Development Of Holistic Processing Research Articles

Development of a 10 g/L process for a difficult-to-express multispecific antibody format using a holistic process development approach

Ichnos has developed a multi-specific antibody platform based on the BEAT® (Bispecific engagement by antibodies based on the T-cell receptor) interface. The increased complexity of the bi- and multi-specific formats generated with this platform makes these molecules difficult-to-express proteins compared to standard monoclonal antibodies (mAbs). This report describes how expression limitations of a bi-specific bi-paratopic BEAT antibody were improved in a holistic approach. An initial investigation allowed identification of a misbalance in the subunits composing the BEAT antibody as the potential root cause. This misbalance was then addressed by a signal peptide optimization, and the overall expression level was increased by the combination of two vector design elements on a single gene vector. Further improvements were made in the selection of cell populations and an upstream (USP) platform process was applied in combination with a cell culture temperature shift. This allowed titer levels of up to 6 g/L to be reached with these difficult-to-express proteins. Furthermore, a high-density seeding process was developed that allowed titers of around 11 g/L for the BEAT antibody, increasing the initial titer by a factor of 10. The approach was successfully applied to a tri-specific antibody with titer levels reaching 10 g/L. In summary, a platform process for difficult-to-express proteins was developed using molecular biology tools, cell line development, upstream process optimization and process intensification.

Social experience drives the development of holistic face processing in paper wasps.

Most recognition is based on identifying features, but specialization for face recognition in some taxa relies on a different mechanism, termed 'holistic processing' where facial features are bound together into a gestalt which is more than the sum of its parts. Although previous work suggests that extensive experience may be required for the development of holistic processing, we lack experiments that test how age and experience interact to influence holistic processing. Here, we test how age and experience influence the development of holistic face processing in Polistes fuscatus paper wasps. Previous work has shown that P. fuscatus use facial patterns to individually identify conspecifics and wasps use holistic processing to discriminate between conspecific faces. We tested face processing in three groups of P. fuscatus: young (1-week-old), older, experienced (2-weeks-old, normal experience), and older, inexperienced (2-weeks-old, 1 week normal social experience and 1 week social isolation). Older, experienced wasps used holistic processing to discriminate between conspecific faces. In contrast, older inexperienced wasps used featural rather than holistic mechanisms to discriminate between faces. Young wasps show some evidence of holistic face processing, but this ability was less refined than older, experienced wasps. Notably, wasps only required 2 weeks of normal experience to develop holistic processing, while previous work suggests that humans may require years of experience. Overall, P. fuscatus wasps rapidly develop holistic processing for conspecific faces. Experience rather than age facilitates the transition between featural and holistic face processing mechanisms.

Design and optimization of membrane chromatography for monoclonal antibody charge variant separation.

The manufacturing scale implementation of membrane chromatography to purify monoclonal antibodies has gradually increased with the shift in industry focus toward flexible manufacturing and disposable technologies. Membrane chromatography are used to remove process-related impurities such as host cell proteins (HCPs) and DNA, leachates, and endotoxins, with improved productivity and process flexibility. However, application of membrane chromatography to separate product-related variants such as charge variants has not gained major traction due to low-binding capacity. The work reported here demonstrates that a holistic process development strategy to optimize static binding (pH and salt concentration) and dynamic process (membrane loading, flowrate, and gradient length) parameters can alleviate the capacity limitations. The study employed high throughput screening tools and scale-down membranes for intermediate and polishing purification of the model monoclonal antibody. An optimized process consisting of anion exchange and cation exchange membrane chromatography reduced the acidic variants present in Protein A eluate from 89.5% to 19.2% with 71% recovery of the target protein. The membrane chromatography process also cleared HCP to below limit of detection with 6- to 30-fold higher membrane loading, compared to earlier reported values. The results confirm that membrane chromatography is effective in separating closely related product variants when supported by a well-defined process development strategy.

Towards Autonomous Operation by Advanced Process Control—Process Analytical Technology for Continuous Biologics Antibody Manufacturing

Continuous manufacturing opens up new operation windows with improved product quality in contrast to documented lot deviations in batch or fed-batch operations. A more sophisticated process control strategy is needed to adjust operation parameters and keep product quality constant during long-term operations. In the present study, the applicability of a combination of spectroscopic methods was evaluated to enable Advanced Process Control (APC) in continuous manufacturing by Process Analytical Technology (PAT). In upstream processing (USP) and aqueous two-phase extraction (ATPE), Raman-, Fourier-transformed infrared (FTIR), fluorescence- and ultraviolet/visible- (UV/Vis) spectroscopy have been successfully applied for titer and purity prediction. Raman spectroscopy was the most versatile and robust method in USP, ATPE, and precipitation and is therefore recommended as primary PAT. In later process stages, the combination of UV/Vis and fluorescence spectroscopy was able to overcome difficulties in titer and purity prediction induced by overlapping side component spectra. Based on the developed spectroscopic predictions, dynamic control of unit operations was demonstrated in sophisticated simulation studies. A PAT development workflow for holistic process development was proposed.

Holistic process development to mitigate proteolysis of a subunit rotavirus vaccine candidate produced in Pichia pastoris by means of an acid pH pulse during fed-batch fermentation.

To meet the challenges of global health, vaccine design and development must be reconsidered to achieve cost of goods as low as 15¢ per dose. A new recombinant protein‐based rotavirus vaccine candidate derived from non‐replicative viral subunits fused to a P2 tetanus toxoid CD4(+) T cell epitope is currently under clinical development. We have sought to simplify the existing manufacturing process to meet these aims. To this end, we have taken a holistic process development approach to reduce process complexity and costs while producing a product with the required characteristics. We have changed expression system from Escherichia coli to Pichia pastoris, to produce a secreted product, thereby reducing the number of purification steps. However, the presence of proteases poses challenges to product quality. To understand the effect of fermentation parameters on product quality small‐scale fermentations were carried out. Media pH and fermentation duration had the greatest impact on the proportion of full‐length product. A novel acidic pH pulse strategy was used to minimize proteolysis, and this combined with an early harvest time significantly increased the proportion of full‐length material (60–75%). An improved downstream process using a combination of CIEX and AIEX to further reduce proteases, resulted in maintaining product quality (95% yield).

Downstream Process Synthesis for Microbial Steroids.

Systematic and holistic process development, considering upstream and downstream processing simultaneously, is crucial for designing effective and economical production processes. Based on a multiphasic fermentation process, where oil is added to improve substrate solubility, a systematic approach for the purification of microbial steroids is presented. The design methodology incorporates expert knowledge in the form of heuristics to generate different downstream processing alternatives for processing the complex, multiphasic fermentation broth, and recovering the target steroid precursor androst-4-ene-3,17-dione (androstenedione; AD). The resulting alternative tree of different purification techniques is evaluated using scouting experiments to check the performance of each technique. Purification steps such as extraction, adsorption-desorption, and precipitation seem most promising and have been investigated in detail. Each process step selected is optimized and connected to a process route for recovering AD.

From development to aging: Holistic face perception in children, younger and older adults

Few published reports examine the development of holistic face processing across the lifespan such that face-specific processes are adequately differentiated from general developmental effects. To address this gap in the literature, we used the complete design of the composite paradigm (Richler & Gauthier, 2014) with faces and non-face control objects (watches) to investigate holistic processing in children (8–10years), young adults (20–32years) and older adults (65–78years). Several modifications to past research designs were introduced to improve the ability to draw conclusions about the development of holistic processing in terms of face-specificity, response bias, and age-related differences in attention. Attentional focus (narrow vs. wide focus at study) influenced the magnitude of the composite effect without eliminating holistic face processing in all age groups. Young adults showed large composite effects for faces, but none for watches. In contrast, older adults and children showed composite effects for both faces and watches, although the effects for faces were larger. Our findings suggest that holistic processing, as measured by the composite effect, might be moderated by less efficient attentional control in children and older adults. The study also underscores the importance of including comparable complex objects when investigating face processing across the lifespan.

Holistic processing as a hallmark of perceptual expertise for nonface categories including Chinese characters

Holistic processing has been associated with perceptual expertise in different domains involving faces, cars, fingerprints, musical notes, English words, etc. Curiously Chinese characters are regarded as an exception, as indicated by reduced holistic processing found for experts with the Chinese writing system as compared with novices. We revisit the issue and examine one type of holistic processing, the obligatory attention to all parts of an object, using the composite paradigm from face perception literature. Chinese readers (experts) and non-Chinese readers (novices) matched the target halves of two characters while ignoring the irrelevant halves. We introduced differential response deadlines for experts and novices in order to match their performance level and to avoid ceiling performance for experts. Both experts and novices showed holistic processing, irrespective of the character structure (left-right or top-bottom) or presentation sequence (sequential or simultaneous matching). Experts' holistic processing also showed some sensitivity to the amount of experience with the characters, as it was larger for characters than noncharacters in some situations. Novices, however, did not show a systematic difference, suggesting that their effects were more related to their inefficient decomposition of a novel, complex pattern into parts. The current results, together with other recent findings of holistic processing for English words and musical notes, indicate that the development of holistic processing is not restricted to faces and objects. Instead it may be a general marker of expertise across a wider domain of visual discrimination than previously thought, including alphabetic and nonalphabetic writing systems.

Individuation experience predicts other-race effects in holistic processing for both Caucasian and Black participants

Same-race (SR) faces are recognized better than other-race (OR) faces, and this other-race effect (ORE) is correlated with experience. SR faces are also processed more holistically than OR faces, suggesting one possible mechanism for poorer performance on OR faces. Studies of object expertise have shown that individuating experiences are necessary for holistic processing to develop; yet thus far no studies have investigated the role of quality of experience and the ORE for holistic processing. In the present study, we found a strong negative correlation between a self-report of individuating experience and the ORE in holistic processing in both Caucasian and Black participants, indicating that the more individuating experience a person has, the less ORE in holistic processing. This confirms the critical role of individuating experience in development of holistic processing for faces and suggests that quality of experience is a key determinant of the manner in which OR faces are processed.

Holistic Processing of Words Modulated by Reading Experience

Perceptual expertise has been studied intensively with faces and object categories involving detailed individuation. A common finding is that experience in fulfilling the task demand of fine, subordinate-level discrimination between highly similar instances is associated with the development of holistic processing. This study examines whether holistic processing is also engaged by expert word recognition, which is thought to involve coarser, basic-level processing that is more part-based. We adopted a paradigm widely used for faces – the composite task, and found clear evidence of holistic processing for English words. A second experiment further showed that holistic processing for words was sensitive to the amount of experience with the language concerned (native vs. second-language readers) and with the specific stimuli (words vs. pseudowords). The adoption of a paradigm from the face perception literature to the study of expert word perception is important for further comparison between perceptual expertise with words and face-like expertise.

Early maturity of face recognition: No childhood development of holistic processing, novel face encoding, or face-space

Historically, it was believed the perceptual mechanisms involved in individuating faces developed only very slowly over the course of childhood, and that adult levels of expertise were not reached until well into adolescence. Over the last 10 years, there has been some erosion of this view by demonstrations that all adult-like behavioural properties are qualitatively present in young children and infants. Determining the age of maturity, however, requires quantitative comparison across age groups, a task made difficult by the need to disentangle development in face perception from development in all the other cognitive factors that affect task performance. Here, we argue that full quantitative maturity is reached early, by 5–7 years at the latest and possibly earlier. This is based on a comprehensive literature review of results in the 5-years-to-adult age range, with particular focus on the results of the few previous studies that are methodologically suitable for quantitative comparison of face effects across age, plus three new experiments testing development of holistic/configural processing (faces versus objects, disproportionate inversion effect), ability to encode novel faces (assessed via implicit memory) and face-space (own-age bias).

A cross-syndrome study of the development of holistic face recognition in children with autism, Down syndrome, and Williams syndrome

We report a cross-syndrome comparison of the development of holistic processing in face recognition in school-aged children with developmental disorders: autism, Down syndrome, and Williams syndrome. The autism group was split into two groups: one with high-functioning children and one with low-functioning children. The latter group has rarely been studied in this context. The four disorder groups were compared with typically developing children. Cross-sectional trajectory analyses were used to compare development in a modified version of Tanaka and Farah’s part–whole task. Trajectories were constructed linking part–whole performance either to chronological age or to several measures of mental age (receptive vocabulary, visuospatial construction, and the Benton Facial Recognition Test). In addition to variable delays in onset and rate of development, we found an atypical profile in all disorder groups. These profiles were atypical in different ways, indicating multiple pathways to, and variable outcomes in, the development of face recognition. We discuss the implications for theories of face recognition in both atypical and typical development, including the idea that part–whole and rotation manipulations may tap different aspects of holistic and/or configural processing.

Holistic processing for faces and cars in preschool‐aged children and adults: evidence from the composite effect

The current study compared the development of holistic processing for faces and non-face visual objects by testing for the composite effect for faces and frontal images of cars in 3- to 5-year-old children and adults in a series of four experiments using a two-alternative forced-choice recognition task. Results showed that a composite effect for faces was present as early as 3 1/2 years, and none of the age groups tested showed signs of a composite effect for cars. These findings provide the first demonstration that holistic processing is already selective for faces in early childhood, and confirm existing evidence that sensitivity to holistic information in faces does not increase from 4 years to adulthood.