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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. [ABSTRACT FROM AUTHOR]

Viral clearance is a critical aspect of biopharmaceutical manufacturing process validation. To determine the viral clearance efficacy of downstream chromatography and filtration steps, live viral 'spiking' studies are conducted with model mammalian viruses such as minute virus of mice (MVM). However, due to biosafety considerations, spiking studies are costly and typically conducted in specialized facilities. In this work, we introduce the concept of utilizing a non-infectious MVM virus-like particle (MVM-VLP) as an economical surrogate for live MVM during process development and characterization. Through transmission electron microscopy, size exclusion chromatography with multi-angle light scattering, chromatofocusing, and a novel solute surface hydrophobicity assay, we examined and compared the size, surface charge, and hydrophobic properties of MVM and MVM-VLP. The results revealed that MVM and MVM-VLP exhibited nearly identical physicochemical properties, indicating the potential utility of MVM-VLP as an accurate and economical surrogate to live MVM during chromatography and filtration process development and characterization studies. [ABSTRACT FROM AUTHOR]

Compared to small molecule process analytical technology (PAT) applications, biotechnology product PAT applications have certain unique challenges and opportunities. Understanding process dynamics of bioreactor cell culture process is essential to establish an appropriate process control strategy for biotechnology product PAT applications. Inline spectroscopic techniques for real time monitoring of bioreactor cell culture process have the distinct potential to develop PAT approaches in manufacturing biotechnology drug products. However, the use of inline Fourier transform infrared (FTIR) spectroscopic techniques for bioreactor cell culture process monitoring has not been reported. In this work, real time inline FTIR Spectroscopy was applied to a lab scale bioreactor mAb IgG3 cell culture fluid biomolecular dynamic model. The technical feasibility of using FTIR Spectroscopy for real time tracking and monitoring four key cell culture metabolites (including glucose, glutamine, lactate, and ammonia) and protein yield at increasing levels of complexity (simple binary system, fully formulated media, actual bioreactor cell culture process) was evaluated via a stepwise approach. The FTIR fingerprints of the key metabolites were identified. The multivariate partial least squares (PLS) calibration models were established to correlate the process FTIR spectra with the concentrations of key metabolites and protein yield of in-process samples, either individually for each metabolite and protein or globally for all four metabolites simultaneously. Applying the 2nd derivative pre-processing algorithm to the FTIR spectra helps to reduce the number of PLS latent variables needed significantly and thus simplify the interpretation of the PLS models. The validated PLS models show promise in predicting the concentration profiles of glucose, glutamine, lactate, and ammonia and protein yield over the course of the bioreactor cell culture process. Therefore, this work demonstrated the technical feasibility of real time monitoring of the bioreactor cell culture process via FTIR spectroscopy. Its implications for enabling cell culture PAT were discussed. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

The size, heterogeneity, and biological production process of protein therapeutics like monoclonal antibodies create unique challenges for their analysis and regulation compared with small molecules. Complete structural characterization of a molecule 1000-fold heavier than aspirin is no small feat. Biological post-translational modifications such as glycosylation further complicate their characterization and regulation. Even approved protein therapeutics are known to contain multiple structural variants in differing amounts. Structural modification occurs during production and storage as well as within patients after administration. Thus, the goals of manufacturers and regulators are to control and characterize this heterogeneity, not take on the impossible task of eliminating it. The aim of this review is to describe the structural heterogeneities known to occur with immunoglobulin G (IgG), note current detection and analytical strategies, establish their causes, and define their potential effects on the ultimate safety, purity, and potency of antibody therapeutics when known. [ABSTRACT FROM AUTHOR]

The article focuses on biomanufacturing and highlights the areas where innovations can be incorporated. It highlights that further innovation can improve product quality and upstream processes productivity. Innovations can also have profound effect on the production in downstream processing and reports that certain unit processes are operating close to their physical limits.

The Tla region of the BALB/c mouse major histocompatibility complex contains at least 20 class I genes. The function of the products of these genes is unknown, but recent evidence demonstrates that some Tla region gene products could be involved in presentation of antigens to γδ T cells. We have generated a set of polymerase chain reaction (PCR) oligonucleotide primers and hybridization probes that permit us to specifically amplify and detect expression of 11 of the 20 BALB/c Tla region genes. cDNA prepared from 12 adult and fetal tissues and from seven cell lines was analyzed. In some cases, northern blot analysis or staining with monoclonal antibodies specific for the Tla-encoded thymus leukemia (TL) antigen were used to confirm the expression pattern of several of the genes as determined by PCR. Some Tla region genes, such as T24 and the members of the T10/ T22 gene pair, are expressed in a wide variety of tissues in a manner similar to the class I transplantation antigens. The members of the TL antigen encoding gene pair, T3/ T18, are expressed in only a limited number of organs, including several sites enriched for γδ T cells. Other Tla region genes, including T1, T2, T16, and T17, are transcriptionally silent from the T8/ T20 gene pair do not undergo proper splicing. In general, sites that contain γδ T lymphocytes have Tla region transcripts. The newly identified pattern of expression of the genes analyzed in sites containing γδ T cells further extends the list of potential candidates for antigen presentation to γδ T cells. [ABSTRACT FROM AUTHOR]

The major histocompatibility complex (MHC) of the BALB/c mouse contains three genes encoding classical class I molecules, as well as at least 32 nonclassical class I genes. Although much is known about the genes encoding the classical class I molecules, the majority of the nonclassical genes have not been characterized. This report describes a newly identified nonclassical class I gene, Thy19.4, which contains an open reading frame and resembles in several regards the genes encoding classical class I molecules. The similarities include shared amino acid sequence motifs which suggest that the putative Thy19.4 molecule may assume a tertiary structure similar to that of the classical class I molecules, as well as widespread transcription in a variety of tissues. However, unlike the classical class I genes, the Thy19.4 gene maps approximately 1 cm distal to the Tla region of the MHC, in the same region as the gene encoding the Hmt element of the maternally transmitted antigen. [ABSTRACT FROM AUTHOR]

The mouse major histocompatibility complex is composed of several genes arranged into the K, D, Qa, and Tla regions. The D region of the BALB/c mouse includes genes D2, D3, and D4, in addition to H-2Dand H-2L. We have determined the DNA sequence of the D2gene and compared it with the known sequences of several class I genes. The exon/intron structure of the D2gene is similar to other class I genes. It also contains similar 5′ regulatory elements. A frameshift occurs in exon seven, resulting in a gene product with a truncated cytoplasmic tail. To examine the surface expression of the D2 molecule, we generated an exon-shuffled construct containing the promoter and exons 1-3, encoding the signal peptide, α1, and α2 external domains of the D2gene linked to exons 4-8, encoding the α3, transmembrane and cytoplasmic domains, of the H-2Dgene. The construct was transfected into mouse L cells, and a protein was detected at the cell surface by a monoclonal antibody (mAb) specific for the α3 domain of H-2D, as well as by other class I-specific mAbs. Although D2 is expressed at low levels, it may be a functional class I gene that most probably evolved from a Qa region gene. [ABSTRACT FROM AUTHOR]