Updated: Dec 12, 2021
Welcome to My Blog series in preformulation for drug development
In this sixth blog on preformulation, I will talk about drug excipient compatibility for biologics. In addition to the characterization of the active pharmaceutical ingredient (API), to stabilize a drug product, you need to know your formulation excipients, their compatibility with your API and with each other. A great emphasis is on buffers and sugars. Stay tuned the next blog series and follow clearviewpharmallc.com/blog.
The performance of the final dosage form (bioavailability and stability) or manufacturability are dependent on the excipients, their concentration and interaction with the API and each other. No longer can excipients be regarded simply as inert or inactive ingredients, as described in ICH Q8(R2) Pharmaceutical Development. It is important to know their properties, safety, handling, and regulatory aspects.
Once we know how the API falls apart, we can select the excipients that can stabilize and deliver it. A list of commonly used excipients in protein formulation has been reported (Capelle, M. A. H. et all. Eur. J. Parm. Biopharm. 2007, 65, 131). Just because some of these excipients were approved by FDA, it does not mean that they can be used indiscriminately (e.g., glucose is a reducing sugar). Also, most surprises during drug product stability come from excipients, particularly from the impurities in excipients, as stated before in the preformulation blog for small molecules.
The recommended approach for drug-excipient compatibility is: (1) Have a goal for a robust simple formulation (do not just copy); (2) Chose first from the list of approved excipients; (3) Assess their chemical structures in context to your drug (needs knowledge of functional groups reactivity); (4) Narrow down to those excipients that are less likely to react; (5) Use low API : excipient ratio and match the prospective formulation; (6) Allow more physical interactions in solid state mixture (amorphous); (7) Look for excipient – excipient interactions (chemical and physical); (8) Experimental design (DOE) is encouraged.
The first information you need to know from API degradation is the pH of maximum stability, which leads to the selection of the formulation buffer. For biologics, the formulation buffer is a must. The water has no ability to resist change of pH; CO2 from air equilibrated with water changes the pH from 7 to 5.7. It is important to know that some buffers may create problems. Phosphates are Ca‑dependent and Ca salts precipitate. Citrates are chelators, while Tris is a reactive buffer. Buffers, like Histidine or Bis-Tris need to be assessed at the pH of maximum stability within ±1 pH unit in 0.5 pH increments. Perform experiments at different buffer concentrations and ionic strengths at 40 ℃. Choose the lowest possible concentration (aggregates form at high µ).
It has been established in the last couple of years that the buffer does not only maintain the appropriate pH of the solution at which the protein is stable in its native structure but can influence the stability of the protein itself (Brudar, S.; Hribar-Lee, B. Biomolecules 2019, 9, 65). If the buffer molecules preferentially bind to the native state of the protein, this would stabilize the protein, while in the cases where buffer molecules bind selectively to the denatured state, this would lead to destabilization of the protein native structure. Buffers recognized as protein stabilizers were Tris, acetate, HEPES and cacodylate; however, Tris is a reactive buffer and should be avoided, unless there are sufficient data to justify its use. The primary function of the buffer is, and always will be the pH control around the protein of interest. Common error alert: Never use a buffer outside its buffering capacity. Always adjust the pH of the stock so that is correct when diluted. When making buffers by the titration method, always start with the buffer species that has the lowest charge (ionic strength consideration).
Sugars are exceptionally good stabilizers of proteins, and they are considered as cosolvents. The reason is that they can replace the water molecule without disturbing the three-dimensional structure of the protein due to their similar solvent properties. The mechanism of sugar’s stabilization is the preferential interaction with the protein and exclusion of other solvents around it, thus prohibiting unfolding. The best choice in protein formulation are non-reducing sugars like sucrose and trehalose. However, trehalose crystallizes during lyophilization, sucrose remains amorphous, but trehalose has higher glass transition temperature (Tg ≈ 80 °C). The exceptional stabilization effect of trehalose on thermal stability of proteins is attributed to the surface tension effect.
The recommended ratio of sugar/protein is 360:1 mol/mol which is approximately 1:1 w/w (depending on the protein's molecular weight); however, this number is usually decreased as the dosage form requires. At a 1:1 w/w sugar/protein ratio and a drug load of 40-50%, there is no room for other required excipients (e.g., buffers). Therefore, there should be a fine line between stabilization from sugars and buffers. In this case, I recommend choosing the protein-buffer ratio first (minimum buffer concentration for stability), followed by sugar addition to achieve an acceptable stability profile of the drug product. Other excipients may contribute to the drug load that needs also to be taken into consideration.
Almost all proteins are adsorbed to a certain extent to surfaces (glass, plastic, metal) and both manufacturing and laboratory analysis had to deal with this property. In addition, the protein solutions at the water/air interface leads to aggregation. To overcome the physical and chemical instability induced by interface interactions, surfactants are being used in protein formulations as anti-absorption excipients. The preference is for nonionic surfactants to avoid extra contribution to the ionic species (e.g., buffers) that may induce aggregation or precipitation. Poloxamer 188/407 and polysorbate 20 and 80 are commonly used in protein formulation. The manufacturing process of these excipients involve ethylene oxide and residues of peroxide at ppm level is always present in their certificate of analysis (COA). The same assessment pertains to other ethylene oxide-based excipients (e.g., povidone, crospovidone). Depending on the protein liability to oxidation, it is important to assess the level of peroxide that affect the stability of your drug product. The information comes from the forced degradation studies that by now should be in place.
Peroxides can be removed with activated alumina (Al2O3); however, a customer set specification for excipients may increase the cost of goods (COG). The good news is that different lots of peroxide-based excipients have different peroxide content. In this case chose the lot with the lowest peroxide content and/or add antioxidants. Always test different lots of the same excipient.
Antioxidants help protect against oxidation by scavenging oxygen for themselves. Ascorbic acid is commonly used, but citric acid is preferred being also a pH adjuster. However, some citrates (Fe3+) cause discoloration. Consider for an antioxidant a sacrificing molecule like methionine. The choice of antioxidant is dictated by the liability of the API to oxidation.
Generally accepted formulations
The current accepted formulations for therapeutic proteins are based on the approved excipients. The choice of excipients is based on their physicochemical properties, the forced degradation studies, and performance requirements of the API. At this stage, the degradation mechanisms such as aggregation, oxidation, and deamidation need to be understood. Aggregation is complex and involves unfolding that can be induced by either physical or chemical factors. To prevent aggregation, use nonionic surfactants as excipients of choice, control the pH and temperature, as well as the physical stress during manufacturing (e.g., freeze-thaw, lyophilization, agitation). Oxidation can be controlled by minimizing the air exposure and eliminating/trapping peroxide impurities from excipients. Antioxidants are commonly used; the addition of free methionine to the formulation will result in its preferential oxidation for the benefit of the methionine residues in the API (we need to know the site of oxidation for the right antioxidant). Deamidation can be controlled by pH control (acid-base catalyzed reaction) or protein engineering, replacing asparagine (Asn) residues with other amino acids. Reducing the water content in protein formulation is recommended. Isomerization involve deamidation but some excipients like glycerol or sucrose increase the reaction rate. Lyophilized drug product is a popular choice but developing a stable lyophilized product can be tricky and time consuming.
As a candidate biotherapeutic moves into clinical development from Phase 1 to Phase 3, the formulation studies support development of formulations used for dose-finding range (different doses and drug loads) and/or the development of a drug product for an alternative route of administration. The formulation of different doses may run into solubility problems, and it is important to notify the formulators and analytical method development team of the projected doses early in the formulation and analytical development. The manufacturing processes for clinical drug supply may also give feed-back on processability and often the formulation needs to be adjusted accordingly. The feed-back should also come from the preformulation and analytical/QC teams because some degradation products may be induced during manufacturing (e.g., aggregates, oxidation products, photodegradation products).
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