Parag Kolhe

Frozen State Storage Instability of a Monoclonal Antibody: Aggregation as a Consequence of Trehalose Crystallization and Protein Unfolding

ABSTRACTPurposeTo investigate the cause of unexpected and erratic increase in aggregation during long-term storage of an IgG2 monoclonal antibody in a trehalose formulation at −20°C.MethodsFrozen matrix was sampled, stored frozen at various temperatures and analyzed by SEC over time.ResultsAggregation increased with time at −20°C but not at −40°C or −10°C. The cause of the instability was the crystallization of freeze-concentrated trehalose from the frozen solute when the storage temperature exceeds the glass transition temperature of the matrix (−29°C). Crystallization at −20°C deprives the protein of the cryoprotectant, leading to a slow increase in aggregation. Storage at −10°C also leads to crystallization of trehalose but no increase in aggregation. It is hypothesized that significantly higher mobility in the matrix at −10°C allows protein molecules that are unfolded at the ice interface on freezing to refold back before significant aggregation can occur. In contrast, lack of mobility at −40°C prevents crystallization, refolding, and aggregation.ConclusionsAggregation in the frozen state when stored above the glass transition temperature is a consequence of balance between rate of crystallization leading to loss of cryoprotectant, rate of aggregation of the unfolded protein molecules, and rate of refolding that prevents aggregation.

Impact of freezing on pH of buffered solutions and consequences for monoclonal antibody aggregation

Freezing of biologic drug substance at large scale is an important unit operation that enables manufacturing flexibility and increased use‐period for the material. Stability of the biologic in frozen solutions is associated with a number of issues including potentially destabilizing pH changes. The pH changes arise from temperature‐associated change in the pKas, solubility limitations, eutectic crystallization, and cryoconcentration. The pH changes for most of the common protein formulation buffers in the frozen state have not been systematically measured. Sodium phosphate buffer, a well‐studied system, shows the greatest change in pH when going from +25 to −30°C. Among the other buffers, histidine hydrochloride, sodium acetate, histidine acetate, citrate, and succinate, less than 1 pH unit change (increase) was observed over the temperature range from +25 to −30°C, whereas Tris‐hydrochloride had an ∼1.2 pH unit increase. In general, a steady increase in pH was observed for all these buffers once cooled below 0°C. A formulated IgG2 monoclonal antibody in histidine buffer with added trehalose showed the same pH behavior as the buffer itself. This antibody in various formulations was subject to freeze/thaw cycling representing a wide process (phase transition) time range, reflective of practical situations. Measurement of soluble aggregates after repeated freeze–thaw cycles shows that the change in pH was not a factor for aggregate formation in this case, which instead is governed by the presence or absence of noncrystallizing cryoprotective excipients. In the absence of a cryoprotectant, longer phase transition times lead to higher aggregation.

Development of Biotechnology Products in Pre-filled Syringes: Technical Considerations and Approaches

A monoclonal antibody (mAb) product development case study is presented to address some of the issues faced during developing a pre-filled syringe (PFS) product for a biotherapeutic. In particular, issues involving incompatibility with silicone oil and a stability-based approach for selection of PFS barrel and tip cap components have been discussed. Silicone spiking studies followed by exposure to agitation stress or accelerated temperature conditions were used to check for incompatibilities of the mAb with silicone oil, a necessary product contact material in PFS. In addition, screening studies to compare various closure materials as well as syringe barrel processing methods were used to select the optimum closure materials as well as the correct syringe processing method. Results indicate that the model mAb formulation used was sensitive to high levels of silicone oil especially under accelerated temperature conditions resulting in formation of protein–silicone particles in the solution for samples that were spiked with the silicone oil. Agitation stress did not have any significant impact on the quality attributes tested. Samples stored in syringe barrels that were processed with sprayed-on silicone had higher levels of subvisible particles as compared to those that were processed with the baked-on process. The tip cap comparability study resulted in one tip cap material having superior compatibility among the three that were tested. The quality attribute that was most impacted by the tip cap materials was mAb oxidation. An approach for evaluation of primary packaging components during the development of pre-filled syringe presentations for biotechnology-based compounds has been highlighted.

Comparative Evaluation of Disodium Edetate and Diethylenetriaminepentaacetic Acid as Iron Chelators to Prevent Metal -Catalyzed Destabilization of a Therapeutic Monoclonal Antibody

Understanding the effect of metal chelators with respect to their ability to inhibit metal-catalyzed degradation in biologic products is a critical component for solution formulation development. Two metal chelators, disodium edetate (Na(2)EDTA) and diethylenetriaminepentaacetic acid (DTPA), were evaluated for their ability to stabilize IgG2 mAb in solution formulations spiked with various levels of iron. Real-time stability attributes such as oxidation, soluble aggregate formation, deamidation, and fragmentation demonstrated that DTPA was equivalent to Na(2)EDTA with respect to inhibiting iron-induced degradation over the range of iron concentrations studied. When sufficient chelator was present to stoichiometrically complex trace iron contamination, both Na(2)EDTA and DTPA exhibited the capacity to reduce protein degradation. However, substoichiometric ratios of both chelators were unable to inhibit the degradation induced by free iron ions, which were found to bind weakly to the mAb. This bound iron did not measurably alter the secondary or the tertiary structure of the mAb but appeared to decrease its intrinsic thermodynamic stability, probably by causing subtle perturbations in the tertiary structure. These destabilization effects were not observed when the chelators were present at stoichiometric ratios highlighting the feasibility of using DTPA as an alternate trace metal chelator to Na(2)EDTA in biologic protein formulations.

Protein and solute distribution in drug substance containers during frozen storage and post‐thawing: A tool to understand and define freezing–thawing parameters in biotechnology process development

Active pharmaceutical ingredient for biotechnology‐based drugs, commonly known as drug substance (DS), is often stored frozen for longer shelf‐life. Freezing DS enhances stability by slowing down reaction rates that lead to protein instability, minimizes the risk of microbial growth, and eliminates the risk of transport‐related stress. High density polyethylene bottles are commonly used for storing monoclonal antibody DS due to good mechanical stress/strain resistant properties even at low temperatures. Despite the aforementioned advantages for frozen storage of DS, this is not devoid of risks. Proteins are known to undergo ice–water surface denaturation, cryoconcentration, and cold denaturation during freezing. A systematic investigation was performed to better understand the protein and solute distribution along with potential of aggregate formation during freeze and thaw process. A significant solute and protein concentration gradient was observed for both frozen and thawed DS bottles. In case of thawed DS, cryoconcentration was localized in the bottom layer and a linear increase in concentration as a function of liquid depth was observed. On the other hand, for frozen DS, a “bell shaped” cryoconcentration distribution was observed between the bottom layers and centre position. A cryoconcentration of almost three‐fold was observed for frozen DS in the most concentrated part when freezing was conducted at −20 and −40 °C and 2.5‐fold cryoconcentration was observed in the thawed DS before mixing. The information obtained in this study is critical to design freeze thaw experiments, storage condition determination, and process improvement in manufacturing environment.

Molecular attributes of conjugate antigen influence function of antibodies induced by anti-nicotine vaccine in mice and non-human primates

Anti-nicotine vaccines aim to prevent nicotine entering the brain, and thus reduce or eliminate the reward that drives nicotine addiction. Those tested in humans to date have failed to improve quit rates over placebo, possibly because antibody (Ab) responses were insufficient to sequester enough nicotine in the blood in the majority of subjects. We have previously shown in mice that the carrier, hapten and linker used in the nicotine conjugate antigen each influence the function (nicotine-binding capacity) of the Ab induced. Herein we have evaluated immunogenicity in mice of 27 lots of NIC7-CRM, a conjugate of 5-aminoethoxy-nicotine (Hapten 7) and a mutant nontoxic form of diphtheria toxin (CRM197), that differed in three antigen attributes, namely hapten load (number of haptens conjugated to each molecule of CRM197), degree of conjugate aggregation and presence of adducts (small molecules attached to CRM197 via a covalent bond during the conjugation process). A range of functional responses (reduced nicotine in the brain of immunized animals relative to non-immunized controls) were obtained with the different conjugates, which were adjuvanted with aluminum hydroxide and CpG TLR9 agonist. Trends for better functional responses in mice were obtained with conjugates having a hapten load of 11 to 18, a low level of high molecular mass species (HMMS) (i.e., not aggregated) and a low level of adducts and a more limited testing in cynomolgus monkeys confirmed these results. Thus hapten load, conjugate aggregation and presence of adducts are key antigen attributes that can influence Ab function induced by NIC7-CRM.

The Effect of Shipping Stresses on Vaccine Re-dispersion Time

A case study is presented for a vaccine drug product (DP) that showed variable re-dispersion times between syringes within a given DP lot and between different DP lots when shipped from the manufacturing site to the receiving site. A simulated shipping study was designed to understand the effect of individual shipping stresses on re-dispersion time and product quality. Shipping stresses simulating shock/drop, aircraft, and truck vibrations were applied separately to 3 syringe orientations, namely tip up, tip down, and tip horizontal (TH). Results from the simulated shipping study showed that shock/drop reduced re-dispersion time while truck and aircraft vibrations increased re-dispersion time in the tip down orientation. The dissimilar effects of different shipping stresses on re-dispersion resulted in the observed intra and inter DP lot variability in re-dispersion time. Shipping stresses did not impact re-dispersion in the TH or tip up orientation. No vaccine product quality attributes or physical properties were affected by shipping stresses. Actual shipping results correlated well with simulated shipping data. Because re-dispersion time was influenced mainly by shipping stress and syringe orientation, the mitigation measure to reduce end-user re-dispersion time was to implement the TH orientation for DP syringes during shipment and storage.

Bulk Protein Solution: Freeze–Thaw Process, Storage and Shipping Considerations

Protein drug substance is typically frozen to enable manufacturing flexibility through prolonging the shelf life of drug substance and providing better biochemical stability. The process of freezing and thawing of bulk protein solutions poses several challenges. It is important to understand the process and define mitigation strategies to address these challenges. Just not the process but the choice of storage container can have an impact on stability and downstream operation of formulation during drug product process. Therefore, understanding the options currently available in terms of drug substance storage containers and how to balance the need based on specific scenarios is critical. Storage temperature is as important as the container the drug substance is stored at. Furthermore, shipping drug substance in frozen state requires thorough understanding of logistics and dependence of shipping temperature on stability. This chapter fundamentally looks at the freezing and thawing process, discusses phenomenon of cryoconcentration in various containers and mitigation strategies, consequence of not choosing appropriate storage temperature, and provides considerations for storage and shipping of drug substance.

Ex Situ and In Situ Characterization of Vaccine Suspensions in Pre-Filled Syringes

Ex situ and in situ techniques were used to characterize the suspended phase over time for a vaccine drug product supplied in syringes. Micro-computed tomography was used to characterize the suspended sediment in situ in the syringe, while traditional techniques such as particle size distribution, charge (zeta potential), settling rate, and front-faced fluorescence were used to characterize the suspension ex situ. In addition, analytical chemical measurements were conducted in parallel during the course of the study. The ex situ and in situ techniques together with the chemical analyses provided different sets of data, but all leading to the same conclusion that the older, hard to re-disperse vaccine product syringes were similar in product quality attributes (both physical and chemical) to the freshly made, easy to re-disperse syringes. Longer re-dispersion time with age was not a result of any altered physical or chemical attributes of the product but simply because of the distance travelled by the sediment from the neck of the syringe barrel deeper into the bore of the syringe over time under the influence of gravity in the tip down orientation, making it harder for the continuous external phase to access the sediment in the bore and enable easy re-dispersion.