Temperature Shift for Recombinant Protein Yield in Yeast
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Solution Overview
Problem
Current methods for optimizing recombinant protein production in yeast, such as Pichia pastoris, are largely empirical and fail to efficiently address the interplay of various parameters like media composition, culture density, and temperature, leading to suboptimal yields and purity of proteins like antibodies.
Innovation Solution
A method involving a temperature shift during cell culture, where the temperature is changed from a first temperature to a second temperature, typically between 20°C and 34°C, to enhance protein production and reduce the abundance of product-associated variants with aberrant properties, such as incorrect molecular weight or disulfide bonds.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional empirical methods are used to optimize recombinant protein production in yeast, then the production process can be maintained with standard protocols, but the yield and purity of proteins remain suboptimal due to inability to efficiently address parameter interplays
Solution Approach 1:
The patent applies parameter changes by implementing a temperature shift from a first temperature (e.g., 20-30°C) to a second temperature (e.g., 31-37°C) during the production phase of yeast culture. This temperature parameter modification optimizes both protein yield and purity by addressing the interplay between growth conditions and protein expression, resolving the contradiction between productivity and manufacturing precision that conventional fixed-temperature methods cannot address
2Productivity
If multiple parameters such as media composition, culture density, and temperature are optimized simultaneously, then protein production efficiency improves, but the complexity of optimization process increases
Solution Approach 1:
The patent segments the culture process into distinct phases: a first phase at a first temperature for cell growth and biomass accumulation, and a second phase at a second temperature for protein expression. This temporal segmentation allows independent optimization of each phase, reducing the complexity of simultaneously optimizing multiple parameters while improving overall production efficiency
3Quantity of substance
If temperature is maintained at a constant first temperature during production, then cell growth is supported, but protein yield and purity are suboptimal
Solution Approach 1:
The patent implements periodic action by alternating temperature conditions between two distinct temperature levels during the production cycle. The first temperature (e.g., 20-30°C) supports cell growth and biomass accumulation, while the second temperature (e.g., 31-37°C) optimizes protein expression and quality. This periodic temperature variation resolves the contradiction between maintaining cell biomass and achieving high protein yield
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach increases the yield of recombinant proteins by up to 30% and decreases the relative abundance of variants with aberrant properties, improving the overall purity and stability of proteins like antibodies.
Implementation Method 1
A method involving a temperature shift during cell culture, where the temperature is changed from a first temperature to a second temperature, typically between 20°C and 34°C, to enhance protein production
Data Source
AI summary
Methods for producing heterologous proteins are disclosed. In particular, the present disclosure provides improved methods of producing desired proteins, including multi-subunit proteins such as antibodies, with a higher yield and improved purity. In exemplary embodiments, the transformed cells are a yeast, e.g., methylotrophic yeast such as Pichia pastoris.