Case study and application of process analytical technology (PAT) towards bioprocessing: Use of tryptophan fluorescence as at‐line tool for making pooling decisions for process chromatography

Process analytical technology (PAT) has been gaining momentum in the biopharmaceutical community due to the potential for continuous real time quality assurance resulting in improved operational control and compliance. Two imperatives for implementing any PAT tool are that “variability is managed by the process” and “product quality attributes can be accurately and reliably predicted over the design space established for materials used, process parameters, manufacturing, environmental, and other conditions.” Recently, we have been examining the feasibility of applying different analytical tools to bioprocessing unit operations. We have previously demonstarted that commercially available online‐high performance liquid chromatography and ultra performance liquid chromatography systems can be used for analysis that can facilitate real‐time decisions for column pooling based on product quality attributes (Rathore et al., 2008 a,b). In this article, we review an at‐line tool that can be used for pooling of process chromatography columns. We have demonstrated that our tryptophan fluorescence method offers a feasible approach and meets the requirements of a PAT application. It is significantly faster than the alternative of fractionation, offline analysis followed by pooling. Although the method as presented here is not an online method, this technique may offer better resolution for certain applications and may be a more optimal approach as it is very conducive to implementation in a manufacturing environment. This technique is also amenable to be used as an online tool via front face fluorescence measurements done concurrently with product concentration determination by UV. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Isoform-Selective Interaction of the Adaptor Protein Tks5/FISH with Sos1 and Dynamins

The adaptor protein Tks5/FISH (tyrosine kinase substrate 5/five SH3 domains, hereafter termed Tks5) is a crucial component of a protein network that controls the invasiveness of cancer cells and progression of Alzheimer's disease. Tks5 consists of an amino-terminal PX domain that is followed by five SH3 domains (SH3A-E), and two different splice variants are expressed. We identified son of sevenless-1 (Sos1) as a novel binding partner of Tks5 and found colocalization of Tks5 with Sos1 in human epithelial lung carcinoma (A549) cells and in podosomes of Src-transformed NIH 3T3 cells. We observe synergistic binding of SH3A and SH3B to Sos1 when peptide arrays are used, indicating that the tandem SH3A and SH3B domains of Tks5 can potentially bind in a superSH3 binding mode, as was described for the homologous protein p47phox. These results are further corroborated by pull-down assays and isothermal titration calorimetry showing that both intact SH3 domains are required for efficient binding to the entire proline-rich domain of Sos1. The presence of a basic insertion between the SH3A and SH3B domains in the long splice variant of Tks5 decreases the affinity to Sos1 isoforms about 10-fold as determined by analytical ultracentrifugation. Furthermore, it leads to an alteration in the recognition of binding motifs for the interaction with Sos1: While the insertion abrogates the interaction with the majority of peptides derived from the proline-rich domains of Sos1 and dynamin that are recognized by the short splice isoform, it enables binding to a different set of peptides including a sequence comprising the splice insertion in the long isoform of Sos1 (Sos1_2). In the absence of the basic insertion, Tks5 was found to bind a range of Sos1 and dynamin peptides including conventional proline-rich motifs and atypical recognition sequences. Hereby, the tandem SH3 domains in Tks5 employ two distinct types of binding modes: One class of peptides is recognized by single SH3 domains, whereas a second class of peptides requires the presence of both domains to bind synergistically. We conclude that the tandem SH3A and SH3B domains of Tks5 constitute a versatile module for the implementation of isoform-specific protein-protein interactions.     

The recent history and population structure of five Mandarina snail species from subtropical Ogasawara (Bonin Islands, Japan)

The effect of Pleistocene climate change on the organisms of tropical and subtropical regions is rather poorly understood. We therefore studied the land snail genus Mandarina (Bradybaenidae) of oceanic Ogasawara (Bonin Islands, Japan), with the aim of using population genetic data to understand their recent history. Our analysis of a mitochondrial 16S ribosomal RNA region from more than 600 snails in five ground-living species suggests that populations on the small islands of Mukoujima, Anejima, Imotojima and Meijima, as well as on the low-lying southern and central parts of Hahajima, have probably undergone recent bottlenecks followed by subsequent expansions. Except between the main island of Hahajima and Mukouijima, there is almost no evidence for gene flow among islands even though the islands were connected repeatedly by land bridges through the Pleistocene. Within islands the population structure is severe, suggestive of a long-term, low level of gene flow (F(ST) is frequently greater than 0.5 among geographically close populations). Finally, there is a marked genetic patchiness, meaning that genetically close populations are sometimes separated by genetically distant populations. These patterns could be a consequence of expansion from bottlenecks, low active dispersal and founder effects caused by rare long-distance migrants. Unfortunately, the exact nature of the refugia and bottlenecks remains unknown because the palaeoclimate of this region is poorly understood. Dating the population size changes is also challenging because the molecular clock is uncertain. We suggest, however, that arid conditions or deforestation induced by decreased atmospheric CO(2) may have been the main factor in determining population size.     

Short-term impact of disturbance on genetic diversity and structure of Indonesian populations of the butterfly Drupadia theda in East Kalimantan

We investigated the short‐term impact of disturbance on genetic diversity and structure of the tropical butterfly Drupadia theda Felder (Lepidoptera: Lycaenidae). Populations were sampled from five landscapes in East Kalimantan (Borneo, Indonesia) which were differentially disturbed by selective logging and the 1997/1998 El Niño Southern Oscillation (ENSO)‐induced drought and fires. Sampling occurred before (in 1997) and after the forest fires (in 1998, 1999, 2000, and 2004). Drupadia theda populations underwent serious population size reductions following the 1997/1998 ENSO event. For a total of 208 individuals, we sequenced a 509‐bp segment of mtDNA containing the control region plus the 5’ end of the 12S rDNA gene. Haplotype diversity in D. theda populations ranged from 0.468 to 0.953. Just after the 1997/1998 ENSO event, number of recorded individuals and genetic diversity were very low in D. theda populations sampled in the two severely burned areas and in a small pristine forest fragment that was surrounded by burned forest and thereby affected by drought. Interestingly, higher levels of genetic diversity were observed in logged forest compared to proximate pristine forest. After 1998, the genetic composition within the three ENSO‐disturbed areas diverged. In the twice‐burned forest, the genetic diversity in 1999 already approached pre‐fire levels, while it remained nearly unchanged in proximate once‐burned forest. Our data suggest that the 1997/1998 ENSO‐induced drought and fires caused massive reductions in the genetic diversity of D. theda and that population recoveries were linked to their geographical position relative to patches of unburned forest (and thus to source populations).     

Integrin alphaIIbbeta3:ligand interactions are linked to binding-site remodeling

This study tested the hypothesis that high-affinity binding of macromolecular ligands to the alphaIIbbeta3 integrin is tightly coupled to binding-site remodeling, an induced-fit process that shifts a conformational equilibrium from a resting toward an open receptor. Interactions between alphaIIbbeta3 and two model ligands-echistatin, a 6-kDa recombinant protein with an RGD integrin-targeting sequence, and fibrinogen's gamma-module, a 30-kDa recombinant protein with a KQAGDV integrin binding site-were measured by sedimentation velocity, fluorescence anisotropy, and a solid-phase binding assay, and modeled by molecular graphics. Studying echistatin variants (R24A, R24K, D26A, D26E, D27W, D27F), we found that electrostatic contacts with charged residues at the alphaIIb/beta3 interface, rather than nonpolar contacts, perturb the conformation of the resting integrin. Aspartate 26, which interacts with the nearby MIDAS cation, was essential for binding, as D26A and D26E were inactive. In contrast, R24K was fully and R24A partly active, indicating that the positively charged arginine 24 contributes to, but is not required for, integrin recognition. Moreover, we demonstrated that priming--i.e., ectodomain conformational changes and oligomerization induced by incubation at 35 degrees C with the ligand-mimetic peptide cHarGD--promotes complex formation with fibrinogen's gamma-module. We also observed that the gamma-module's flexible carboxy terminus was not required for alphaIIbbeta3 integrin binding. Our studies differentiate priming ligands, which bind to the resting receptor and perturb its conformation, from regulated ligands, where binding-site remodeling must first occur. Echistatin's binding energy is sufficient to rearrange the subunit interface, but regulated ligands like fibrinogen must rely on priming to overcome conformational barriers.     

Biosimilars: Key regulatory considerations and similarity assessment tools

A biosimilar drug is defined in the US Food and Drug Administration (FDA) guidance document as a biopharmaceutical that is highly similar to an already licensed biologic product (referred to as the reference product) notwithstanding minor differences in clinically inactive components and for which there are no clinically meaningful differences in purity, potency, and safety between the two products. The development of biosimilars is a challenging, multistep process. Typically, the assessment of similarity involves comprehensive structural and functional characterization throughout the development of the biosimilar in an iterative manner and, if required by the local regulatory authority, an in vivo nonclinical evaluation, all conducted with direct comparison to the reference product. In addition, comparative clinical pharmacology studies are conducted with the reference product. The approval of biosimilars is highly regulated although varied across the globe in terms of nomenclature and the precise criteria for demonstrating similarity. Despite varied regulatory requirements, differences between the proposed biosimilar and the reference product must be supported by strong scientific evidence that these differences are not clinically meaningful. This review discusses the challenges faced by pharmaceutical companies in the development of biosimilars.     

Introgression in peripheral populations and colonization shape the genetic structure of the coastal shrub Armeria pungens

The coastal shrub Armeria pungens has a disjunct Atlantic-Mediterranean distribution. The historic range expansion underlying this distribution was investigated using the nuclear internal transcribed spacer region, three plastid regions (namely trnL-F, trnS-fM and matK) and morphometric data. A highly diverse ancestral lineage was identified in southwest Portugal. More recently, two areas have been colonized: (1) Corsica and Sardinia, where disjunct Mediterranean populations have been established as a result of the long-distance dispersal of Portuguese genotypes, and (2) the southern part of the Atlantic range, Gulf of Cadiz, where a distinct lineage showing no genetic differentiation among populations occurs. Genetic consequences of colonization seem to have been more severe in the Gulf of Cadiz than in Corsica-Sardinia. Although significant genetic divergence is associated with low plastid diversity in the Gulf of Cadiz, in Corsica-Sardinia, the loss of plastid haplotypes was not accompanied by divergence from disjunct Portuguese source populations. In addition, in its northernmost and southernmost populations, A. pungens exhibited evidence for ancient or ongoing introgression from sympatric congeners. Introgression might have created novel genotypes able to expand beyond the latitudinal margins of the species or, alternatively, these genotypes may be the result of surfing of alleles from other species in demographic equilibrium into peripheral populations of A. pungens. Our results highlight the evolutionary significance of genetic drift following the colonization of new areas and the key role of introgression in range expansion.     

Strong gene flow and lack of stable population structure in the face of rapid adaptation to local temperature in a spring-spawning salmonid, the European grayling (Thymallus thymallus)

Gene flow has the potential to both constrain and facilitate adaptation to local environmental conditions. The early stages of population divergence can be unstable because of fluctuating levels of gene flow. Investigating temporal variation in gene flow during the initial stages of population divergence can therefore provide insights to the role of gene flow in adaptive evolution. Since the recent colonization of Lake Lesjaskogsvatnet in Norway by European grayling (Thymallus thymallus), local populations have been established in over 20 tributaries. Multiple founder events appear to have resulted in reduced neutral variation. Nevertheless, there is evidence for local adaptation in early life-history traits to different temperature regimes. In this study, microsatellite data from almost a decade of sampling were assessed to infer population structuring and its temporal stability. Several alternative analyses indicated that spatial variation explained 2-3 times more of the divergence in the system than temporal variation. Over all samples and years, there was a significant correlation between genetic and geographic distance. However, decomposed pairwise regression analysis revealed differing patterns of genetic structure among local populations and indicated that migration outweighs genetic drift in the majority of populations. In addition, isolation by distance was observable in only three of the six years, and signals of population bottlenecks were observed in the majority of samples. Combined, the results suggest that habitat-specific adaptation in this system has preceded the development of consistent population substructuring in the face of high levels of gene flow from divergent environments.     

Zinc binding to the Tyr402 and His402 allotypes of complement factor H: possible implications for age-related macular degeneration

The Tyr402His polymorphism of complement factor H (FH) with 20 short complement regulator (SCR) domains is associated with age-related macular degeneration (AMD). How FH contributes to disease pathology is not clear. Both FH and high concentrations of zinc are found in drusen deposits, the key feature of AMD. Heterozygous FH is inhibited by zinc, which causes FH to aggregate. Here, zinc binding to homozygous FH was studied. By analytical ultracentrifugation, large amounts of oligomers were observed with both the native Tyr402 and the AMD-risk His402 homozygous allotypes of FH and both the recombinant SCR-6/8 allotypes with Tyr/His402. X-ray scattering also showed that both FH and SCR-6/8 allotypes strongly aggregated at > 10 μM zinc. The SCR-1/5 and SCR-16/20 fragments were less likely to bind zinc. These observations were supported by bioinformatics predictions. Starting from known zinc binding sites in crystal structures, we predicted 202 putative partial surface zinc binding sites in FH, most of which were in SCR-6. Metal site prediction web servers also suggested that SCR-6 and other domains bind zinc. Predicted SCR-6/8 dimer structures showed that zinc binding sites could be formed at the protein-protein interface that would lead to daisy-chained oligomers. It was concluded that zinc binds weakly to FH at multiple surface locations, most probably within the functionally important SCR-6/8 domains, and this explains why zinc inhibits FH activity. Given the high pathophysiological levels of bioavailable zinc present in subretinal deposits, we discuss how zinc binding to FH may contribute to deposit formation and inflammation associated with AMD.     

Origin and taxonomic status of the Palearctic population of the stem borer Sesamia nonagrioides (Lefèbvre) (Lepidoptera: Noctuidae)

The major pest of maize in Mediterranean Europe, the stem borer Sesamia nonagrioides (Lefèbvre) (Lepidoptera: Noctuidae), has a fragmented distribution, north and south of the Sahara. The present study aimed: (1) to clarify the uncertain taxonomic status of the Palearctic and sub‐Saharan populations which were first considered as different species and later on as subspecies (Sesamia nonagrioides nonagrioides and Sesamia nonagrioides botanephaga) and (2) to investigate the origin of the Palearctic population which extends from Spain to Iran, outside what is considered typical for this mainly tropical genus. We reconstructed the evolutionary history of both populations using one nuclear and two mitochondrial genes. The sub‐Saharan taxon was fragmented in two isolated populations (West and East) whose mitochondrial genes were distant by 2.3%. The Palearctic population was included in the East African clade and its genes were close or identical to those of a population from Central Ethiopia, where the species was discovered for the first time. Similarly, in Africa, the alleles of the nuclear gene were distributed mainly in two West and East clades, whereas some Palearctic alleles belonged to the West clade. The Palearctic population originated therefore from East and West Africa and is the progeny of the cross between these two African populations. The main species concepts were in agreement, leading to the conclusion that the three populations are still conspecific. In the surveyed regions, the species therefore does not include two subspecies but three isolated populations. The Palearctic population suffered from severe bottlenecks that resulted in the fixation of one East African mitochondrial genome and the large reduction in its genetic diversity compared to the African populations. The data suggest that natural colonization of the Palearctic region was more plausible than human introduction. The allelic distribution of the Palearctic population was similar to that of species that survived the last glaciation. It is concluded that the African populations expanded during the last interglacial, crossed the Sahara and mixed in North Africa where fixation of the East mitochondrial genome occurred. The species then colonized Europe westward through only one eastern entrance. The coalescent‐based estimate of the time to the ancestor of the Palearctic population was 108 000 years, which is consistent with this scenario. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 103 , 904–922.