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Preformulation for Biologics (1)

Updated: Dec 12, 2021

Welcome to My Blog series in preformulation for drug development

In this fifth blog on preformulation, I will talk about biologics. To stabilize a drug product, you need to know your active pharmaceutical ingredient (API) and the formulation excipients for biologics, including buffers and sugars. In the next blog series, I am going to talk more about formulation development of biologics (stay tuned and follow If you like this blog, please re-post it.

Preformulation for Biologics is Different from Small Molecules

Biologics need to be manufactured, shipped, stored, and delivered to the patient, while minimizing their degradation. One of the most concerning degradation pathways is aggregation, leading to immunogenicity that may be triggered with the potential to cause adverse effects in patients. Analytical methods must be developed and validated. Once there is confidence in the analytical data, you may start the formulation development. To stabilize your active (API), you need to know how it falls apart under different stressed conditions. It is also important to understand how various excipients succeed or fail to stabilize the API during manufacturing and on storage.

The “platform” formulations for drug product or platform analytical methods are only starting points and must be qualified and/or redeveloped for each individual molecule. The selection of the lead formulation is based on stability studies and other performance attributes. Parallel to accelerated stability, stressed stability studies are recommended to shorten the development time. It is important to establish the cause of instability.

The stress conditions usually are milder for biologics vs. small molecules. The first use of the forced degradation of the API is for the stability indicating analytical method development, a must to have tool for the assessment of different formulations. There are three commonly modes of denaturation and degradation: aggregation, oxidation, and deamidation. Depending on the complexity of the biomolecule, other types of degradation can take place: isomerization, fragmentation, disulfide scrambling, N- and O-glycosylation, clipping (C-terminal lysine truncation), and pyroglutamate formation.

Sample Preparation May Lead to Degradation

Sample preparation for analysis may lead to degradation. Adjusting pH with NaOH/HCl > 0.1% may generate degradation. It is also recommended to work under slightly reducing conditions (10 mM reducing agent). Most biologics are photo sensible. Therefore, it is important to know the photo liability of the molecule that can occur in the hood, where samples in solution are exposed to fluorescent light.

During sample preparation, exposure to air can also lead to aggregates. Oxidation due to molecular oxygen is called autooxidation.

The pH – Rate/stability Profile Determines the pH of Maximum Stability

For the maximum stability of biologics, the formulation buffer is crucial. The experiments should be carefully designed to avoid false interpretation of degradation that is not pH related (e.g., photodegradation during sample preparation). Proteins are more stable at their isoelectric point (pI), but the pKa of amino acids and therefore pI’s, shift with ionic strength; a formulation loaded with ionic species should be avoided and buffer concentration should be minimized.

A common practice is to target only the highest reasonable temperature; however, setting up stability samples at three temperatures (from the same stock solution) does not require too much effort and they can serve as back up samples in case the reaction is too fast. It also allows to follow the appearance and disappearance of degradation species that may not be able to be monitored at higher temperatures. It is important to focus on the primary degradation products as they will appear first on long time and accelerated stability studies. The kinetics of degradation is an additional tool for impurity ID and elucidation of the degradation mechanism.

Understanding Forced Degradation Is Key to a Robust Formulation

In forced degradation we are looking for the major degradation pathways: aggregation, oxidation, deamidation, and photolysis.

Aggregation is one of the most scrutinized degradation types for biologics because aggregates may induce an immunogenic response with safety consequences. Aggregation may occur at almost all stages of the drug development, during freeze-thaw, agitation, air/light exposure, and on exposure to chemicals. Traces of formaldehyde from excipients may cause aggregation; they are particularly good cross‑linkers and they may be present as impurities in excipients.

The percent of ionization has also implication on the stability of proteins. The cysteine residue is frequently involved in aggregation (as well as disulfide scrambling). Cysteine, Cys-SH has a pKa of 8.5. At pH 7.4 the thiol group is 7.4% in the ionized form that can lead to S-S bond formation (if changes in conformation occur, e.g., unfolding). Therefore, formulations with pH >7 should be avoided. Size exclusion chromatography (SEC) is the standard analytical technique, but orthogonal techniques like analytical ultracentrifugation (AUC) in also used.

Oxidation. Degradation by oxidation most likely occurs at the side chains involving histidine, tryptophan, tyrosine, methionine, and cysteine groups. Traces of peroxides as well as the molecular oxygen from air (autooxidation) affect the chemical stability and subsequently the physical stability (unfolding, aggregation). Tryptophan can undergo photooxidation, while methionine is oxidized by atmospheric oxygen. The experiments should be conducted at the optimized pH, constant ionic strength, and at 25 ℃. Check the certificate of analysis (COA) of the excipients for peroxide content. Note: For ANVISA (Brazil) submission, at the API level, oxidation must be conducted with initiators as well: O2 + Azobisisobutyronitrile (AIBN), acetonitrile/water 80:20 v/v at RT. A good start is to target the lowest level of H2O2 found in the COA of excipients or, if data is not available, use 0.01% and increase it to a maximum of 0.1% H2O2.

Deamidation of Asn and Gln residues is an acid and base catalyzed hydrolysis reaction and can lead to aggregation and isomerization (Asp → isoAsp). The extent of size variants and charge variants can affect the potency and purity of the drug product (DP). Sometimes, the deamidation does not affect the active site and this is reflected in the activity assays.

Photodegradation. Most proteins degrade upon exposure to UV and/or visible light. In addition to the forced degradation studies, it is important to establish if the manufacturing and laboratory conditions will significantly impact the stability of the DP during compounding or analysis. It is recommended the use of UV and visible light at 1 and 2 x ICH conditions as well as 2 times the fluorescent light exposure of the longest manufacturing unit operation time. Perform experiments with control (amber glass or aluminum foil protection of samples). Before any analysis is performed, check if degradation occurs during sample preparation (samples exposed to fluorescent light in the hood and aluminum foil protected in dimmed light hood) and take appropriate steps.

Metal ion catalysis. Many therapeutic proteins are inhibited by one or two types of metal ions. Therefore, the effect of Cu(II), Mg(II), Ca(II), Zn(II), Fe(II), Fe(III), Co(III), and Cr(III) at the working pH (constant ionic strength) is recommended to be studied with EDTA as control. Use 0.05 M Fe(III) sulfate; 0.05 M Cu(II) sulfate at 30 - 40 ℃ to satisfy ANVISA regulations. Check COA of the excipients for heavy metals. If the COA reports the heavy metal as a total Pb, ask for individual metal content analysis from the vendor.

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