Dark, opaque walls reduce light oxidation.

Air-tight containers or argon atmosphere reduces air oxidation.

Tips for storage-refrigeration:

• Low temperature reduces microbial growth and metabolism.
• Low temperature reduces thermal or spontaneous denaturation.
• Low temperature reduces adsorption.
• Freezing is best for long-term storage.
• Freeze/Thaw can denature proteins.

Resources requirements for initial protein formulation development:

• Purified protein: representative of manufacturing process; sufficient quantity to cover dose bracket, formulation variables and stress conditions, minimum complication by impurity (precipitation, degradation like proteolytic cleavage). It can be of non-GMP quality. Examples: purified bulk, sample from final purification process.
• Qualified excipients: pharmaceutically acceptable quality, manufacturers with qualified production procedures and sufficient scale, specifications on critical impurities, quality that can be carried on to clinical studies.

1. Access to fill finish facility:
   • Capability to sterilize container/closure components
   • Fill/finish under aseptic environment
   • Head-space purge system, drying equipment
2. Analytical instruments:
   • Structural analyses
   • Concentration determination
   • Chromatographic analyses
   • Electrophoresis
   • Bioassays
   • Other micro-characterization
3. Facility to accommodate stability studies:
   • Controlled temperature and relative humidity
   • Controlled light exposure
   • Devices to provide controlled agitation
4. Information for designing formulation studies:
   • Clinical indication: site of treatment (self-administration, office visit, hospital), methods of delivery, concomitant medication, competition
   • Patient population: age, strength, capability to manipulate devices

1. Oxidation: oxygen purge, peroxide spike. For storage, excipient stability impurity. Problems: oxidations, inactivation.
2. Humidity: 0-100% RH. For storage, container integrity, powder. Problems: moisture content, moisture related degradations.
3. Mechanical stresses: vortex agitation, shear-stress (3000 s^-1). Problems: precipitation, aggregation, recovery loss.
4. Other denaturants: impurities, pH, denaturing excipients. Problems: precipitation, aggregation, recovery loss.

• Column chromatography: most physical and chemical degradations, excipients impurities, leachates. Examples: HPLC, FPLC (lower pressure than HPLC, so no metal parts in contact with the solution), low pressure LC, size-exclusion, reversed-phase, ion-exchange, hydrophobic, affinity columns; coupled with UV, fluorescence, RI and other

analytical instruments as detectors.

• Electrophoresis: degradations with changes in size and/or charges.
  • Examples: SDS-PAGE, native PAGE, isoelectric focusing, capillary electrophoresis, etc.
• Spectroscopy: structural changes, chemical modifications of side groups.
  • Examples: CD, fluorescence, FTR, UV, Raman, NMR.
• Thermal analysis: protein structure, lyophilized cake structure, powder characterization. To know the Tmof the protein to know the stress conditions that can be used.
  • Examples: differential scanning calorimetry, thermogravimetric analysis, thermomechanicalo analysis, etc.
• Light scattering/turbidity: aggregation, precipitation, MW determination. Stay below melting T.