1. INTRODUCTION:

Immunity permits the body to greater defend itself against illness produced by the definite microorganism. Immunity may take place on its own, or by vaccination¹. Immunization is a simple preventive service². Vaccination has made the greatest contribution to global health. The initiation of multitudinous vaccination programs worldwide is undoubtedly one of the greatest public health achievements of modern times³. A vaccine typically contains an agent that resembles a disease-causing microorganism and is often made from weakened or killed forms of the microbe or its toxins⁴. The vaccines currently used are killed vaccines, attenuated vaccines, toxoids, subunit vaccines, DNA vaccines, and combined vaccines⁵. Mucosal route vaccines are also developed for more patient compliance ⁶.

However, the mobility to distribute vaccines to outlying areas of the world has been challenged by unavoidable exposure to many stresses such as temperature, light, and agitation that can produce various physicochemical modifications and may result in loss of vaccine safety and effectiveness because it is biological formulations, vaccines are sensitive to many environmental stress conditions⁷. Therefore the major motive of vaccine formulation development is to develop a safe and immunogenic composition that can defeat the many challenges and stresses exposed during manufacturing, storage, and distribution³. The stability-indicating capability was established by forced degradation experiments⁷.

Forced degradation study (FD) studies (stress testing) are an intrinsic part of pharmaceutical product development⁸. Stress degradation (forced degradation) is a process that involves degradation conditions more severe than accelerated conditions and thus generates degradation products that can be examined to determine the stability of the molecule⁹. Forced degradation studies comprise the influence of temperature, oxidation, neutrality, photolysis, and susceptibility to acidic and basic hydrolysis through a considerable range of pH values¹⁰. Stress degradation studies are a necessary step in vaccine formulation development. studies are used to,

· Facilitate the development of analytical methodology, to gain a better understanding of active pharmaceutical ingredient (API) and drug product (DP) stability for the establishment of degradation pathways, differentiate degradation products, related drug products.

· Illustrate the structure of degradation products.

· To determine the intrinsic ability of a drug substance in the formulation.

· To reveal the degradation mechanisms such as hydrolysis, oxidation, thermolysis, or photolysis of the drug's substance and drug product.

· To understand the chemical profile of drug molecules and generate highly stable formulations.

· To make a degradation profile related to that of what would be observed in a formal stability study under ICH conditions and solve stability-related issues¹¹.

According to FDA guidance, phase III of the regulatory submission process is the prominent time for stress degradation studies. Forced degradation in the pre-clinical phase or phase I of a clinical trial is encouraged, which provides sufficient time for identifying degradation products, structure elucidation, and optimizing the stress conditions. Improvement in the manufacturing process and proper selection of stability-indicating analytical procedures is obtained by early stress degradation studies¹². The regulatory guidelines ICH provides general guidance about stress degradation studies but there are little to no practical examples of how to set up adequate stress conditions and appropriate acceptance criteria. Vaccine developers are thus left with numerous gaps for interpretation, including suitability and selection of stress conditions, the timing of stress studies, and the extent of degradation. Guidelines roles are in Table no.1

A primary challenge in vaccine formulation development is to find out the mechanism of degradation accountable for the instability of each constituent in the formulation. In vaccines, there are mainly two mechanisms of degradation that are mainly responsible for product instability^1.Shown in figure:1 mechanism of degradation.

2. MECHANISM OF DEGRADATION:

2.1 Chemical mechanism of degradation:

2.1.1 Deamination of Protein:

Deamination is the most ordinary chemical degradation pathway proteins and peptides. Due to deamination reduction of biological activity can happen. Deamination is the cause of increased immunogenicity in therapeutic proteins significantly formulated in aluminum hydroxide (AH) adjuvant at neutral pH. In un-adjuvanted Bacillus, antigen MS techniques were used successfully used²³. Asparagine (Asparagine) deamination can be captured by analytical methods that differentiate proteins by electrostatic charge²⁴.

2.1.2 Non-enzymatic cleavage of peptide bonds:

Lower pH conditions are suitable for the non-enzymatic cleavage of peptide bonds and can be generally seen at elevated temperatures while pursuing stress studies⁷. Under accelerated conditions, Aspartate hydrolysis has been seen for ribonuclease and human epidermal growth factor²⁴.

2.1.3 Disulfide exchange:

At alkaline and neutral pH conditions, Disulfide exchange can be observed in proteins. This reaction can alternate structure, conformational instability, and, ultimately, change the antigenicity of protein antigens²⁴. The correct formation of disulfide pairings is a key requirement for the correct folding of virus-like particles and the development of immunogenicity against hepatitis B major surface antigen²⁶.

2.1.4 Oxidation:

2.1.4.1 Protein oxidation:

Although oxidation can take place in any protein-containing methionine, histidine, tryptophan, tyrosine, or cysteine, it occurs more frequently in sulfur-containing amino acids²⁴. The detection of protein oxidation is typically conducted by reverse-phase high-performance liquid chromatography (RP-HPLC), it can be detected by matrix-assisted laser desorption ionization (MALDI)-a time of flight MS. Strong oxidants such as H₂O₂have been proposed as physiologically relevant means for producing inactivated viral vaccines. The use of H₂O₂for the inactivation of viruses has been recently reported as a new approach for the development of vaccines²⁷.

2.1.4.2 Phospholipids oxidation:

Attenuated enveloped viruses and attenuated bacteria composed of Phospholipids and also be present in the vaccine as components of liposomes in the formulation of adjuvants. Phospho lipid oxidation can be monitored by the simplest technique is ultraviolet (UV) spectroscopy by which the formation of conjugated dienes resulting from oxidation of polyunsaturated fatty acids is detected at 233nm²⁹.Besides, phospholipid oxidation can be monitored by RP-HPLC with chemiluminescence detection and liquid chromatography–MS that facilitate a complete mapping of oxidized Phospholipid species in biological and pharmaceutical samples³⁰.

2.4.1.3 Nucleic acid oxidation:

Nucleic acids can be found as active ingredients in the vaccine formulation, such as plasmid deoxyribonucleic acid (DNA), immunostimulatory molecules (such as cytosine–phosphate–guanine oligodeoxynucleotide molecules), or as part of the genome of an attenuated virus or bacterium. Oxidation of genomic DNA can be a potential cause of inactivation and loss of potency in live attenuated vaccines^24,29.

2.1.5 Chemical degradation of conjugated vaccine:

Conjugated vaccines are produced by the covalent attachment of a bacteria-derived polysaccharide to a carrier protein to convert a normally T-cell independent immunogen into a T-cell dependent immunogen. In some cases, depolymerization of polysaccharides and liberation of oligomers from the conjugate can be catalyzed by aluminum salts. This was observed for the Haemophilus influenza type B conjugate vaccine in the presence of AH²³.

2.2 Physical mechanisms of degradation:

2.2.1 Aggregation:

Aggregation in biological products poses many threats and its effects can be detrimental during any stage of the product lifetime, including protein refolding, product purification, sterilization, shipping, and storage ¹⁸. It is observed in other components of the vaccine formulation, such as inorganic adjuvant salts, adjuvant emulsions, or whole attenuated microorganisms. Antimicrobial preservatives commonly used in multidose vaccines can also have an impact on aggregation. For the case of aluminum salt adjuvants, exposure of the vaccine to subzero temperatures is a well-known factor that induces a significant increase in the particle size and sedimentation rate. The extent of aluminum salt flocculation appears to depend on several factors, including a charge on the particles at the formulation pH, excipients, ionic strength of formulation, and concentration of aluminum adjuvant²⁵.

2.2.2 Denaturation:

Denaturation can be induced by multiple factors such as exposure to high or low temperatures, chemicals, and high pressure. Temperature-induced denaturation is typically irreversible due to aggregation and has been reported during stress degradation studies in many vaccine candidates including recombinant protein antigens, virus-like particles³¹, and live attenuated microorganisms. Denaturation with viral receptors in cells can result in a decrease in vaccine stability³⁰.

2.2.3 Adsorption to surfaces:

During bioprocessing, vaccines are in contact with multiple surfaces. It is not uncommon to observe the adsorption of vaccine components to surfaces, such as glass, plastic, and stainless steel containers, rubber stoppers, and prefilled syringes. Shedding material from prefilled syringes has been reported to cause adsorption and physical instability of monoclonal antibodies and vaccine antigens³⁰.

3. METHODS FOR STRESS TESTING ANALYSIS:

3.1 Potency testing:

To measure vaccine stability, a quantification test for potency is necessary for biologically active components of the vaccine¹. Regulatory agencies require that vaccines that are for market use, be tested for potency to infuse defensive immunity after immunization. For live, attenuated vaccines and also for inactivated vaccines In vivo potency assay are done, although biological assays are often used. While forced degradation studies of vaccines are condemnatory for set up the robustness and suitability³³.

3.2 Physicochemical testing:

Structural data of protein antigens can be found out from given methodologies, analytical, spectroscopy, and immunoassay⁷.

3.3 Mass spectrometry (MS):

A sensitive and accurate method to detect the molecular weight of a biological molecule is MS. Epitope identity, antigen structure, and antigen processing can all be analyzed by using this method. Various methods of MS ionization manners that can be used for antigen identification also involve MALDI, electrospray ionization, and tandem mass spectrometry (MS/MS)²⁹. MS/MS obtain structural details of peptide series and favors extortionate fragmentation. For detection of non-enzymatic cleavage and distinguished the cleft area in the certain antigen liquid chromatography/MS can be used³³.

3.4 Differential scanning calorimetry (DSC):

Differential scanning is based on the difference in heat applied to a cell containing a sample proposing to a cell containing the buffer. The thermal stability of various vaccine constituents such as proteins, nucleic acids, and viral particles can be analyzed by thermally induced transitions discovered by differential scanning calorimetry (DSC). DSC was satisfyingly used to distinguished the pH sensitivity of Norwalk virus-like molecules vaccine,³¹polio and influenza viruses, and replica antigens soak up to aluminum salt adjuvants. DSC can also be applied for conducting pre-formulation, excipient screening, and stability assessments³⁵.

3.5 Circular Dichroism (CD):

Circular dichroism (CD) is used to differentiate between the secondary and tertiary structure of proteins additionally for spotting nucleic acid. CD is applied to identify the change in the structure of biologics due to degradation by various factors. CD has shown to be a useful technology when analyzing the consequence of formaldehyde concentrations on the conformational stability of diphtheria toxins and toxoids⁷.

3.6 Fluorescence spectrometry (FS):

Fluorescence spectroscopy (FS) is an analytical method based on fluorescent amino acids frequently used to distinguished the conformational stability of various proteins. The folded protein was analyzed by using tetanus toxoid, stored at low pH conditions in the form of polymeric microspheres. By this method, stress degradation of antigens at low concentrations can be detected in a couple of hours using low volumes of material³⁶.

3.7 Fourier transform infrared spectroscopy (FTIR):

Fourier transform infrared spectroscopy (FTIR) is a method used for identification of the complexes such as viruses. For proteins, amide regions 1, 2, and 3 signals are monitored for secondary structure. For DNA-based vaccines, vibrational bands in which the position of peaks are determined from nucleic acid bases and phosphate groups are used to estimate the secondary structure. High protein concentration is required for this technique, which may be difficult to attain when conducting FD studies in the liquid state ^[39]. This technology was used to assess the secondary structure of lyophilized tetanus toxoid. FTIR can be used to detect the secondary structure of proteins when adsorbed to aluminum adjuvants since optical turbidity of the adjuvant suspension does not affect the measurements³⁷.

3.8 Liquid chromatography:

High-performance liquid chromatography (HPLC) is a common tool used to separate analytes based on their size, charge, hydrophobicity, or affinity. Size exclusion chromatography is a method used regularly to determine aggregation behavior²⁵ for the characterization and stability of protein antigens, conjugated vaccines, as well as viral vaccines. In the case of polysaccharide-conjugated vaccines, quantification and catalytic depolymerization of polysaccharides in the presence of AH can be monitored by high-performance anion-exchange chromatography also can separate antigens and degradation products in vaccine preparations²³.

3.9 Electrophoretic techniques:

Polyacrylamide gel electrophoresis (PAGE) is widely used for the analysis of protein-based vaccine components. These techniques can be used as a starting point to study protein fragmentation due to nonenzymatic cleavage, disulfide exchange, and aggregation. Antigens can be identified in gels by blotting and detection with antibodies, a technique referred to as Western blotting. Western blotting is considered to be a qualitative assay in which antigens in a complex mixture can be identified to be present and their molecular weight confirmed²⁴.

3.10 Immunochemical testing:

In vaccines, antigenicity is defined as the ability of an antigen to bind to a specific antibody. It is often expressed as a measurement of concentration and is distinct from antigen concentration. The majority of antigenicity measurements are conducted using the enzyme-linked immunosorbent assay (ELISA) technique for vaccine formulations. Apart from ELISA, immunochemical tests such as radioimmunoassay, nonlabeled immunoassays, and biosensor analysis can also be used to determine antigenicity. ELISA is a method used in industry and academia to determine the antigenicity of a vaccine^24,25.

4. METHODS OF FORCED DEGRADATION:

Stress degradation studies involve the subjection of vaccine samples that are considered a specimen of the manufacturing procedure for the drug substance or the drug product to several external stress conditions, including heat, humidity, freeze/thaw, acid/base hydrolysis, oxidation, and light⁷. Stress degradation studies require less time. Vaccine formulation robustness is tremendously important because there are various exterior stresses during production, filling, storage and transports. The data obtained from studies can help with the evolution of stability-indicating assays, formulation draft, selecting normal storage conditions, and detecting the duration for time out of law temperature condition while manufacturing. It is highly believed in the industry to inchoate stress studies on the drug substance and the drug product before Phase I development, despite ICH guidelines not indicating a regulatory requirement for these studies until at least clinical Phase III development. There is good reasoning to initiate these studies before Phase I, most appreciably in that they aid in the development of stability-indicating methods where potential degradation products can be identified and understand about degradation pathways its mechanisms, and stabilizer analysis ^[22].A general format for conducting FD studies and stress testing is shown in table 2.

4.1 Thermal stress:

Vaccines are often stored under refrigerated conditions (2°C to 8°C). Most of the aqueous formulated vaccines have been created for low-temperature storage. However, under either elevated or subzero temperatures, the stability of the vaccine may be compromised. The most common stress condition used in the companies is thermal stress, which causes physical destructions such as altering structure and aggregation of proteins, as well as chemical damage, such as dissociation of polysaccharides from the protein carrier in polysaccharide conjugated vaccines²⁸. For live-attenuated virus vaccines exposed to high temperatures are can degrade and reduced stability by exposed to this combined damage. All of these changes in the vaccine can be examine while pursuing studies under elevated temperatures.³⁰

The selection of an appropriate temperature for thermal stress testing depends on certain cases. As a rough guidance, stress temperatures of at least a few degrees to 10° below the melting temperature (Tm) can be studied. Stability conclusions are often impossible if the temperature conditions are studied at or above the Tm, where severe aggregation and/or protein unfolding may occur. Moreover, degradation products may form that are less relevant to the product degrading in a natural environment. Techniques such as DSC, CD, FTIR, and intrinsic and extrinsic FS, can be used to determine the Tm of a protein.⁷

In combination with thermal stress, extreme pH studies can perform in evaluating whether or not the vaccine component has a particular instability as a function of pH, Different buffering agents, hydrochloric acid, and/or sodium hydroxide solutions are generally used for carrying out the effect of pH on vaccine degradation. Stress studies designed for extreme acidic or basic pH are typically initiated in-room and at elevated temperatures (50°C–70°C) for up to 7 days. Alternatively, thermal denaturation experiments can be conducted, DSC, and FS to study the effect of pH on the Tm of the antigens. For example, such studies were conducted in the case of measles,^[31]the Norwalk virus, the respiratory syncytial virus,^30,and the hookworm vaccine.

4.2 Freeze-thaw stress:

Freeze-thaw studies are conducted to assess the vaccine twice–water interfaces in addition to low temperatures, which could lead to nonstable the product. Freeze-thaw studies are necessary to support any temperature excursions that may occur during storage or shipment of the product, as well as to identify any potential excipients that may help to stabilize the product when frozen for either storage or during the lyophilization process. When designing the freeze-thaw experiment, factors to consider include the rate of freezing, concentration, and container geometry.³⁸

It has been observed that freeze-thawing may induce aggregates and proteinaceous particles for protein-based vaccines. High protein concentrations may be more resistant to surface-induced aggregation. Therefore, studies on protein antigens need to be evaluated on a case-by-case basis on factors, such as interfacial stresses (protein/ice surface), crystallization of formulation excipients, phase separation, and pH shifts. Furthermore, the freezing rate may also affect the concentration gradients of a vaccine, whereby faster freezing rates generally result in fewer changes in the concentration gradient.⁷

Other variables that may influence vaccine stability are cooling rate, the temperature at the beginning and end of the freezing process, and filling volume. The cooling rate is one of the most important factors. This parameter deals with controlling the amount and size of ice crystals, as well as the dimension of the interfacial area between ice and freeze-concentrated solutions.

The cooling rate can be classified as either slow (,1°C/minute), intermediate (∼5°C/minute), and fast (10°C–900°C/minute). The cooling rate compared to the actual temperature of the product within the container can be different. These differences could be due to the nature of the product, filling volume, the container used, and formulation composition. For example, when conducting fast freezing, liquid nitrogen is primarily used, which may lead to many small ice crystals and a larger interfacial area. This may increase the instability of the protein depending on the sensitivity of the protein towards surface-induced denaturation. Besides, thawing rates that include thawing temperatures and agitation can also influence the stability of the formulations.³⁹

4.3 Mechanical stress:

Mechanical stress is a common stress that vaccine components are exposed to during manufacturing, transportation, and final administration. Stress factors (i.e., shaking, shearing, liquid filtration, and filling under pressure) must be assessed during preformulation/formulation development. These factors can have significant detrimental effects on the vaccine. It is consequently critical to test formulation robustness against these stress factors⁴.

Surfactants and excipients are added to the formulations to help reduce surface-induced aggregation due to mechanical stress. Stress testing such as agitation (shaking or stirring), pumping, vertexing, sonication, and special shearing, are all used to investigate the effects on vaccine formulations that may include surfactants. During stress studies, protein antigens could exhibit physical and chemical instability. The manufacturing process and experimental design give relevance to the different stresses required for testing⁷.

During the manufacturing process, proteins are exposed to shear (ie, pumping, filtration, and mixing). However, the literature indicates that, in most cases, shear stress is not a major factor concerning aggregation (even at high shear rates of 250,000 seconds-1).93 Conversely, mechanical stress testing experiments have shown that the air-liquid interfaces causing aggregation and/or particle formation can have a great impact on the formulation. Furthermore, severe aggregation during agitation of vials with greater air-headspace compared to less air-headspace has been observed. Foaming of protein after agitation studies affects the air-liquid interface and can potentially induce oxidation.³⁸

The experimental design for mechanical stress studies is dependent on the stage of development and the condition being studied. Stirring and pumping stresses can be assessed to understand the manufacturing processes (i.e., mixing or filling of the drug product material). Based on the representative manufacturing step, parameters should be selected accordingly.

4.4 Light stress:

There are many situations where vaccines are exposed to light. These include exposure to artificial or sunlight during production, shipment, storage, and administration in the clinic. Most vaccines on the market indicate on the label, “protect from direct light,” and are distributed with appropriate secondary packaging to ensure the vaccine is protected from direct and long-term exposure to light⁷.

The current ICH guideline, Q1B,3 should be reviewed when designing a photostability study for new vaccine products. The guideline recommends using a combination of a cool white fluorescent lamp with a near-UV lamp. The ICH Q1B makes a distinction between “force degradation testing studies” and “confirmatory testing studies.” It recommends the conditions used for confirmatory studies to predict the stability of the product. The conditions include exposure to at least 1.2 million lux hours of visible light (400–800 nm) and at least 200 W hour/m2 of UV light (320–400 nm). Any significant observable changes under these confirmatory conditions result in a product recommendation to be protected from direct light and have adequate packaging. In the case of FD studies, the guideline does not recommend specific conditions that increase understanding of vaccine photosensitivity and elucidate degradation pathways. However, a variety of exposure conditions higher than those recommended for confirmatory testing can be applied. Identifying parameters, such as exposure time during manufacturing, fill/finish, and clinical step up and wavelength of the light source can help with designing the photostability study. Besides, proper controls such as light-protected samples are essential.¹³

4.5 Oxidation stress:

Protein oxidation is a common phenomenon that requires investigation during formulation development. It can be caused by a variety of factors such as the presence of peroxides (ie, using less pure polysorbate), metal ions from stainless steel surfaces, residues of aseptic agents (used in isolators), and light⁷. Oxidation stress is often used to gather product knowledge by elucidating degradation pathways and kinetics or assay development. ICH Q1A recommends that the oxidation of the drug substance be assessed; however, it does not define the conditions that should be used⁴.

Oxidation can either be site-specific or non-site-specific. In the case of site-specific oxidation, amino acid residuals such as His, Met, Cysteine, Tyrosine, and Tryptophan are highly sensitive to oxidation. On the other hand, site-specific reactions include photo-oxidation and oxidation, involving free radicals or ROS. Common oxidation stress studies include using low levels of H₂O₂ (0.01%–0.1%), tertbutyl hydroperoxide (0.1%–1%), or ozone at ambient temperatures. The European Pharmacopeia describes an oxidation procedure using 0.005% (v/v) H₂O₂ for 20 hours at 37°C. Forced oxidation in proteins can also be conducted by incubating samples with increasing concentrations of H₂O₂ under normal or accelerated temperatures. As an example, RP-HPLC results for the forced oxidation of a protein antigen conducted at room temperature. At higher H₂O₂concentrations, a complete shift to lower retention times concerning the normal retention time of the protein was observed. These results are compatible with Met oxidation, as degradation products are known to confer higher polarity to the molecule, which may explain the low retention time of the oxidized protein⁷.

The concentration of H₂O₂ is not the only factor affecting oxidation, as the pH of the solution can also influence the amount of oxidation. For example, met residues can become accessible due to pH-induced conformational changes, consequently affecting the degree of oxidation. Furthermore, a combination of certain metal ions, such as Fe (III) or Cu (II), have been used as oxidation agents and can be used when designing oxidation protocols. Oxidation of amino acids makes the protein molecule more hydrophilic, and therefore, chromatographic methods may be used to monitor the oxidation of intact protein as an analytical tool.³⁹

5. CONCLUSION:

Stress degradation studies play an important role in the formulation development of vaccines also due to is lack of specific guidelines for the stress degradation studies for vaccines. Vaccines are very sensitive formulation to environmental conditions, storage, and transport and passthrough various stress. So that for effective and stable formulation development stress studies are an important tool for more stable formulation development. It is better to start degradation studies earlier in the drug development process to have sufficient time to gain more information about the stability of the molecule, it can be helpful for better formulation and good packaging. A properly designed and executed forced degradation study would generate an appropriate sample for the development of the stability-indicating method.

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Q1B	Photostability testing of new drug substances and products ¹³	Guides on how to perform photostability studies to support the submission for NDA registration applications for new molecular entities.
Q2(R1)	Validation of analytical procedures: text and methodology ¹⁴	Stress degradation studies are essential for analytical method validation to demonstrate test specificity.
Q3A(R2)	Impurities in new drug substances¹⁵	Suggest the use of appropriate stress conditions for validation of analytical procedures applies to new chemical substances there is no suggestion about Biological/biotechnological substances.
Q3B(R2)	Impurities in new drug products ¹⁶	Suggest the use of appropriate stress conditions for validation of analytical procedures applies to new chemical products there is no suggestion about Biological/biotechnological products.
Q5C	Stability testing of biotechnological/biological products ¹⁷	Gives an idea about accelerated and stress conditions to support the establishment of the expiration date and to encourage product comparability. Selection of stress conditions to be conducted on a basis of different cases.
Q6B	Test procedures and acceptance criteria for biotechnological/biological products ¹⁸	Forced degradation studies can serve to understand process-related degradants/impurities; justify and rationalize to meet the needs of setting up the acceptance criteria based on potency and the category of product-related impurities.
Q8(R2):	Pharmaceutical development ¹⁹	Forced degradation studies usually include extended variations (ie, temperature, pH, light, shear), container closure system compatibility, and suitability studies under normal and stressed storage conditions.
Q11	Development and manufacture of drug substance ²⁰	Forced degradation studies will give product knowledge and fulfill quality needs.
WHO/KFDA	Workshop on Stability Evaluation of Vaccines, Seoul, Republic of Korea ²¹	It indicates the details about how to perform stability study and glimpse of stability indicating study.

Stress type	Applied in	Attenuated microorganisms	Recombinant protein	Immunostimulator/ adjuvant
Thermal	Thermal denaturation pH range 3–9 (ex, citric phosphate buffer) Ramping temperature 10°C–95°C
Thermal	Drug substance, Drug product	Up to 45°C, up to 4 weeks (varies depending on headspace and if oxidation is expected)	Up to 45°C, up to 4 weeks (varies depending on headspace and if oxidation is expected)	40°C–70°C, up to 12–24 weeks
Freeze-thaw stress	Drug substance, Drug product	3–5 cycles of controlled freezing (-20°C or -70°C) and thawing at room temperature
Mechanical stress	Drug product	Stepwise increase up to 350 rpm for 3.5 hours	350 rpm for up to 72 hours	350 rpm for up to 72 hours
Photodegradation	Drug product	Stepwise increase to confirmatory conditions. (ex, 600 klux/hour visible light, 600 W/hour/m2 UV)	1.2 million lux hours visible, 200 wh/m2 UV	1.2 million lux hours visible, 200 wh/m² UV
Oxidation	Drug substance, Drug product	0.001%–0.01% H₂O₂, room temperature	0.01%–0.1% H₂O₂, 25°C–60°C	0.01%–0.1% H₂O₂, 45°C–60°C
acidic/basic conditions	Drug substance, Drug product	Isothermal incubation 0.1–1.0 N HCl/NaOH, 1–7 days (up to 45°C)	Isothermal incubation 0.1–1.0 N HCl/NaOH,1–7 days (25°C to 60°C)	Isothermal incubation 0.1–1.0 NaCl/NaOH, 1–7 days (25°C to 75°C)
Thermal/ humidity	Drug product	45°C–70°C/ 75% relative humidity	45°C–70°C/ 75% relative humidity	45°C–70°C/ 75% relative humidity