The biotherapeutic market, including monoclonal antibodies (mAbs), is growing rapidly due to their specificity and sensitivity to treating various cancers, autoimmune, and neurodegenerative diseases [1]. Despite their runaway success, product stability during different stages of development, manufacturing, storage, and administration remains a concern [2], [3]. The proteinaceous nature of mAbs makes them susceptible to instability, which could be (1) physical instability: aggregation or denaturation; or (2) chemical instability: deamidation, oxidation, fragmentation, glycation, isomerization, hydrolysis, and Maillard reaction.

These instabilities depend on several factors, including temperature, pH, concentration, excipients, salts, oxygen, light, shaking, and shear [2], [4]. Generally, as per the current guidelines, stability studies implemented by the pharmaceutical industry are proposed to fulfil the licensing requirements [5]. Stability studies include various conditions encountered during product development and manufacturing using orthogonal tools. Stability studies postproduction and during clinical usage have not been extensively considered. Thus, a systematic approach should be encouraged for considering in-use (practical stability) or extended stability. Practical stability refers to the conditions encountered in clinical practice, both voluntarily defined or accidentally, in addition to the stability studies under conventional conditions [5]. Extended stability includes the stability study of the reconstitutes and diluted drug for storage periods beyond those approved from the regulatory authorities. Various conditions faced during postproduction handling and administration of the drug include temperature excursions [6], [7] (storage at room temperature or below the recommended temperature, freeze–thaw cycles), exposure to light [8], [9], [10] (exposure to sunlight while transferring drug from hospital pharmacies to the patient, exposure to fluorescent or LED lights in the clinic or ambient light which can include an ultraviolet component usually in the range of 320–400 nm), mechanical stress [11], [12] (agitation, repeated back and forth movement of the syringe plunger, dropping of vial) and combination of these stress factors [4], [13].

In recent years, the concept of pharmacy-based centralized preparation of mAb infusions, including batch preparations for dose banding or filling ambulatory devices for continuous infusions, is being practiced. Dose banding has been proposed as an effective way to prepare for chemotherapy. [14]. Dose banding is defined as: “A system where chemotherapy doses, calculated by body surface area (BSA) or other means, are fitted to pre-defined dose ranges or ‘bands.’ A standard dose (mid-point of the band) is provided for each band using standard, pre-made syringes or infusion bags, which can be used separately or in combination. These can be prepared by the hospital pharmacy or purchased from a commercial source.” Dose banding reduces outpatient waiting time, reduces the burden on hospital staff, reduces medication error, and reduces drug wastage. Hence, efficiently extended infusion stability (28–84 days) for safe dose banding is required. Apart from dose banding, unacceptable failure of cold storage during transportation of diluted drugs in remote areas is known to occur. It is essential to include additional data comprising practical and extended stability studies with the conventional stability studies at the time of regulatory filing. Many investigations have been reported, including stability studies of reconstituted and diluted mAbs maintained for a few days at 2–8 °C or room temperature [15], [16], [17], [18]. Those studies showed prolonged physicochemical stability but did not investigate all aspects [2]. During practical and extended stability studies, different orthogonal tools should include monitoring of both physical and chemical instabilities, as both can impact the drug product’s half-life, binding affinity, and efficacy. Apart from monitoring the level of aggregates during practical and extended stability, it is critical to monitor the charge heterogeneity of the product as well, as charge heterogeneity, including acidic and basic variants, can impact stability, binding, and efficacy depending on the site(s) of the modifications. For instance, deamidation of asparagine has been reported as an acidic variant that, when present in a complementary determining region (CDR), may reduce the binding affinity and efficacy of the product [19]. It has also been reported that at low pH, deamidation can cause a change in hydrophobicity, resulting in self-association or aggregation of a drug product (DP) [20]. Similarly, methionine oxidation in the Fc region eluted in the basic variant and impacted the binding to neonatal Fc receptor (FcRn) and half-life of the product [21], [22]. Methionine and tryptophan oxidation are known to disrupt protein local conformation, promoting aggregation [23].

Besides the physical and chemical instabilities, practical stability studies should consider the effect of diluent (i.e., normal saline and isotonic dextrose).The dilution of trastuzumab with saline may lead to a drop in pH as low as 5.2 [18]. In mildly acidic solution (above pH 4), aspartic acid (Asp) isomerizes to form a succinimide intermediate, which is susceptible to hydrolysis to generate iso-aspartate (iso-Asp) and aspartate at a ratio of 3:1. It has been reported that under slightly acidic conditions, the rate of succinimide accumulation HC Asp102 in IgG1 and LC Asp30 in IgG2 is higher than its hydrolysis [24], [25]. Further, it has been reported that the formation of iso-aspartate may result in loss of biological activity and may induce an immunogenic response [26], [27], [28], [29], [30]. Many studies show that the succinimide intermediate, either due to Asparagine (Asn) deamidation or Asp isomerization, decreases binding affinity and potency [30], [31]. Another degradation pathway at mildly acidic pH and elevated temperature is fragmentation at the C-terminus of an Asp residue [32], a dominating pathway at pH below 3 with the rate of cleavage at Asp decreasing with an increase in the pH. Between pH 3–5, the peptide bond cleavage at Asp occurs at a comparable rate with Asp isomerization, whereas above pH 5, Asp hydrolysis is negligible. In a study, fragmentation at the C-terminus of an Asp has been reported in the CDR3 region of an IgG2 in a mildly acidic buffer and elevated temperature [33]. Thus, monitoring and controlling the formation of iso-aspartate and succinimide intermediate is necessary.

In the present study, we investigate the effect of elevated temperature and a slight shift in the pH towards acidic values upon dilution in saline. We observed that storing reconstituted and diluted mAb at high temperatures resulted in an increase in the basic variants. Further, even a slight shift in pH towards the acidic range fuels further increase in basic variants. Next, individual basic variants were characterized to monitor and locate chemical modifications, and to assess their impact on physicochemical properties and binding activity. Basic variants identified were predominantly due to heavy chain (HC) Asp102 modification into iso-Asp and succinimide intermediate. Size variant analysis of the variants showed the presence of aggregates mainly in the basic peak, and fragments were present in both main and basic variants. There was no significant difference in the secondary and tertiary structure of the basic variants compared to the main specie and control. Though, the human epidermal growth factor receptor 2 (HER2) binding affinity was reduced with the basic variants compared to the main and control. On the other hand, there was no significant change in binding affinity for neonatal Fc receptor (FcRn). Our study highlights the importance of including the effect of high temperature and changes in pH during practical stability study so as to mimic postproduction handling during pneumatic tube transfer, dose banding, and unexceptional failure of cold storage during transportation to remote areas.