Predicting the Stability of Lyophilized Human Serum Albumin Formulations Containing Sucrose and Trehalose Using Solid-State NMR Spectroscopy: Effect of Storage Temperature on 1H T1 Relaxation Times.

In a lyophilized protein/disaccharide system, the ability of the disaccharide to form a homogeneous mixture with the protein and to slow the protein mobility dictates the stabilization potential of the formulation. Human serum albumin was lyophilized with sucrose or trehalose in histidine, phosphate, or citrate buffer. 1H T1 relaxation times were measured by solid-state NMR spectroscopy and were used to assess the homogeneity and mobility of the samples after zero, six, and twelve months at different temperatures. The mobility of the samples decreased after 6 and 12 months storage at elevated temperatures, consistent with structural relaxation of the amorphous disaccharide matrix. Formulations with sucrose had lower mobility and greater stability than formulations with trehalose.

Investigation of the Solid-State Interactions in Lyophilized Human G-CSF Using Hydrogen-Deuterium Exchange Mass Spectrometry.

Hydrogen/deuterium exchange mass spectrometry (HDX-MS) previously elucidated the interactions between excipients and proteins for liquid granulocyte colony stimulating factor (G-CSF) formulations, confirming predictions made using computational structure docking. More recently, solid-state HDX mass spectrometry (ssHDX-MS) was developed for proteins in the lyophilized state. Deuterium uptake in ssHDX-MS has been shown for various proteins, including monoclonal antibodies, to be highly correlated with storage stability, as measured by protein aggregation and chemical degradation. As G-CSF is known to lose activity through aggregation upon lyophilization, we applied the ssHDX-MS method with peptide mapping to four different lyophilized formulations of G-CSF to compare the impact of three excipients on local structure and exchange dynamics. HDX at 22 °C was confirmed to correlate well with the monomer content remaining after lyophilization and storage at -20 °C, with sucrose providing the greatest protection, and then phenylalanine, mannitol, and no excipient leading to progressively less protection. Storage at 45 °C led to little difference in final monomer content among the formulations, and so there was no discernible relationship with total deuterium uptake on ssHDX. Incubation at 45 °C may have led to a structural conformation and/or aggregation mechanism no longer probed by HDX at 22 °C. Such a conformational change was observed previously at 37 °C for liquid-formulated G-CSF using NMR. Peptide mapping revealed that tolerance to lyophilization and -20 °C storage was linked to increased stability in the small helix, loop AB, helix C, and loop CD. LC-MS HDX and NMR had previously linked loop AB and loop CD to the formation of a native-like state (N*) prior to aggregation in liquid formulations, suggesting a similar structural basis for G-CSF aggregation in the liquid and solid states.

Reference Standards to Support Quality of Synthetic Peptide Therapeutics.

PURPOSE: Peptides are an important class of therapeutics. Their quality is evaluated using a series of analytical tests, many of which depend on well-characterized reference standards to determine identity, purity, and strength. OBJECTIVE: Discuss approaches to producing peptide reference standards, including vialing, lyophilization, analytical testing and stability studies. METHODS: Case studies are used to illustrate analytical approaches to characterize reference standards, including methods for value assignment, content uniformity, and identity testing. Methods described include NMR, mass spectrometry, and chromatography techniques for identity testing and HPLC and GC methods for assessing peptide content and impurities. RESULTS: This report describes the analytical strategy used to establish peptide reference standard and illustrates how results from multiple labs are integrated to assign a value to the final lyophilized vial. A two-step process for value assignment is described, which uses a mass balance approach to assign a quantitative value to a bulk peptide material. The bulk material is then used as a standard to assign a final value to the vialed material. Testing to confirm peptide identity and to ensure consistency of the vialed material is also described. Considerations for addressing variability, identifying outliers, and implementing stability studies are also presented. CONCLUSION: The methods and case studies described provide a benchmark for best practices in establishing the preparation, analytical testing, handling, and storage of peptide reference standards for the pharmaceutical industry. Some peptide features, such as chiral or isobaric amino acids, may require additional techniques to ensure a full characterization of the peptide reference standard.

NMR Reveals Functionally Relevant Thermally Induced Structural Changes within the Native Ensemble of G-CSF.

Structure-function relationships in proteins refer to a trade-off between stability and bioactivity, molded by evolution of the molecule. Identifying which protein amino acid residues jeopardize global or local stability for the benefit of bioactivity would reveal residues pivotal to this structure-function trade-off. Here, we use 15N-1H heteronuclear single quantum coherence (HSQC) nuclear magnetic resonance (NMR) spectroscopy to probe the microenvironment and dynamics of residues in granulocyte colony-stimulating factor (G-CSF) through thermal perturbation. From this analysis, we identified four residues (G4, A6, T133, and Q134) that we classed as significant to global stability, given that they all experienced large environmental and dynamic changes and were closely correlated to each other in their NMR characteristics. Additionally, we observe that roughly four structural clusters are subject to localized conformational changes or partial unfolding prior to global unfolding at higher temperature. Combining NMR observables with structure relaxation methods reveals that these structural clusters concentrate around loop AB (binding site III inclusive). This loop has been previously implicated in conformational changes that result in an aggregation prone state of G-CSF. Residues H43, V48, and S63 appear to be pivotal to an opening motion of loop AB, a change that is possibly also important for function. Hence, we present here an approach to profiling residues in order to highlight their potential roles in the two vital characteristics of proteins: stability and bioactivity.

Impact of Formulation Choices on the Freeze-Drying of an Interleukin-6 Reference Material.

Formulation is critical to successful delivery of lyophilized biologics. We have compared the impact of buffer choice and the addition of sodium chloride (a formulant often viewed as unfavorable for freeze-drying applications) on the outcome of trial lyophilization of an interleukin-6 reference material. While phosphate buffer was a preferred choice and yielded well-formed cakes associated with fair recovery of biological activity, the resultant residual moisture content was high (2-4% w/w). By inclusion of isotonic levels of NaCl, the freeze-dried appearance and process were not impaired, but the residual moisture delivered was considerably reduced to levels <1% w/w. We postulate that this is due to the presence of a more open-cake structure and support this with evidence from thermal analysis and scanning electron microscopy. This work illustrates the importance of wide ranging empirical investigation of formulation options in order to optimize freeze-drying outcomes for biologics.

Protein Engineering and HDX Identify Structural Regions of G-CSF Critical to Its Stability and Aggregation.

The protein engineering and formulation of therapeutic proteins for prolonged shelf-life remain a major challenge in the biopharmaceutical industry. Understanding the influence of mutations and formulations on the protein structure and dynamics could lead to more predictive approaches to their improvement. Previous intrinsic fluorescence analysis of the chemically denatured granulocyte colony-stimulating factor (G-CSF) suggested that loop AB could subtly reorganize to form an aggregation-prone intermediate state. Hydrogen deuterium exchange mass spectrometry (HDX-MS) has also revealed that excipient binding increased the thermal unfolding transition midpoint (Tm) by stabilizing loop AB. Here, we have combined protein engineering with biophysical analyses and HDX-MS to reveal that increased exchange in a core region of the G-CSF comprising loop AB (ABI, a small helix, ABII) and loop CD packed onto helix B and the beginning of loop BC leads to a decrease in Tm and higher aggregation rates. Furthermore, some mutations can increase the population of the aggregation-prone conformation within the native ensemble, as measured by the greater local exchange within this core region.

Shelf-Life Extension of Fc-Fused Single Chain Fragment Variable Antibodies by Lyophilization.

Generation of sequence defined antibodies from universal libraries by phage display has been established over the past three decades as a robust method to cope with the increasing market demand in therapy, diagnostics and research. For applications requiring the bivalent antigen binding and an Fc part for detection, phage display generated single chain Fv (scFv) antibody fragments can rapidly be genetically fused to the Fc moiety of an IgG for the production in eukaryotic cells of antibodies with IgG-like properties. In contrast to conversion of scFv into IgG format, the conversion to scFv-Fc requires only a single cloning step, and provides significantly higher yields in transient cell culture production than IgG. ScFv-Fcs can be effective as neutralizing antibodies in vivo against a panel of pathogens and toxins. However, different scFv fragments are more heterologous in respect of stability than Fab fragments. While some scFv fragments can be made extremely stable, this may change due to few mutations, and is not predictable from the sequence of a newly selected antibody. To mitigate the necessity to assess the stability for every scFv-Fc antibody, we developed a generic lyophilization protocol to improve their shelf life. We compared long-term stability and binding activity of phage display-derived antibodies in the scFv-Fc and IgG format, either stored in liquid or lyophilized state. Conversion of scFv-Fcs into the full IgG format reduced protein degradation and aggregation, but in some cases compromised binding activity. Comparably to IgG conversion, lyophilization of scFv-Fc resulted in the preservation of the antibodies' initial properties after storage, without any drop in affinity for any of the tested antibody clones.

Investigating Alternative Container Formats for Lyophilization of Biological Materials Using Diphtheria Antitoxin Monoclonal Antibody as a Model Molecule.

When preparing biological reference materials, the stability of the lyophilized product is critical for long-term storage, particularly in order to meet WHO International Standards, which are not assigned expiry dates but are expected to be in use for several decades. Glass ampoules are typically used by the National Institute for Biological Standards and Control (NIBSC) for the lyophilization of biological materials. More recently, a clear need has arisen for the filling of smaller volumes, for which ampoules may not be optimal. We investigated the use of plastic microtubes as an alternative container for small volume fills. In this study, a recombinant diphtheria antitoxin monoclonal antibody (DATMAB) was used as a model molecule to investigate the suitability of plastic microtubes for filling small volumes. The stability and quality of the dried material was assessed after an accelerated degradation study using a toxin neutralization test and size exclusion HPLC. While microtubes have shown some promise in the past for use in the lyophilization of some biological materials, issues with stability may arise when more labile materials are freeze-dried. We demonstrate here that the microtube format is unsuitable for ensuring the stability of this monoclonal antibody.

The Principles of Freeze-Drying and Application of Analytical Technologies.

Freeze-drying is a complex process despite the relatively small number of steps involved, since the freezing, sublimation, desorption, and reconstitution processes all play a part in determining the success or otherwise of the final product qualities, and each stage can impose different stresses on a product. This is particularly the case with many fragile biological samples, which require great care in the selection of formulation additives such as protective agents and other stabilizers. Despite this, the process is widely used, not least because once any such processing stresses can be overcome, the result is typically a significantly more stable product than was the case with the starting material. Indeed, lyophilization may be considered a gentler method than conventional air-drying methods, which tend to apply heat to the product rather than starting by removing heat as is the case here. Additionally, due to the high surface area to volume ratio, freeze-dried materials tend to be drier than their conventionally dried counterparts and also rehydrate more rapidly. This chapter provides an overview of freeze-drying (lyophilization) of biological specimens with particular reference to the importance of formulation development, characterization, and cycle development factors necessary for the commercial exploitation of freeze-dried products, and reviews the recent developments in analytical methods which have come to underpin modern freeze-drying practice.

HDX and In Silico Docking Reveal that Excipients Stabilize G-CSF via a Combination of Preferential Exclusion and Specific Hotspot Interactions.

Assuring the stability of therapeutic proteins is a major challenge in the biopharmaceutical industry, and a better molecular understanding of the mechanisms through which formulations influence their stability is an ongoing priority. While the preferential exclusion effects of excipients are well known, the additional presence and impact of specific protein-excipient interactions have proven to be more elusive to identify and characterize. We have taken a combined approach of in silico molecular docking and hydrogen deuterium exchange-mass spectrometry (HDX-MS) to characterize the interactions between granulocyte colony-stimulating factor (G-CSF), and some common excipients. These interactions were related to their influence on the thermal-melting temperatures (Tm) for the nonreversible unfolding of G-CSF in liquid formulations. The residue-level interaction sites predicted in silico correlated well with those identified experimentally and highlighted the potential impact of specific excipient interactions on the Tm of G-CSF.

Impact of controlled vacuum induced surface freezing on the freeze drying of human plasma.

During the freezing step of a typical freeze drying process, the temperature at which nucleation is induced is generally stochastically distributed, resulting in undesired within-batch heterogeneity. Controlled nucleation techniques have been developed to address this problem; these make it possible to trigger the formation of ice crystals at the same time and temperature in all the batch. Here, the controlled nucleation technique known as vacuum induced surface freezing is compared to spontaneous freezing for the freeze drying of human plasma, a highly concentrated system commonly stored in a dried state. The potency of Factor VIII (FVIII), a sensitive, labile protein present in plasma, and the reconstitution time of the dried cakes are evaluated immediately after freeze drying, and after 1, 3, 6 or 9 months storage at different degradation temperatures. We show that the application of controlled nucleation significantly reduces the reconstitution time and in addition helps to improve FVIII stability.

The Influence of Moisture Content and Temperature on the Long-Term Storage Stability of Freeze-Dried High Concentration Immunoglobulin G (IgG).

High protein concentration products for targeted therapeutic use are often freeze-dried to enhance stability. The long-term storage stability of freeze-dried (FD) plasma-derived Immunoglobulin G (IgG) from moderate to high concentrations (10-200 mg/mL) was assessed. Monomer content, binding activity and reconstitution times were evaluated over a 12-month period under accelerated and real-term storage conditions. In the first case study it was shown that FD IgG from 10 to 200 mg/mL had minimal monomer/activity losses at up to ambient temperature after 12 months of storage. However, at 45 °C the sucrose-to-protein ratio played a significant impact on IgG stability above 50 mg/mL. All IgG concentrations witnessed moisture ingress over a 12-month period. The impact of moisture ingress from environmental exposure (between 0.1% and 5% w/w moisture) for IgG 50 mg/mL was assessed, being generated by exposing low moisture batches to an atmospheric environment for fixed time periods. Results showed that at -20 °C and 20 °C there was no significant difference in terms of monomer or antigen-binding activity losses over 6 months. However, at 45 °C, there were losses in monomer content, seemingly worse for higher moisture content samples although model binding activity indicated no losses. Finally, the difference between a low moisture product (0.1-1% w/w) and a moderately high moisture (3% w/w) product generated by alternative freeze-drying cycles, both stoppered under low oxygen headspace conditions, was evaluated. Results showed that at -20 °C and 20 °C there was no difference in terms of binding activity or monomer content. However, at 45 °C, the low moisture samples had greater monomer and binding activity losses than samples from the highest moisture cycle batch, indicating that over-drying can be an issue.

Vacuum-Induced Surface Freezing for the Freeze-Drying of the Human Growth Hormone: How Does Nucleation Control Affect Protein Stability?

In the present work, the effect of controlled nucleation on the stability of human growth hormone (hGH) during freeze-drying has been investigated. More specifically, the vacuum-induced surface freezing technique has been compared to conventional freezing, both with and without an annealing step. Size exclusion chromatography and cell-based potency assays have been used to characterize the formation of soluble aggregates and the biological activity of hGH, respectively. The results obtained indicate that controlled nucleation has a positive effect on both cycle performance and product homogeneity because of the formation of bigger ice crystals, and characterized by a narrower dimensional distribution. From the point of view of hGH stability, we observed that vacuum-induced surface freezing is not detrimental to the biological activity of the protein, or aggregate formation. In addition, the effect of 2 different formulations, including trehalose or cellobiose, on protein preservation was also considered for this study.

Freeze-drying cycle optimization for the rapid preservation of protein-loaded liposomal formulations.

Technology such as the use of microfluidics to generate liposomes has been well researched, yet the stabilisation of liposomal formulations is a major challenge to their greater implementation. To the best of our knowledge, this is the first study investigating the use of 96 well plates to freeze-dry ovalbumin (OVA) loaded neutral (DMPC:Chol and DSPC:Chol), anionic (DSPC:Chol:PS) and cationic (DSPC:Chol:DOTAP) liposomes. Through the use of high throughput screening, a freeze drying cycle was optimised; ramp freezing from from 4 °C to -45 °C, followed by primary drying at -30 °C and secondary drying at 30 °C under a vacuum of 0.1 mBar. These parameters maintained liposome physicochemical properties, with the liposomes remaining below 100 nm and were homogenous (polydispersity index of less than 0.2 post rehydration). Minimal leakage of the OVA protein was observed, with almost 100% OVA remaining encapsulated post rehydration of the formulations. Here we have identified a simple method that allows for the rapid screening and freeze-drying of a range of liposomal formulations.

The influence of the closure format on the storage stability and moisture content of freeze-dried influenza antigen.

Low moisture content is seen as crucial to achieving long term stability of freeze dried biologics and reference materials. Highly hygroscopic freeze-dried material are susceptible to moisture ingress over time which can lead to degradation and loss of biological potency. This study compared vials with unprocessed stoppers, vials with vacuum-oven dried stoppers and glass ampoules in order to determine the superior long term storage format in terms of moisture ingress and potency. B/Phuket influenza antigen was chosen as the model biological standard and the lyophilized antigen was stored at -20, 25 and 45 °C over a 1 year period. Ampoules had no significant moisture change across all storage temperatures as would be anticipated. Moisture content results at -20 °C showed no significant differences between ampoules, vials with vacuum-oven dried stoppers and vials with unprocessed stoppers over 12 months. Vials with vacuum-oven dried stoppers performed similarly to ampoules at -20 °C and 20 °C, but had a small increase in moisture content after 6 months at 45 °C. Vials with unprocessed stoppers preformed the worst and exhibited the largest moisture ingress after just 3 months at both 20 °C and 45 °C. Single radial immunodiffusion (SRD) potency assays showed at -20 °C and 20 °C there was no significant difference between all closure formats. At 45 °C there was a drop in potency for all closure formats, but ampoules and vials with vacuum-oven dried stoppers retained higher potency than vials with unprocessed stoppers. Thus, while ampoules are still considered to be the gold standard format for long term storage stability, using vials with vacuum-oven dried stoppers provides comparable stability and moisture integrity at -20 °C and 20 °C storage.

Measuring the specific surface area (SSA) of freeze-dried biologics using inverse gas chromatography.

The specific surface area (SSA) of freeze-dried biologics (FD) is usually measured via a Brunauer-Emmett-Teller (BET) analysis of volumetric nitrogen adsorption isotherms. However, this technique has accuracy limitations for materials <0.5 m2/g, requires dry samples, must be measured at 77 K and has slow sample preparation times (drying/degassing). Inverse gas chromatography (IGC) is chromatographic characterization technique which can be used to analyse the SSA (down to ≈0.1 m2/g) of various solid-state materials including powders using organic molecules such as octane at ambient temperatures/pressure for a range of relative humidities. This study presents a comprehensive comparison between the N2 BET adsorption versus octane BET adsorption using IGC methods for determining the SSA's for a range of freeze dried biological materials. These include IgG 5% w/w, an influenza antigen standard, sucrose 5% w/w and trehalose 5% w/w. IGC provided comparable SSA values to the N2 BET method with better reproducibility (lower RSDs %). Large variations in average SSA within manufactured FD batches were observed for both IGC and volumetric determinations. IGC was also used to measure the change in SSA with increasing humidity, with FD cakes shrinking and losing their structural integrity with increasing moisture content. Such information highlights the importance of moisture content in determining the physical stability of FD cakes as exemplified by their SSA. In conclusion, IGC is a suitable alternative method for determining the SSA of freeze-dried biological materials which are generally strongly dependent on their moisture content.

Development of a stable chemically cross-linked erythropoietin dimer for use in the quality control of erythropoietin therapeutic products.

Erythropoietin (EPO) is a glycoprotein hormone which promotes red cell replenishment and is also a global biotherapeutic medicine widely used to treat anaemia resulting, for example, from chemotherapy. Requirements of the European Pharmacopoeia stipulate that the level of dimer must be quantified in clinical EPO products (with a limit of 2%). Quantification is hampered by the lack of reference preparations containing stable measurable levels of EPO dimer, but the reproducible generation of a stable dimerised EPO preparation is challenging. We describe here the development of a lyophilised, chemically cross-linked EPO preparation, which has good stability and may be used for calibration and system suitability assurance for the size exclusion chromatographic separation of EPO preparations. Graphical abstract.

Selective Stabilization and Destabilization of Protein Domains in Tissue-Type Plasminogen Activator Using Formulation Excipients.

Multidomain biotherapeutic proteins present additional behavioral and analytical challenges for the optimization of their kinetic stability by formulation. Tissue-type plasminogen activator (tPA) comprises six protein domains that exhibit a complex and pH-dependent thermal unfolding profile, due to partially independent domain unfolding. Here we have used tPA as a model for evaluating the relationships between various thermal unfolding and aggregation parameters in multidomain proteins. We show that changes in the thermal unfolding profile of tPA were parametrized by the overall thermal midpoint transition temperature, Tm, and the Van't Hoff entropy for unfolding, Δ Svh, which is a measure of unfolding cooperativity. The kinetics of degradation at 45 °C, leading to aggregation, were measured as rates of monomer and activity loss. These two rates were found to be coincident at all pH. Aggregation accelerated at pH 4 due to the early unfolding of the serine protease N-terminal domain (SP-N), whereas at pH 5-8, the fraction unfolded at 45 °C ( f45) was <1%, resulting in a baseline rate of aggregation from the native ensemble. We used a Design of Experiments (DoE) approach to evaluate how formulation excipients impact and control the thermal unfolding profile for tPA and found that the relative stability of each of the tPA domains was dependent on the formulation. Therefore, the optimization of formulations for complex multidomain proteins such as tPA may need to be multiobjective, with careful selection of the desired attributes that improve stability. As aggregation rates (ln v) correlated well to Tm ( R2 = 0.77) and Δ Svh ( R2 = 0.71) but not Tagg ( R2 = 0.01), we analyzed how formulation excipients and pH would be able to optimize Tm and Δ Svh. Formulation excipient behaviors were found to group according to their combined impact on Tm and Δ Svh. The effects of each excipient were often selectively stabilizing or destabilizing to specific tPA domains and changed the stability of particular domains relative to the others. The types of mechanism by which this could occur might involve specific interactions with the protein surface, or otherwise effects that are mediated via the solvent as a result of the different surface hydrophobicities and polarities of each domain.

Towards international standardization of immunoassays for Müllerian inhibiting substance/anti-Müllerian hormone.

RESEARCH QUESTION: Is formulated and lyophilized, recombinant human Müllerian inhibiting substance, also known as anti-Müllerian hormone (AMH), suitable for the preparation of a WHO international standard to calibrate AMH immunoassays? DESIGN: The AMH content of a trial preparation, coded SS-581, was determined by five laboratories using seven immunoassay methods. Participants were requested to report the content of the preparation in terms of their method calibrators through the measurement of a minimum of five concentrations in the linear part of the dose-response curve. Participants were also asked to measure, concomitantly, a panel of six serum samples containing AMH at concentrations of 0.1-13.0 ng/ml. RESULTS: Across all assays, including two automated assays in development, the geometric mean content was 361.76 ng/ampoule with a geometric coefficient of variation (GCV%) of 39.95%. When measured by immunoassays that were commercially available at the time of the study, the mean content was 423.08 ng/ampoule, with a GCV% of 26.67%. The inter-method geometric means of five serum samples with an AMH concentration >0.3 ng/ml and measured concomitantly with dilutions of SS-581 varied with a range of GCV% of 14.90-22.35%, which may reflect the use of serum sample value transfer to calibrate current immunoassays, some of which use non-human AMH calibrators. The AMH in trial preparation SS-581 was shown to be biologically active in the Müllerian duct regression assay. CONCLUSIONS: A reference material prepared using human recombinant AMH is a promising candidate for the preparation of an international standard for AMH for immunoassays calibrated to recombinant human AMH.

Quantification of Müllerian Inhibiting Substance/Anti-Müllerian Hormone polypeptide by isotope dilution mass spectrometry.

Measurement of serum concentrations of Müllerian inhibiting substance (MIS), also known as anti-Müllerian Hormone (AMH) by immunoassay is gaining clinical acceptance and widespread use for the diagnosis of ovarian conditions and for prediction of the response to ovarian stimulation protocols as part of assisted reproductive therapies. Provision of an International Standard to harmonize immunoassay methods is required. It is desirable for the content of a future International Standard to be assigned in mass units for consistency with the units reported by current methods. Isotope dilution mass spectrometry (IDMS), a physicochemical method with traceability to the SI (Système International d'Unités) unit of mass, is a candidate approach to provide orthogonal data to support this mass assignment. Here, we report on the development of an IDMS method for quantitation of AMH using three peptides from different regions of the AMH monomer as surrogates for the measurement of AMH. We show the sensitivity and linearity of the standard peptides and demonstrate the reproducibility and consistency of the measurement amongst the three peptides for determining the AMH content in buffered preparations and in trial preparations of recombinant AMH, lyophilised in the presence of an excess of bovine casein.