Cell Line Development

GS Clonal Selection of Stable Cell Lines

Reach Our Mammalian Experts:

mammalian@lonza.com

  • Introduction

    The Foundation of Your Process

    Lonza's Cell Culture & Process Development team has over 25 years of experience in the development of production processes for animal cell products. A wide range of cell types, (e.g. CHO, hybridomas, various mouse myeloma and human cell lines) have been used to produce monoclonal antibodies (mAbs) and other recombinant proteins using a variety of expression systems.

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  • Cell Line Construction

    Our customers benefit from more than 30 years of cell culture and regulatory compliance experience during which more than 500 GMP cell lines have been constructed. Of these cell lines, 36 have received regulatory approval and now support commercial product supply. This represents more than 35% of all commercial antibody-producing cell lines.

    The GS Xceed® Gene Expression System produces a wide variety of product types including: standard format monoclonal antibodies, bispecifics, novel glycoproteins and other complex molecules. Our cell line development services are available in both our Slough (UK) and Singapore sites.

    A key factor in reducing the production costs of biopharmaceuticals is the development of cell lines that produce a high yield of product with the desired critical quality attributes. Yields of up to 10 grams/liter have been achieved using the GS System in chemically-defined, animal component (CDACF) medium. Our cell line selection strategy has been designed to identify those clones that are highly productive. They are also selected to grow well in suspension culture in our fed-batch fermentation platform process. Selecting the cell lines in the commercial production process enables significant time savings in later stages of development.

    Our scientists have developed cell line construction processes that deliver cGMP material in 14 months from the receipt of DNA sequence. Using rapid methods for generating clonal cell lines, we can also deliver cGMP material from uncloned cell lines for early phase use in 11 months from receipt of DNA sequence. Material for proof-of-concept studies from pooled transfectants can be delivered in four months. 

    Options for Cell Line Construction (CLC)

    We offer a flexible approach to cell line construction that matches your priorities in terms of timelines, yield optimization and risk.  Our team will discuss options in detail to determine the best scope to  fit your project requirements. We are able to offer customized combinations of the following services.

    Full Scope CLC

    Full Scope CLC is suitable for complex proteins which are not standard format antibodies. This service includes comprehensive clone generation and selection with supporting analytics to ensure high yielding cell lines that fit with the GS Xceed® Platform manufacturing process. Lead cell line candidates can be selected at 16 weeks from transfection.

    LightpathTM CLC

    Lightpath™ CLC is applicable for standard format antibodies and Fc-fusion proteins that have a low risk manufacturability profile. Manufacturability assessment is performed with a rapid in silico evaluation to ensure that it is appropriate for this offering.  Lightpath™ CLC is designed to rapidly and cost effectively progress your candidates to a stable, commercially viable cell line. Lead cell line candidates can be selected at 12 weeks from transfection.

    Multiplex CLC  

    Multiplex CLC is designed to progress multiple candidates or variants to the Research Cell Bank (RCB) stage. This offering progresses multiple candidate cell lines simultaneously so that the most suitable can be selected for the final lead.  More data on variants/candidates can be generated and analyzed with Multiplex CLC to support your decision-making process for the best candidate. Lead candidate cell lines for up to four candidates/variants can be selected at 12 weeks from transfection. Multiplex CLC can save 4 months on an overall development program compared to a traditional biologics development approach.

  • Mammalian Cell Banking

    A dedicated cGMP unit within our licensed UK manufacturing facility is used to create master and working cell banks for subsequent manufacturing use. The cell banks are characterized in accordance with FDA and European regulatory agency requirements. For safety and security considerations, dual-site storage of all cell banks are maintained between our US and UK facilities.

  • Protein Sequence Variant Analysis

    A protein sequence variant is an unintended amino acid sequence change within a region of a product. Any biological expression system is prone to generation of these variants, which may affect (inter alia) product efficacy and immunogenicity. Traditionally, detailed analysis of product variants is performed during Phase II/III of development once cell line and manufacturing process has been fully defined. This is usually performed as part of the reference standard characterisation studies which are required for regulatory submissions.  However this traditional, reactive approach provides no effective options should an issue be identified – either the process must be redeveloped or a different cell line taken forward. These options carry significant unforeseen costs and timeline impact.  

    In contrast, incorporation of advanced analytical tools into cell line development enables early, proactive detection of protein variants. Risk factors that would otherwise undermine a project can be managed before they become an issue, supporting a Quality by Design approach to development and ensuring your program stays on track. The same analytics may also be leveraged for later development activities (e.g. to support process changes such as scale-up for late-phase clinical or commercial supply).  

    Since variants may be generated during translation of mRNA in the ribosome, effective detection must be performed at the protein level rather than by DNA or mRNA sequencing alone. Lonza’s Protein Sequence Variant Analysis (PSVA) platform uses class-leading mass spectrometry methods including Electron-Transfer Dissociation and Ultra-High-Resolution Orbitrap mass detection. Our scientists apply advanced informatics to rapidly detect and quantify any amino acid substitutions in panels of candidate cell lines, or across processes.  

    This technology allows consistent detection of variants across the entire product sequence. Variants at levels as low as 0.004% have been confidently identified. In one example, shown in Figure 1, a differential response for a specific protein region is clearly observed by comparative analysis of potential manufacturing cell lines. Sequencing of this region reveals a lysine to asparagine substitution has occurred in 0.1% of the product made by one candidate cell line. Lonza provides risk assessment tools to manage these variants.

    Figure 1 shows a 3-dimensional visualisation of mass spectrometry data (high zoom), the presence of an amino acid sequence variant can be clearly observed by the presence of a signal in cell line B (outlined in red), in comparison to the same region in cell line A. Despite the presence of over one million discrete features in the dataset for each cell line, this difference can be rapidly discriminated. No prior knowledge of the identity of this species is required to determine that there is a difference between cell lines.

     

    Figure 1: Example Data for Detection of an Amino Acid Sequence Variant

     

    Once a difference in cell lines is detected, the identity of the peak must be determined. This identification is performed by tandem mass spectrometry (MS/MS).  Figure 2 below shows an example MS/MS spectra for a low level variant (0.1% abundance relative to the expected sequence).  Peaks colored in red or blue match to the peptide sequence shown in the top left of the figure which has been identified at greater than 99% confidence level.  It can be determined that the asparagine (N) residue marked at position three in this peptide has been substituted for the intended lysine (K) residue.  

      

    Figure 2: High-confidence Identification of the Variant

    When used at the cell line selection stage, Lonza's Protein Sequence Variant Analysis conclusively de-risks clinical candidates.