Publications by authors named "Paul Matejtschuk"

31 Publications

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

Pharmaceutics 2021 Nov 17;13(11). Epub 2021 Nov 17.

Standardization Science Section, National Institute of Biological Standards and Control, MHRA, Blanche Lane, South Mimms, Potters Bar EN6 3QG, Hertfordshire, UK.

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.
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http://dx.doi.org/10.3390/pharmaceutics13111948DOI Listing
November 2021

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

Mol Pharm 2020 12 28;17(12):4637-4651. Epub 2020 Oct 28.

Department of Biochemical Engineering, University College London, Gower Street, London WC1E 6BT, United Kingdom.

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 () 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 of G-CSF.
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http://dx.doi.org/10.1021/acs.molpharmaceut.0c00877DOI Listing
December 2020

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

Methods Mol Biol 2021 ;2180:99-127

National Institute for Biological Standards and Control (NIBSC), Potters Bar, UK.

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.
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http://dx.doi.org/10.1007/978-1-0716-0783-1_3DOI Listing
March 2021

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

Int J Pharm 2020 May 31;582:119290. Epub 2020 Mar 31.

Department of Applied Science and Technology, Politecnico di Torino, 24 corso Duca degli Abruzzi, Torino 10129, Italy.

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.
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http://dx.doi.org/10.1016/j.ijpharm.2020.119290DOI Listing
May 2020

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

Pharmaceutics 2020 Mar 27;12(4). Epub 2020 Mar 27.

Surfaces and Particle Engineering Laboratory, Department of Chemical Engineering, Imperial College London, SW7 2AZ, UK.

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% / 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% /) and a moderately high moisture (3% /) 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.
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http://dx.doi.org/10.3390/pharmaceutics12040303DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7238084PMC
March 2020

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

Int J Pharm 2020 Jan 6;573:118722. Epub 2019 Nov 6.

Standardisation Science, National Institute for Biological Standards & Control (NIBSC), Blanche Lane, South Mimms, Potters Bar EN6 3QG, United Kingdom. Electronic address:

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.
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http://dx.doi.org/10.1016/j.ijpharm.2019.118722DOI Listing
January 2020

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

Vaccine 2019 07 2;37(32):4485-4490. Epub 2019 Jul 2.

Surfaces and Particle Engineering Laboratory, Department of Chemical Engineering, Imperial College London, SW7 2AZ, United Kingdom.

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.
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http://dx.doi.org/10.1016/j.vaccine.2019.06.070DOI Listing
July 2019

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

Eur J Pharm Biopharm 2019 Sep 22;142:216-221. Epub 2019 Jun 22.

Surfaces and Particle Engineering Laboratory, Department of Chemical Engineering, Imperial College London, SW7 2AZ, United Kingdom. Electronic address:

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 m/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 m/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 N 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 N 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.
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http://dx.doi.org/10.1016/j.ejpb.2019.06.026DOI Listing
September 2019

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

J Pharm Sci 2020 01 17;109(1):254-263. Epub 2019 Apr 17.

Standardisation Science Section, Analytical and Biological Sciences Division, National Institute for Biological Standards and Control, Blanche Lane, Medicines & Healthcare Products Agency, South Mimms, Potters Bar, Hertfordshire EN6 3QG, UK. Electronic address:

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.
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http://dx.doi.org/10.1016/j.xphs.2019.04.014DOI Listing
January 2020

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

Anal Bioanal Chem 2019 May 10;411(13):2755-2758. Epub 2019 Apr 10.

National Institute for Biological Standards & Control (NIBSC), Blanche Lane, South Mimms, Potters Bar, Hertfordshire, EN6 3QG, UK.

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.
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http://dx.doi.org/10.1007/s00216-019-01768-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6522647PMC
May 2019

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

Mol Pharm 2019 02 9;16(2):744-755. Epub 2019 Jan 9.

Department of Biochemical Engineering , University College London , Gower Street , London WC1E 6BT , U.K.

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, T, and the Van't Hoff entropy for unfolding, Δ S, 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 ( f) 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 T ( R = 0.77) and Δ S ( R = 0.71) but not T ( R = 0.01), we analyzed how formulation excipients and pH would be able to optimize T and Δ S. Formulation excipient behaviors were found to group according to their combined impact on T and Δ S. 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.
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http://dx.doi.org/10.1021/acs.molpharmaceut.8b01024DOI Listing
February 2019

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

Reprod Biomed Online 2018 Nov 5;37(5):631-640. Epub 2018 Sep 5.

Biotherapeutics Division, NIBSC, South Mimms, Potters Bar Hertfordshire, UK.

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.
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http://dx.doi.org/10.1016/j.rbmo.2018.08.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6302068PMC
November 2018

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

Anal Biochem 2018 11 6;560:50-55. Epub 2018 May 6.

National Institute for Biological Standards and Control, South Mimms, Potters Bar, EN6 3QG, UK.

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.
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http://dx.doi.org/10.1016/j.ab.2018.05.006DOI Listing
November 2018

Humidity induced collapse in freeze dried cakes: A direct visualization study using DVS.

Eur J Pharm Biopharm 2018 Jun 3;127:29-36. Epub 2018 Feb 3.

Surfaces and Particle Engineering Laboratory, Department of Chemical Engineering, Imperial College London, SW7 2AZ, United Kingdom. Electronic address:

Maintaining low moisture content is seen as crucial to sustaining long term stability in freeze dried (FD) cakes as higher moisture could lead to cake collapse, degradation and a loss of biological potency. Using a combination of gravimetric data and video images captured from a Dynamic Vapour Sorption instrument the onset humidity Collapse Point (RH), the humidity onset Crystallisation (RH) and onset Glass Transition (RH) points for a series of freeze dried cakes at 10, 25 and 40 °C have been determined. The moisture sorption behavior with respect to cake collapse and other morphological phase transitions are reported for a two freeze drying excipients and one product formulation; sucrose, trehalose (both 5% w/w) and an influenza antigen (A/Wisconsin/15/2009 H3N2 NYMCX-183, formulated with 1.1% w/w sucrose). Stability maps for all three formulations tested were reported as a function of %RH and temperature using the methods described in this work, thus the direct visualization of collapse behavior for any FD cake can now be standardized and routinely determined, facilitating the formulation of FD products with improved stability and storage performance.
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http://dx.doi.org/10.1016/j.ejpb.2018.02.003DOI Listing
June 2018

T-Values and Unfolded Fraction Can Predict Aggregation Rates for Granulocyte Colony Stimulating Factor Variant Formulations but Not under Predominantly Native Conditions.

Mol Pharm 2018 01 28;15(1):256-267. Epub 2017 Nov 28.

Department of Biochemical Engineering, University College London , London WC1E 7JE, U.K.

Protein engineering and formulation optimization strategies can be taken to minimize protein aggregation in the biopharmaceutical industry. Short-term stability measures such as the midpoint transition temperature (T) for global unfolding provide convenient surrogates for longer-term (e.g., 2-year) degradation kinetics, with which to optimize formulations on practical time-scales. While successful in some cases, their limitations have not been fully evaluated or understood. T values are known to correlate with chemical degradation kinetics for wild-type granulocyte colony stimulating factor (GCSF) at pH 4-5.5. However, we found previously that the T of an antibody Fab fragment only correlated with its rate of monomer loss at temperatures close to the T. Here we evaluated T, the fraction of unfolded protein (f) at temperature T, and two additional short-term stability measures, for their ability to predict the kinetics of monomer and bioactivity loss of wild-type GCSF and four variants, at 37 °C, and in a wide range of formulations. The GCSF variants introduced one to three mutations, giving a range of conformational stabilities spanning 7.8 kcal mol. We determined the extent to which the formulation rank order differs across the variants when evaluated by each of the four short-term stability measures. All correlations decreased as the difference in average T between each pair of GCSF variants increased. The rank order of formulations determined by T was the best preserved, with R-values >0.7. T-values also provided a good predictor (R = 0.73) of the aggregation rates, extending previous findings to include GCSF variant-formulation combinations. Further analysis revealed that GCSF aggregation rates at 37 °C were dependent on the fraction unfolded at 37 °C (f), but transitioned smoothly to a constant baseline rate of aggregation at f < 10. A similar function was observed previously for A33 Fab formulated by pH, ionic strength, and temperature, without excipients. For GCSF, all combinations of variants and formulations fit onto a single curve, suggesting that even single mutations destabilized by up to 4.8 kcal mol, are insufficient to change significantly the baseline rate of aggregation under native conditions. The baseline rate of aggregation for GCSF under native conditions was 66-fold higher than that for A33 Fab, highlighting that they are a specific feature of each native protein structure, likely to be dependent on local surface properties and dynamics.
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http://dx.doi.org/10.1021/acs.molpharmaceut.7b00876DOI Listing
January 2018

Preservation and stability of cell therapy products: recommendations from an expert workshop.

Regen Med 2017 07 19;12(5):553-564. Epub 2017 Jul 19.

Fraunhofer-Institute for Biomedical Engineering, Sulzbach, Germany.

If the field of regenerative medicine is to deliver therapies, rapid expansion and delivery over considerable distances to large numbers of patients is needed. This will demand efficient stabilization and shipment of cell products. However, cryopreservation science is poorly understood by life-scientists in general and in recent decades only limited progress has been made in the technology of preservation and storage of cells. Rapid translation of new developments to a broader range of cell types will be vital, as will assuring a deeper knowledge of the fundamental cell biology relating to successful preservation and recovery of cell cultures. This report presents expert consensus on these and other issues which need to be addressed for more efficient delivery of cell therapies.
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http://dx.doi.org/10.2217/rme-2017-0073DOI Listing
July 2017

The importance of formulation in the successful lyophilization of influenza reference materials.

Biologicals 2015 Mar 20;43(2):110-6. Epub 2015 Jan 20.

Standardisation Science, NIBSC, Medicines & Healthcare Products Regulatory Agency, Blanche Lane, South Mimms, Potters Bar, EN6 3QG, UK. Electronic address:

Lyophilized Influenza antigen reference reagents are a critical resource in the quality control of influenza vaccines. A standard formulation has been used successfully at NIBSC for many years however, following the unexpected occurrence of a collapsed appearance in a particular batch a study was carried out to establish the impact of the sugar concentration in the formulation using modulated differential scanning calorimetry (mDSC) and nuclear magnetic resonance spectroscopy (NMR). There was a correlation between the presence and size of the mDSC eutectic temperature events and the freeze dried appearance of the cakes, which became progressively worse with increasing amounts of sugar. NMR spectroscopy could be used to positively identify and quantify the sugars in the formulations. MDSC can rapidly predict if the freeze dried appearance will be acceptable so as to assure the successful lyophilization of influenza reference preparations.
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http://dx.doi.org/10.1016/j.biologicals.2014.12.001DOI Listing
March 2015

Differential scanning fluorimetry: rapid screening of formulations that promote the stability of reference preparations.

J Pharm Biomed Anal 2013 Apr 8;77:163-6. Epub 2013 Jan 8.

Standardisation Science, National Institute for Biological Standards and Control, Health Protection Agency, South Mimms, Potters Bar Herts, UK.

When formulating a biopharmaceutical protein, its stability in the liquid state is critical. In addition, when preparing biological reference materials the stability, both when lyophilised and after reconstitution, needs to be determined. In order to optimise the stability in aqueous conditions (as indicated by Tmelt or denaturation point) the impact of different excipient choices should be evaluated. Micro differential scanning calorimetry is a well established method for these applications but can be time consuming even when an autosampler is used. Differential scanning fluorimetry (DSF) is a novel technique which measures the fluorescence of a dye when bound to the hydrophobic regions of a denatured protein. We have investigated these techniques for their suitability using alpha-1-protease inhibitor (A1PI) as a model system and found similar trends in terms of the impact of different excipients by both methods. DSF is a promising method and has advantages in terms of speed and quantities of biological material required and can be performed using a PCR instrument.
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http://dx.doi.org/10.1016/j.jpba.2013.01.006DOI Listing
April 2013

Freeze drying formulation using microscale and design of experiment approaches: a case study using granulocyte colony-stimulating factor.

Biotechnol Lett 2012 Apr 21;34(4):641-8. Epub 2011 Dec 21.

Department of Biochemical Engineering, The Advanced Centre for Biochemical Engineering, University College London, Torrington Place, London, WC1E 7JE, UK.

The lyophilization of proteins in microplates, to assess and optimise formulations rapidly, has been applied for the first time to a therapeutic protein and, in particular, one that requires a cell-based biological assay, in order to demonstrate the broader usefulness of the approach. Factorial design of experiment methods were combined with lyophilization in microplates to identify optimum formulations that stabilised granulocyte colony-stimulating factor during freeze drying. An initial screen rapidly identified key excipients and potential interactions, which was then followed by a central composite face designed optimisation experiment. Human serum albumin and Tween 20 had significant effects on maintaining protein stability. As previously, the optimum formulation was then freeze-dried in stoppered vials to verify that the microscale data is relevant to pilot scales. However, to validate the approach further, the selected formulation was also assessed for solid-state shelf-life through the use of accelerated stability studies. This approach allows for a high-throughput assessment of excipient options early on in product development, while also reducing costs in terms of time and quantity of materials required.
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http://dx.doi.org/10.1007/s10529-011-0822-2DOI Listing
April 2012

The first international standard for antibodies to HPV 16.

Vaccine 2011 Sep 19;29(38):6520-6. Epub 2011 Jul 19.

Division of Virology, National Institute for Biological Standards and Control, Health Protection Agency, Blanche Lane, South Mimms, Potters Bar, Herts EN6 3QG, UK.

Current HPV vaccines and vaccine candidates are based on recombinant virus capsid proteins, so called virus-like particles (VLPs). Standardisation of assays for HPV capsid antibody will assist with epidemiology studies and future vaccine development. A World Health Organization international collaborative study was undertaken to assess the suitability of a freeze-dried serum, obtained from women naturally infected with HPV 16 and reactive against HPV 16 only, to serve as the International Standard for antibodies to HPV 16 in immunoassays and pseudovirion neutralisation assays. Eleven laboratories from nine countries participated in the collaborative study in which the candidate (NIBSC code 05/134) was assayed alongside samples from both vaccinees and naturally infected individuals. 05/134 had titres which were comparable to those obtained with serum from a naturally infected individual. Overall the variation between laboratories is similar to that observed in the previous study for samples from naturally infected individuals although slightly wider for sera from vaccinees. 05/134 has been established by the WHO Expert Committee on Biological Standardization as the 1st International Standard for antibodies to HPV 16, human serum, with an assigned potency of 5IUper ampoule.
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http://dx.doi.org/10.1016/j.vaccine.2011.07.007DOI Listing
September 2011

The 2nd International Standard for human granulocyte colony stimulating factor.

J Immunol Methods 2011 Mar 17;367(1-2):63-9. Epub 2011 Feb 17.

Biotherapeutics Group, NIBSC, Blanche Lane, South Mimms, Potters Bar, Hertfordshire, EN6 3QG, UK.

Five candidate preparations of human sequence recombinant granulocyte-colony stimulating factor (G-CSF) were formulated and lyophilized at NIBSC prior to evaluation in a collaborative study for their suitability to serve as a replacement International Standard (IS). The preparations were tested by 13 laboratories using in vitro bioassays. The candidate preparation 09/136 was judged suitable to serve as a replacement international standard based on the data obtained for biological activity and stability. On the basis of the results reported here, the preparation coded 09/136 was established by the WHO Expert Committee on Biological Standardization (ECBS) as the 2nd IS for human G-CSF with an assigned value for G-CSF activity of 95,000 IU/ampoule. Calibration of the 2nd IS is primarily based on the bioassay in use in various laboratories and relies entirely on the estimates calculated relative to the WHO 1st IS for continuity of the IU.
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http://dx.doi.org/10.1016/j.jim.2011.02.005DOI Listing
March 2011

Release of proteolytic activity following reduction in therapeutic human serum albumin containing products: detection with a new neoepitope endopeptidase immunoassay.

J Pharm Biomed Anal 2011 Jan 19;54(1):74-80. Epub 2010 Aug 19.

National Institute for Biological Standards and Control, Health Protection Agency, Division of Bacteriology, South Mimms, Potters Bar, Hertfordshire EN6 3QG, UK.

Botulinum type A toxin (BoNT/A) is defined by its specific endopeptidase cleavage of SNAP25 between Gln(197) and Arg(198) under reducing conditions. The neurotoxin is widely used for therapeutic or cosmetic purposes, but should not contain other toxin serotypes or unwanted protease activities. Using a neoepitope endopeptidase immunoassay, additional cleavage between Arg(198) and Ala(199) was detected with a range of therapeutic BoNT/A products confirming an earlier report of an unidentified proteolytic component. By developing the assay and making it insensitive to BoNT/C1, any activity due to the type C1 toxin was excluded. Therapeutic preparations consist of ng quantities of toxin protein which are typically stabilised by 0.125-30 mg of HSA. An excellent correlation (R(2)=0.993) between HSA content per vial and measured activity was obtained within the therapeutic BoNT/A products tested. No activity was detected in any of the non-albumin formulated preparations, thereby identifying HSA as the source of the unknown protease for the first time. To investigate the cause of this activity, either as an intrinsic molecular activity of albumin or due to an albumin-associated purification contaminant, further studies on a variety of commercial plasma-derived HSA products or recombinant HSA materials free from potential plasma contaminants were carried out. The measured proteolytic levels were highly consistent amongst preparations, and could all be partially inhibited by the presence of zinc and blocked by PKSI-527 and aprotinin. By contrast, the data did not support the role of plasmin, kallikrein, trypsin, α(2)-antiplasmin-plasmin complexes or HSA purification contaminants, PKA (prekallikrein activator) or kallikrein-like activity. Taken together, these findings indicate a new intrinsic proteolytic activity of the albumin molecule revealed under reducing conditions as the source of the unexpected Arg-Ala cleaving activity.
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http://dx.doi.org/10.1016/j.jpba.2010.08.013DOI Listing
January 2011

Establishment of the first World Health Organization International Genetic Reference Panel for quantitation of BCR-ABL mRNA.

Blood 2010 Nov 18;116(22):e111-7. Epub 2010 Aug 18.

National Genetics Reference Laboratory Wessex, Salisbury District Hospital, Salisbury, UK.

Serial quantitation of BCR-ABL mRNA levels is an important indicator of therapeutic response for patients with chronic myelogenous leukemia and Philadelphia chromosome-positive acute lymphoblastic leukemia, but there is substantial variation in the real-time quantitative polymerase chain reaction methodologies used by different testing laboratories. To help improve the comparability of results between centers we sought to develop accredited reference reagents that are directly linked to the BCR-ABL international scale. After assessment of candidate cell lines, a reference material panel comprising 4 different dilution levels of freeze-dried preparations of K562 cells diluted in HL60 cells was prepared. After performance evaluation, the materials were assigned fixed percent BCR-ABL/control gene values according to the International Scale. A recommendation that the 4 materials be established as the first World Health Organization International Genetic Reference Panel for quantitation of BCR-ABL translocation by real-time quantitative polymerase chain reaction was approved by the Expert Committee on Biological Standardization of the World Health Organization in November 2009. We consider that the development of these reagents is a significant milestone in the standardization of this clinically important test, but because they are a limited resource we suggest that their availability is restricted to manufacturers of secondary reference materials.
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http://dx.doi.org/10.1182/blood-2010-06-291641DOI Listing
November 2010

Collaborative study for the calibration of a replacement international standard for diphtheria toxoid adsorbed.

Biologicals 2010 Sep 19;38(5):529-38. Epub 2010 Jun 19.

Division of Bacteriology, National Institute for Biological Standards and Control, Health Protection Agency, Blanche Lane, South Mimms, Potters Bar, Hertfordshire EN6 3QG, United Kingdom.

We present the results of a collaborative study for the characterization of a preparation of diphtheria toxoid adsorbed, and its calibration in terms of the 3rd International Standard (IS) for Diphtheria Toxoid Adsorbed. Calibration was performed using established World Health Organization (WHO) and European Pharmacopoeia (Ph. Eur.) protection models. Two candidate toxoid preparations were included in the study, one of which was adopted as a replacement Ph. Eur. Biological Reference Preparation (BRP, batch 4) in February 2009. The second candidate preparation was found to have a unitage of 213 IU/ampoule based on the calibration by in vivo bioassay in 19 laboratories in 16 countries, and was established as the 4th IS for Diphtheria Toxoid Adsorbed by the WHO Expert Committee on Biological Standardization (ECBS) in October 2009. The study also assessed performance of the replacement standard in mouse and guinea pig serological assays which are used as alternative procedures for diphtheria potency testing. Participants tested both candidate preparations and potency was expressed in relative terms only. Results suggest that the replacement standard is suitable for use as the reference vaccine in serological assays and that the Vero cell assay may be suitable for calibration of future replacement standards.
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http://dx.doi.org/10.1016/j.biologicals.2010.04.001DOI Listing
September 2010

Use of dynamic mechanical analysis (DMA) to determine critical transition temperatures in frozen biomaterials intended for lyophilization.

Cryobiology 2010 Aug 27;61(1):27-32. Epub 2010 Apr 27.

Gearing Scientific Ltd., 4 Springhead, Ashwell, Hertfordshire, SG7 5LL, United Kingdom.

Dynamic mechanical analysis is widely used to determine glass transitions in solid state materials. However, here we demonstrate the application of DMA for the determination of glass transitions (Tg') in the frozen liquid state by means of a steel sample pocket. The use of the pocket allows frozen material to be analysed and glass transition events demonstrated. In addition, it allows weak glass transitions to be detected clearly in some complex formulations where they can be obscured by eutectic and other strong thermal events when other methods such as DSC or DTA are used. Classical excipients (trehalose, lactose, dextran) were analysed and shown to give reproducible Tg' values, though with values slightly higher than those obtained by DSC. Finally, several complex real biological materials, typical of those encountered when freeze drying biological and biopharmaceutical materials, were analysed and the potential value of DMA demonstrated to determine the relevant glass transition temperatures for use in cryobiology and freeze drying.
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http://dx.doi.org/10.1016/j.cryobiol.2010.04.002DOI Listing
August 2010

Rapid optimization of protein freeze-drying formulations using ultra scale-down and factorial design of experiment in microplates.

Biotechnol Bioeng 2009 Dec;104(5):957-64

Department of Biochemical Engineering, The Advanced Centre for Biochemical Engineering, University College London, Torrington Place, London WC1E 7JE, UK.

Retaining biopharmaceutical proteins in a stable form is critical to their safety and efficacy, and is a major factor for optimizing the final product. Freeze-dried formulations offer one route for improved stability. Currently the optimization of formulations for freeze-drying is an empirical process that requires many time-consuming experiments and also uses large quantities of product material. Here we describe a generic framework for the rapid identification and optimization of formulation excipients to prevent loss of protein activity during a lyophilization process. Using factorial design of experiment (DOE) methods combined with lyophilization in microplates a range of optimum formulations were rapidly identified that stabilized lactose dehydrogenase (derived from Lactobacillus leichmanii) during freeze-drying. The procedure outlined herein involves two rounds of factorially designed experiments-an initial screen to identify key excipients and potential interactions followed by a central composite face designed optimization experiment. Polyethylene glycol (PEG) and lactose were shown to have significant effects on maintaining protein stability at the screening stage and optimization resulted in an accurate model that was used to plot a window of operation. The variation of freezing temperatures and rates of sublimation that occur across a microplate during freeze-drying have been characterized also. The optimum formulation was then freeze-dried in stoppered vials to verify that the microscale data was relevant to the effects observed at larger pilot scales. This work provides a generic approach to biopharmaceutical formulation screening where possible excipients can be screened for single and interactive effects thereby increasing throughput while reducing costs in terms of time and materials.
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http://dx.doi.org/10.1002/bit.22448DOI Listing
December 2009

Lyophilization of proteins.

Authors:
Paul Matejtschuk

Methods Mol Biol 2007 ;368:59-72

Standardization Science, National Institute of Biological Standards and Control, Potters Bar, Hertfordshire, UK.

This chapter describes the methods that can be applied to successfully freeze-dry proteins. Laboratory applications are given at small scale, typified by the purification of a protein intermediate as part of the analytical characterization of a protein, and at intermediate scale, as illustrated by the pilot development of a lyophilized protein reference material such as for use in bioassay or immunoassay. Advice on common problems with freeze-drying of proteins is also given.
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http://dx.doi.org/10.1007/978-1-59745-362-2_4DOI Listing
July 2008

Impact of residual moisture and formulation on Factor VIII and Factor V recovery in lyophilized plasma reference materials.

Anal Bioanal Chem 2007 Apr 27;387(7):2503-7. Epub 2006 Oct 27.

National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Hertfordshire, EN6 3QG, UK.

Residual moisture content and formulation are important parameters when preparing lyophilized reference materials containing labile proteins. The protection of Factor VIII and Factor V activities were monitored in a lyophilized plasma preparation following formulation with either no additional excipient, 40 mM Hepes (4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid), 10 mg/mL glycine or a combination of 40 mM Hepes and 10 mg/mL glycine. The preservation of Factor VIII activity during freeze-drying was improved by the addition of either stabiliser and improved most, amongst the options studied, by the addition of both glycine and Hepes. The predicted stability at -20 degrees C and 20 degrees C was estimated using accelerated degradation studies. Although for plasma lyophilized alone there was some benefit from further desiccation over phosphorus pentoxide, resulting in very low moistures, for suitably formulated samples the predicted stability was as good for freeze-dried only samples as for those with further desiccation. This study emphasises the importance of optimum formulation on the stability of lyophilized proteins.
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http://dx.doi.org/10.1007/s00216-006-0855-xDOI Listing
April 2007

Lyophilization of biological standards.

Authors:
Paul Matejtschuk

Cryo Letters 2005 Jul-Aug;26(4):223-30

Centre for Biological Reference Materials, National Institute for Biological Standards & Control, Blanche Lane, South Mimms, Potters Bar, Hertfordshire EN6 3QG, UK.

Freeze drying plays a vital part in the preparation of many biological reference materials. The general approaches that may be adopted in the application of lyophilization to these biomolecules are discussed and the importance of analytical methodology and formulation optimization emphasised. Examples are given from a number of recently processed materials.
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November 2009

A comparison of vials with ampoules for the storage of biological reference materials.

Biologicals 2005 Jun;33(2):63-70

National Institute for Biological Standards & Control, Blanche Lane, South Mimms, Potters Bar, Hertfordshire EN6 3QG, UK.

Lyophilization is a key strategy in the stabilization of biological materials. Protection of the lyophilized material from an oxidizing atmosphere is essential if stability is to be maintained under long term storage. Vials of lyophilized albumin closed by two methods and ampoules of albumin have been examined for moisture and oxygen ingress after storage both under conditions of stress for two months and under thermally accelerated conditions for one year. The results show that the gas and moisture contents of ampoules do not detectably change even under conditions of stress, in contrast to vials for which this study shows clearly detectable moisture ingress and suggests some oxygen ingress. This is consistent with the results of other studies. Thus, although vials may be satisfactory under constrained conditions of temperature and storage for limited periods of time, and are presently used satisfactorily for some working standards, it would be prudent to continue to use ampoules for storage of international reference materials which are intended for indefinite storage and for which stability is an essential requirement.
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http://dx.doi.org/10.1016/j.biologicals.2004.12.002DOI Listing
June 2005
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