Practical stability studies of therapeutic proteins in oncology

Department of Pharmacy,

Henri Mondor University Hospital Group,

AP-HP, Créteil, France

It is of paramount importance for hospital pharmacists to have well-documented data about the real stability of an opened drug formulation, either after reconstitution of a lyophilised product or after dilution in various vehicles. This is even more important for anticancer drugs, which are frequently used at their maximum tolerated dose, have a tightly defined therapeutic range and are very toxic. Unfortunately, either these data are not always available or the published results are old, contradictory and limited to a particular vehicle or a specific bag.

Moreover, the manufacturers frequently quote stability after dilution as being ‘stable for eight or 12 hours’, but not for valid reasons. These short times reflect application of the ‘care principle’ considering possible bacterial contamination or the fact that stability tests were only conducted over a very short period.

As, in most countries, anticancer drugs are prepared by the pharmacy in centralised units under strict aseptic techniques, the only relevant issue is the real chemical stability, which should take into account the various drug concentrations, vehicles and containers used in clinical situations. An advantage of knowing true stability limits is that the centralised units can optimise the workload, the opening hours and the cost balance. Doses prepared in advance can limit the need to work during the weekend. The implementation of a dose-banding strategy is strongly dependent on the stability data. Finally, extending the stability limits of costly drugs such as biologicals, that is, proteins such as monoclonal antibodies (mAbs), would be extremely helpful for both practical and financial reasons. In summary, hospitals pharmacists need to have available the stability data pertinent to their needs in real-life situations. This is what is meant by the in-use (or ‘practical’) stability of drugs.(1,2)

The main problem is the difficulty of assessing the stability of new biotechnology-issued drugs such as mAbs. Indeed, these sensitive products can undergo more complex degradation pathways during the various manipulation steps than do classical drugs. Indeed, in vivo activity of proteins depends not only on their primary structure (sequence) but also on their structure in three-dimensional (3D) space (secondary, tertiary and quaternary structures). Thus, the conformation of a protein could change subtly when exposed to mild chemical or physical stresses such as shaking, small temperature change, variations in ionic strength, light, and exposure to oxygen or to traces of metal.(3,4)

However, it should be emphasised that mAbs are expected to have good stability as compared to other proteins. Indeed, immunoglobulins are normal constituents of the blood and their natural half-lives are approximately three weeks in conditions that are largely unfavourable (37°C, presence of degrading enzymes). Interestingly, the half-life of bevacizumab in the body is also three weeks. Therefore, it could reasonably be considered that an unopened vial of bevacizumab accidentally stored for one-to-two days at room temperature has no reason to be significantly altered. However, as manufacturer’s recommendations require avoiding any rupture of the cold-chain, in the case of the failure of the refrigerator during the week-end, the strict application of the recommendation will imply that all stock of this expensive drug should be wasted. Thus, to give some arguments to hospital pharmacists to avoid this costly practice, we have studied the thermal stability of diluted bevacizumab and cetuximab and demonstrated that no alteration was observed even after three months at 37°C.(5)

Protein instability includes two main types of alteration with several possible pathways: (1) physical instability: aggregation, denaturation or adsorption on surfaces; (2) chemical instability: desamidation, disulfide bond breakage, hydrolysis, isomerisation, non-disulfide crosslinking or deglycosylation. The main causes of instability include temperature (elevation or freezing), formulation pH, adsorption, salt effects, oxidation (associated with metal ions and chelating agents), shaking or shearing and concentration. Many of these conditions can be found during the handling process at the pharmacy or ward level. Unexpected instability can be very subtly induced by simple processes such as filtration due to extractables/leachables from filters.(6)

Therefore, stability assays for therapeutic proteins must involve several specific studies and represent a real analytical challenge. To assess a complete stability profile, most authors agreed that several complementary (orthogonal) methods must be used, including at least three complementary separating methods.(7) The use of such orthogonal methods is also suggested in the current European Medicines Agency (EMA) draft guideline on ‘‘production and quality control of monoclonal antibodies and related substances” and has been also recommended in the guidelines on the practical stability of anticancer drugs, recently published by a group of European oncology pharmacist, under the auspices of the French Society of Oncology Pharmacy (SFPO).(8)

The aggregation of proteins is a major physical instability that could have major implications in terms of efficacy or toxicity. Aggregates formed may be strongly antigenic and therefore loss of efficacy could result from the appearance of neutralising antibodies or the patient could suffer severe immunological reactions. In particular, one of the most underestimated causes of aggregation is mechanical stress: shaking or stirring, shearing (for example, caused by rapid sampling by syringe), exposure to hydrophobic gas interface (bubbling or filtration). It should be considered that aggregation of antibodies is the initial sign of instability regardless of its causes (physical or chemical).

Therefore, the assessment of physical stability is of paramount importance as a valuable primary marker of instability.

Fortunately, a simple method such as turbidimetry can easily estimate the formation of micro-aggregates.(9,10) However, other complementary methods should be used to study more closely the physical stability of a protein. Dynamic light scattering is able to evaluate both soluble and nonsoluble aggregates and can describe time-dependant profiles of particle size distribution. Size exclusion chromatography (SEC) can measure level of monomeric protein and soluble polymeric aggregates. By direct UV spectroscopy after centrifugation, the determination of non-aggregated protein content (absorbance at 279nm) readily permits the calculation of the aggregation ratio. Second-derivative spectra can be useful to detect small modification of tertiary structure (UV) or secondary structure (Fourier Transform Infrared Spectroscopy; FTIR) As an example, in a previous study focusing on the mechanically induced aggregation of the monoclonal cetuximab, we reported the association of these different complementary methods to fully assess its physical stability.(9)

Because of multiple causes of physical instability, the evaluation of the stability of biotherapies should ideally be performed by including stressed conditions typical of ‘daily practice’: rapid injection and rinsing with the production of bubbles into the infusion bag, accidental shaking and transportation by pneumatic network.(3) Indeed, it is critical that pharmacists ensure that handling techniques are not deleterious to the final administered product. As an example, in France, some pharmacy inspection bodies claim that the use of a pneumatic network is not sufficiently safe for antibodies. No study is available to justify their position. Unfortunately, these data on stability in practical situations are almost never available in manufacturer drug information files or only under very generic sentences such as ‘‘avoid shaking’’, permitting all manner of extrapolation or interpretation.

Simple experimental design, such as the stirring test, with concomitant increase exposure of the antibody molecules to the gas/liquid interface, can be done to generally mimic mechanical stresses that can be generated during all of the handling process.(9) Indeed, if a diluted antibody remains stable in these strongly stressed conditions, it can be reasonably concluded that the mechanical stress it undergoes during the sending by pneumatic network will be unable to induce instability. Therefore, the European consensus conference has recommended that physical stability of proteins, especially antibodies, should be evaluated by several complementary methods, including at least turbidimetry and SEC.(8)

Deamidation is considered a common degradation pathway for proteins and peptides, strongly depending on the pH. This reaction generates degradation products and may contribute to immunogenicity. As for the evaluation of physical instability, several complementary methods must be used to assess chemical degradation of proteins. To do this, several chromatographic methods have been used. Ionic exchange chromatography (IEC), particularly cation exchange chromatography, is the gold standard for protein analysis, as desamidation, which is the main thermal-dependent degradation pathway of large molecules such as mAbs (amidic asparagine residue giving aspartic acid residue by hydrolysis), is readily visualised by the appearance of new acidic peaks. In addition, to visualise sub-visible aggregation, SEC can also identify chain scission, and the peptide mapping after reverse-phase HPLC separation of peptides formed by enzymatic treatment can reveal even subtle alteration of the primary structure of proteins (that is, modification of one amino acid). Therefore, the European guidelines recommend that the chemical stability of antibodies must be assessed by a minimum of three separation methods, namely, IEC, SEC and peptide-mapping, but that complementary or alternative methods such as mass spectrometry can also be used.(8)

However, an initial mechanical, chemical or thermal stress can induce a subtle destabilisation of the protein 3D structure without any other visible sign, despite the use of several analytical methods. Indeed, the destabilised protein could be less resistant to a second stress. As an example, a mAb solution, that has been accidentally frozen can appear as unaltered after thawing and remain physically stable after storage at 4°C (that is, no detectable aggregation), but could be less stable than the non-frozen sample during exposure to elevated temperature. This “memory effect” is very subtle but can be easily estimated by the determination of the thermal denaturation curve.

Indeed, the degree of internal or external stabilisation can be estimated by the change in unfolding temperature (Tm) or its surrogate, aggregation temperature(Tagg). Technically, the protein is slowly heated, and reversible, then irreversible, aggregation was monitored by various methods such as heat transfer (differential scanning calorimetry), turbidimetry, determination of the hydrodynamic diameter, FTIR or fluorescence spectra. The curves are recorded for proteins before and after the tested stress and the respective thermodynamics parameters of the thermal transition(s) can be then calculated. Generally speaking, destabilisation was characterised by a decrease in the Tm. Therefore, this method is very fruitful, as it is able to show rapidly if a particular stress can induce destabilisation of a protein, without requiring complex analysis, and is often the only way to demonstrate its “memory effect”. Finally, cycling or sequential experiments such as freezing, thawing, storage at 4°C, an increase to 25°C and then a return to 4°C in order to mimic the possible “real life” of a vial, could be very interesting.

Due to the particular structure of a protein and its activity/3D-structure relationship, the assessment of its biological activity during stability studies could be useful as an ultimate test. Obviously, the most relevant method to test the pharmacological activity should be chosen. ELISA could be a useful method for monoclonal antibodies. However, a complementary test, such as the determination of the cytotoxic activity on cell lines, could be also used, as, for example, in the case of rituximab. Therefore, the determination of the remaining pharmacological activity by a biological assay, albeit specific, can be complementary to a full physicochemical analysis but should not be considered alone as a stability-indicating method, taking into account its inherent analytical variability and its inability to detect low-level degradation products or aggregates, which can induce serious anaphylactic reactions or renal failure.

From our experience, as a general rule in practical situations, and in the absence of published studies concerning the influence of a specific stress on a mAb, the following recommendations can be suggested. Exposure at room temperature for several days (and often much more) is not deleterious, as demonstrated in our lab, for the classically used dilutions of rituximab, bevacizumab, traztuzumab, cetuximab and infliximab. Freezing seems more problematic and more studies should be devoted to this aspect. Similarly, concerning the role of hydrophobic interfaces on the stability of mAb, diluted solutions in bags or syringes should minimise exposure to air/liquid interface (that is, no remaining head space or bubbles). Additional steps such as filtration should be avoided, unless strictly recommended, and if performed, the first millilitre of the filtrate should be rejected to eliminate extractables or leachables.

In conclusion, the practical stability of therapeutic proteins used in oncology, mainly mAbs, is an emerging and very exciting field of research. The outputs of studies testing the stress situations that can occur during the storage and handling of these drugs are of paramount importance for daily practice, to optimise workload, and can strongly reduce unjustified expenses.

• The practical stability of therapeutic proteins used in oncology, mainly monoclonal antibodies (mAbs), is an emerging field of research.
• As a general rule, for currently used mAbs and in the absence of published studies on the influence of a specific stress, good practice recommendations can be suggested.
• Exposure at room temperature for several days is not deleterious.
• Freezing seems more problematic.
• Preparation and handling of diluted solutions in bags or syringes should minimise exposure to air/liquid interface (that is, no remaining head space or bubbles).
• Filtration steps should be avoided.

1. Astier A. The stability of anticancer drugs. EJHP Pract 2007;13:90–3.
2. Hawe A et al. Forced degradation of therapeutic proteins. J Pharm Sci 2012;101:895–913.
3. Manning MC et al. Stability of proteins pharmaceuticals: an update. Pharm Res 2010;27:544–75.
4. Astier A, Pinguet F, Vigneron J, and the SFPO stability group members. The practical stability of anticancer drugs: SFPO and ESOP recommendations. Eur J Oncol Pharm 2010;4(3):4–10.
5. Paul M et al. Thermal stability of two monoclonal antibodies: cetuximab and bevacizumab. Eur J Oncol Pharm 2008;2(1):37.
6. Huang M. Impact of extractables/leachables from filters on stability of proteins formulations. J Pharm Sci 2011;100:4617–30.
7. Staub A et al. Intact protein analysis in the biopharmaceutical field. J Pharm Biopharm Anal 2011;55:810–22.
8. Bardin C et al. Guidelines for the practical stability studies or anticancer drugs: A European consensus. Ann Pharm Fr 2011;69:221–31.
9. Lahlou A et al. Mechanically-induced aggregation of the monoclonal antibody cetuximab. Ann Pharm Fr 2009;67:340–52.
10. Mahler HC et al. Protein aggregation: pathways, induction factors, and analysis. J Pharm Sci 2009;98:2909–34.