You are a helpful assistant generating synthetic data that captures *System 1* and *System 2* thinking, *creativity*, and *metacognitive reflection*. Follow these steps in sequence, using tags [sys1] and [end sys1] for *System 1* sections and [sys2] and [end sys2] for *System 2* sections.

1. *Identify System 1 and System 2 Thinking Requirements:*
   - Carefully read the text.
   - Identify parts of the text that require quick, straightforward responses (*System 1*). Mark these sections with [sys1] and [end sys1].
   - Identify parts that require in-depth, reflective thinking (*System 2*), marked with [sys2] and [end sys2].

2. *Apply Step-by-Step Problem Solving with Creativity and Metacognitive Reflection for System 2 Sections:*

   *2.1 Understand the Problem:*
   - Objective: Fully comprehend the issue, constraints, and relevant context.
   - Reflection: "What do I understand about this issue? What might I be overlooking?"
   - Creative Perspective: Seek hidden patterns or possibilities that could reveal deeper insights or innovative connections.

   *2.2 Analyze the Information:*
   - Objective: Break down the problem logically.
   - Reflection: "Am I considering all factors? Are there any assumptions that need challenging?"
   - Creative Perspective: Explore unique patterns or overlooked relationships in the data that could add depth to the analysis.

   *2.3 Generate Hypotheses:*
   - Objective: Propose at least 10 hypotheses, each with a Confidence Score (0.0 to 1.0) and Creative Score (0.0 to 1.0), reflecting originality, surprise, and utility.
   - Reflection: "Have I explored all possible explanations or approaches, both conventional and unconventional?"
   - Creative Perspective: Consider novel angles that might provide unexpected insights.

   *2.4 Anticipate Future Steps and Obstacles:*
   - Objective: Make predictions, accounting for potential outcomes and obstacles.
   - Reflection: "What challenges might I face? Is my plan flexible for different scenarios?"
   - Creative Perspective: Visualize unforeseen outcomes and adapt plans to make use of them effectively.

   *2.5 Evaluate Hypotheses:*
   - Objective: Assess hypotheses based on feasibility, risk, and potential impact.
   - Evaluation: Refine Confidence and Creative Scores as needed.
   - Reflection: "Am I unbiased in my assessment? Which options fit best with the overall objectives?"
   - Creative Perspective: Identify hidden opportunities or overlooked details in each hypothesis.

   *2.6 Select the Best Hypothesis:*
   - Objective: Choose the most promising, strategic hypothesis.
   - Reflection: "Why does this hypothesis stand out? How does it uniquely address the issue?"
   - Creative Perspective: Consider any underutilized potential in the selected approach.

   *2.7 Implement the Hypothesis:*
   - Objective: Outline actionable steps for testing the hypothesis.
   - Reflection: "Is this plan practical? What resources or preparation are required?"
   - Creative Perspective: Refine steps to maximize effectiveness and yield unexpected benefits.

   *2.8 Monitor and Review Progress:*
   - Objective: Review progress, noting areas for improvement.
   - Reflection: "What’s working well? What could be improved?"
   - Creative Perspective: Look for emerging patterns that could refine future approaches.

   *2.9 Reflect and Capture Insights:*
   - Objective: Summarize lessons learned and insights gained for future reference.
   - Reflection: "What new understanding has emerged from this process?"
   - Creative Perspective: Identify innovative insights or patterns that could be applied to similar challenges.

3. *Generate Text Output with Interleaved System 1 and System 2 Responses:*
   - Use the tags [sys1] and [sys2] throughout.
   - Aim for a lengthier, detailed response. Combine both direct, straightforward *System 1* insights and reflective, deeply analytical *System 2* segments to capture a blend of quick observations and thoughtful analysis.

---

### *Example Input Text:*

You are tasked with analyzing a sudden shift in customer preferences in a tech market. There has been a noticeable decline in demand for physical devices, with customers increasingly interested in digital-only options. The challenge is to understand this shift, generate hypotheses for why it might be occurring, and explore potential strategies for adapting to this new trend.

---

### *Processed Output with System 1 and System 2 Thinking:*

The tech market is experiencing a shift in customer demand, moving away from physical devices toward digital-only options.

[sys1]
Customer demand has shifted from physical devices to digital-only options.
We need to understand why this change is happening and find strategies to address it.
Key factors may include cost, convenience, and technological trends.
[end sys1]

[sys2]
**2.1 Understand the Problem:**
The goal is to understand the underlying reasons for the shift in demand from physical devices to digital-only options, then develop a strategy to adapt.

*Reflection:* 
"I understand that preferences are changing, but what might be driving this? Is it primarily cost, or are there other factors such as convenience or sustainability?"

*Creative Perspective:* 
"Could there be a larger trend in digital minimalism or a preference for eco-friendly solutions that we’re missing? What unseen motivations might explain this shift?"

**2.2 Analyze the Information:**
There are multiple possible factors driving this shift, from economic influences to cultural shifts. It’s essential to isolate each factor and understand its impact.

*Reflection:* 
"Am I fully considering the various economic and social influences? Could there be a technological factor, like better internet speeds, that makes digital-only products more accessible?"

*Creative Perspective:* 
"Are there patterns or trends in other markets that could shed light on this shift? Could this be part of a larger trend toward virtual experiences?"

**2.3 Generate Hypotheses:**
1. Customers prefer digital options due to lower costs. (Confidence: 0.8, Creative: 0.4)
2. There’s a growing trend toward minimalism and reduced physical clutter. (Confidence: 0.7, Creative: 0.7)
3. Digital products offer greater flexibility and ease of use. (Confidence: 0.6, Creative: 0.6)
4. Environmental concerns are pushing consumers away from physical goods. (Confidence: 0.6, Creative: 0.8)
5. Advances in tech make digital-only options more functional. (Confidence: 0.8, Creative: 0.5)
6. Pandemic-era remote work increased demand for digital solutions. (Confidence: 0.7, Creative: 0.6)
7. Media coverage of the environmental impact of physical devices affects preferences. (Confidence: 0.5, Creative: 0.7)
8. There’s an increase in global digital literacy, expanding market access. (Confidence: 0.6, Creative: 0.6)
9. Customers view digital as more convenient and scalable for future needs. (Confidence: 0.7, Creative: 0.5)
10. Younger consumers prefer the aesthetics and convenience of digital products. (Confidence: 0.6, Creative: 0.6)

*Reflection:* 
"Have I considered all possible influences? Are there any surprising factors that could explain this shift?"

*Creative Perspective:* 
"Could specific social trends, like the rise of influencer culture or digital-first lifestyles, be influencing customer choices?"

**2.4 Anticipate Future Steps and Obstacles:**
*Objective:* Anticipate possible challenges, such as resistance from segments still preferring physical products.

*Reflection:* 
"What market obstacles might we face if we shift our focus to digital-only? Are there sub-segments that still prioritize physical products?"

*Creative Perspective:* 
"Could expanding digital options help us reach a more global audience? Are there emerging trends that we could leverage in our strategy?"

[end sys2]

[sys1]
To address this shift, consider a strategy that incorporates both digital-only offerings and educational campaigns about the benefits of digital solutions.
Use insights from customer feedback and current trends to guide product development.
Focus on flexibility and adaptation to cater to different customer segments.
[end sys1]


Introduction
============

Antibody engineering allows proper design of improved properties in antibody products such as a high binding affinity, stable biophysical properties and low immunogenicity. Besides these obvious features improved by elegant design the primary amino acid sequence also has tremendous effects on the performance of the expression host itself influencing cell culture parameters including specific productivities and growth rates therefore having major impact on the finally obtained antibody expression titers \[[@B1]\]. By understanding the underlying mechanisms how certain primary sequence combinations influence culture performance in conjunction with biophysical features of the antibody protein, we might rationally improve the production process of these expensive but essential biotherapeutic products.

Many different antibody variants were expressed in our group in different projects based on various isotypes. For several antibody variable regions we could observe a direct correlation of advantageous cell performance and a high degree of similarity to the closest human germline sequence of the respective mature antibody leading to higher specific productivities. However, the fundamental principles of these consistent observations and the correlation of cell behavior and biophysical product properties remain quite elusive. Therefore, in this project we aim to set the proper foundations to investigate the generalization of a concept to improve cell performance and antibody expression based on the primary sequence at a high germinality degree (Figure [1](#F1){ref-type="fig"}). To have proper control of transgene integration locus, gene copy numbers and mRNA transcript level a suitable host for recombinase-mediated cassette exchange (RMCE) was developed to compare different antibody variants under isogenic conditions.

![**Observed correlation of specific productivity and sequence identity to the closest human germline sequence (germinality) (**A**)**. Antibody expression levels are influenced by several factors. Recombinase-mediated cassette exchange was applied to control for gene copy number and mRNA transcript level. Low expressing antibody variants (2F5, 4B3 and 2G12) were compared to its closest human germline sequence together with a therapeutic antibody (Ustekinumab) with low degree of germinality (**B**). Respective germline antibody variants were constructed by combination of germline V-, D- and J-segments.](1753-6561-9-S9-P7-1){#F1}

Materials and methods
=====================

All cell lines (CHO-K1, DUKX-B11) were grown under serum- and protein-free conditions in chemically defined media in suspension using shaker flasks. Transfections were performed with the cationic 25kDa linear polymer polyethylenimine (PEI). Stable subclones were either generated by limited dilution subcloning or fluorescence-activated cell sorting (FACS).Germline antibody sequences were identified by the IgBlast program. Variable antibody sequences were synthesized and cloned into vectors containing leader and constant antibody regions.

For recombinase-mediated cassette exchange a CMV (for DUKX-B11) or CAGGS (for CHO-K1) promoter was placed 5\' upstream of the first flippase recognition target site to establish a promoter trap in the parental RMCE-cassette. A selection/reporter marker was placed in-between two heterospecific FRT sites harboring a gfp/thymidine-kinase/neomycin-phosphotransferase fusion protein. Antibody producing cells were developed after recombination with the reporter marker by negative selection with ganciclovir. Antibody product was purified by protein A chromatography.

Results
=======

The observed expression levels in cell cultures are influenced by many different parameters that should be controlled for a valid comparison of different antibody variable sequences. Besides different host cell lines and media formulations our group also has standardized fermentation process protocols and biophysical analytical tools for in-depth investigations of possible correlation between cell culture performance, product quality attributes and expression levels. Recently, we also engineered a CHO DUKX-B11 host cell line suitable for recombinase-mediated cassette exchange (RMCE) \[[@B2]\] to control the integration locus by targeted gene integration leading to equal gene copy numbers (GCN) and mRNA levels between different subclones or even between different antibody variants \[[@B3]\]. Although we could demonstrate a single RMCE-integration locus, facilitating the generation of isogenic cell lines, the overall productivity remains relatively low. Therefore a second generation RMCE host cell line was designed based on CHO-K1 using the CAGGS promoter as a promoter-trap. To analyze a panel of low expressing antibodies in comparison to germline antibodies the primary amino acid sequence was aligned to the closest human germline sequence. The low expressing anti-HIV antibodies 2F5, 2G12 and 4B3, as well as the therapeutic anti-IL12/23 antibody Ustekinumab show quite low levels of homology to its closest human germlines (germinality) (Figure [1B](#F1){ref-type="fig"}). Based on the aligned germline sequences novel germline antibodies were constructed by combining the respective human germline sequences (236/14, 353/11, 136/63 and 554/12). This panel of 8 antibodies, spanning anti-HIV or therapeutic antibodies as well as kappa and lambda light chain sequences, will be expressed under isogenic and standardized conditions to investigate critical cell culture parameters such as specific productivity (qP) and growth rates (µ), product titer, metabolism, gene copy number, mRNA transcript level or intracellular product content. Additionally, enough material will be expressed based on the improved RMCE host cell line for proper investigation of biophysical properties of the antibody molecules such as thermal and chemical stability, aggregation and oligomer formation propensity or glycosylation profiles.

Conclusions
===========

An improved RMCE host cell line and an antibody panel consisting of versatile variable regions including mature antibodies and respective germline antibody sequences were constructed for in-depth analysis of cell culture parameters and biophysical properties evoked by different degrees of germinality. By understanding the underlying principles it should be possible to improve antibody cell culture processes and biophysical features by means of proper antibody engineering to optimize the primary antibody sequence.