The ELISA Detectability and Potency of Pegfilgrastim Decrease in Physiological Conditions: Key Roles for Aggregation and Individual Variability.

PEGylated recombinant human granulocyte colony stimulating factor (pegfilgrastim) is used clinically to accelerate immune reconstitution following chemotherapy and is being pursued for biosimilar development. One challenge to overcome in pegfilgrastim biosimilar development is establishing pharmacokinetic (PK) similarity, which is partly due to the degree of PK variability. We herein report that commercially available G-CSF and PEG ELISA detection kits have different capacities to detect pegfilgrastim aggregates that rapidly form in vitro in physiological conditions. These aggregates can be observed using SDS-PAGE, size-exclusion chromatography, dynamic light scattering, and real-time NMR analysis and are associated with decreased bioactivity as reflected by reduced drug-induced cellular proliferation and STAT3 phosphorylation. Furthermore, individual variability in the stability and detectability of pegfilgrastim in human sera is also observed. Pegfilgrastim levels display marked subject variability in sera from healthy donors incubated at 37 °C. The stability patterns of pegfilgrastim closely match the stability patterns of filgrastim, consistent with a key role for pegfilgrastim's G-CSF moiety in driving formation of inactive aggregates. Taken together, our results indicate that individual variability and ELISA specificity for inactive aggregates are key factors to consider when designing and interpreting studies involving the measurement of serum pegfilgrastim concentrations.

A consensus rating method for small virus-retentive filters. II. Method evaluation.

Virus filters are membrane-based devices that remove large viruses (e.g., retroviruses) and/or small viruses (e.g., parvoviruses) from products by a size exclusion mechanism. In 2002, the Parenteral Drug Association (PDA) organized the PDA Virus Filter Task Force to develop a common nomenclature and a standardized test method for classifying and identifying viral-retentive filters. A test method based on bacteriophage PP7 retention was chosen based on developmental studies. The detailed final consensus filter method is published in the 2008 update of PDA Technical Report 41: Virus Filtration. Here, we evaluate the method and find it to be acceptable for testing scaled-down models of small virus-retentive filters from four manufacturers. Three consecutive lots of five filter types were tested (Pegasus SV4, Viresolve NFP, Planova 20N and 15N, Virosart CPV). Each passed the criteria specified in the test method (i.e., >4 log10 PP7 retention, >90% intravenous immunoglobulin passage, and passing integrity/installation testing) and was classified as PP7-LRV4.

A consensus rating method for small virus-retentive filters. I. Method development.

Virus filters are membrane-based devices that remove large viruses (e.g., retroviruses) and/or small viruses (e.g., parvoviruses) from products by a size exclusion mechanism. In 2002, the Parenteral Drug Association (PDA) organized the PDA Virus Filter Task Force to develop a common nomenclature and a standardized test method for classifying and identifying viral-retentive filters. One goal of the task force was to develop a test method for small virus-retentive filters. Because small virus-retentive filters present unique technical challenges, the test method development process was guided by laboratory studies to determine critical variables such as choice of bacteriophage challenge, choice of model protein, filtration operating parameters, target log10 reduction value, and filtration endpoint definition. Based on filtration, DLS, electrospray differential mobility analysis, and polymerase chain reaction studies, a final rating based on retention of bacteriophage PP7 was chosen by the PDA Virus Filter Task Force. The detailed final consensus filter method was published in the 2008 update of PDA Technical Report 41. Virus Filtration.

Chemometrics applications in biotechnology processes: predicting column integrity and impurity clearance during reuse of chromatography resin.

Separation media, in particular chromatography media, is typically one of the major contributors to the cost of goods for production of a biotechnology therapeutic. To be cost-effective, it is industry practice that media be reused over several cycles before being discarded. The traditional approach for estimating the number of cycles a particular media can be reused for involves performing laboratory scale experiments that monitor column performance and carryover. This dataset is then used to predict the number of cycles the media can be used at manufacturing scale (concurrent validation). Although, well accepted and widely practiced, there are challenges associated with extrapolating the laboratory scale data to manufacturing scale due to differences that may exist across scales. Factors that may be different include: level of impurities in the feed material, lot to lot variability in feedstock impurities, design of the column housing unit with respect to cleanability, and homogeneity of the column packing. In view of these challenges, there is a need for approaches that may be able to predict column underperformance at the manufacturing scale over the product lifecycle. In case such an underperformance is predicted, the operators can unpack and repack the chromatography column beforehand and thus avoid batch loss. Chemometrics offers one such solution. In this article, we present an application of chemometrics toward the analysis of a set of chromatography profiles with the intention of predicting the various events of column underperformance including the backpressure buildup and inefficient deoxyribonucleic acid clearance.

Evaluation of electrospray differential mobility analysis for virus particle analysis: Potential applications for biomanufacturing.

The technique of electrospray differential mobility analysis (ES-DMA) was examined as a potential potency assay for routine virus particle analysis in biomanufacturing environments (e.g., evaluation of vaccines and gene delivery products for lot release) in the context of the International Committee of Harmonisation (ICH) Q2 guidelines. ES-DMA is a rapid particle sizing method capable of characterizing certain aspects of the structure (such as capsid proteins) and obtaining complete size distributions of viruses and virus-like particles. It was shown that ES-DMA can distinguish intact virus particles from degraded particles and measure the concentration of virus particles when calibrated with nanoparticles of known concentration. The technique has a measurement uncertainty of ≈20%, is linear over nearly 3 orders of magnitude, and has a lower limit of detection of ≈10(9)particles/mL. This quantitative assay was demonstrated for non-enveloped viruses. It is expected that ES-DMA will be a useful method for applications involving production and quality control of vaccines and gene therapy vectors for human use.

Salt tolerant membrane adsorbers for robust impurity clearance.

Clearance of impurities such as viruses, host cell protein (HCP), and DNA is a critical purification design consideration for manufacture of monoclonal antibody therapeutics. Anion exchange chromatography has frequently been utilized to accomplish this goal; however, anion exchange adsorbents based on the traditional quaternary amine (Q) ligand are sensitive to salt concentration, leading to reduced clearance levels of impurities at moderate salt concentrations (50-150 mM). In this report, membrane adsorbers incorporating four alternative salt tolerant anion exchange ligands were examined for impurity clearance: agmatine, tris-2-aminoethyl amine, polyhexamethylene biguanide (PHMB), and polyethyleneimine. Each of these ligands provided greater than 5 log reduction value (LRV) for viral clearance of phage phiX174 (pI approximately 6.7) at pH 7.5 and phage PR772 (pI approximately 4) at pH 4.2 in the presence of salt. Under these same conditions, the commercial Q membrane adsorber provided no clearance (zero LRV). Clearance of host-cell protein at pH 7.5 was the most challenging test case; only PHMB maintained 1.5 LRV in 150 mM salt. The salt tolerance of PHMB was attributed to its large positive net charge through the presence of multiple biguanide groups that participated in electrostatic and hydrogen bonding interactions with the impurity molecules. On the basis of the results of this study, membrane adsorbers that incorporate salt tolerant anion exchange ligands provide a robust approach to impurity clearance during the purification of monoclonal antibodies.

Characterization of coliphage PR772 and evaluation of its use for virus filter performance testing.

Virus filtration is a key clearance unit operation in the manufacture of recombinant protein, monoclonal antibody, and plasma-derived biopharmaceuticals. Recently, a consensus has developed among filter manufacturers and end users about the desirability of a common nomenclature and a standardized test for classifying and identifying virus-retentive filters. The Parenteral Drug Association virus filter task force has chosen PR772 as the model bacteriophage to standardize nomenclature for large-pore-size virus-retentive filters (filters designed to retain viruses larger than 50 to 60 nm in size). Previously, the coliphage PR772 (Tectiviridae family) has been used in some filtration studies as a surrogate for mammalian viruses of around 50 to 60 nm. In this report, we describe specific properties of PR772 critical to the support of its use for the standardization of virus filters. The complete genomic sequence of virulent phage PR772 was determined. Its genome contains 14,946 bp with an overall G+C content of 48.3 mol%, and 32 open reading frames of at least 40 codons. Comparison of the PR772 nucleotide sequence with the genome of Tectiviridae family prototype phage PRD1 revealed 97.2% identity at the DNA level. By dynamic light-scattering analysis, its hydrodynamic diameter was measured as 82 +/- 6 nm, consistent with use in testing large-virus-retentive filters. Finally, dynamic light-scattering analysis of PR772 preparations purified on CsCl gradients showed that the phage preparations are largely monodispersed. In summary, PR772 appears to be an appropriate model bacteriophage for standardization of nomenclature for larger-pore-size virus-retentive filters.