Achieving rapid, large-scale data acquisition and processing for early-stage decision making

The Growing Need for High-Throughput Analysis

The requirement for high-throughput analyses is becoming an increasingly widespread necessity for the development of biotherapeutics. This need applies to all product types, from small peptides to conjugated proteins and highly heterogeneous, complex glycoproteins.

The Need for High-Throughput Characterization

At BioPharmaSpec we believe that understanding your product at the earliest stage possible, whether in early phase development, or at a stage where large numbers of sample matrices are produced, is critical to gaining the broadest understanding of the product’s structure and physico-chemical properties.

Depending on the analytical tools available, it is not a trivial task to sufficiently characterize a large sample set. Significant investment is required to acquire the instrumentation, scientific staff, software utilities and an understanding to coalesce all data sets.

The goal of high-throughput analysis is to target a particular attribute, or to monitor multiple attributes, through efficient workflow design, data acquisition, and data processing. This enables more detailed and informed decisions that helps to mitigate further risks in the next stages of your production pipeline.

BioPharmaSpec's High-Throughput Methods

BioPharmaSpec has developed the following high-throughput methods to achieve this goal:

  1. Intact mass assessment using on-line LC/ES-MS.
  2. Peptide mapping.
  3. Glycosylation analysis.
  4. Charge profiling using Imaging Capillary IsoElectric Focusing (icIEF).
  5. Secondary structure assessment using RedshiftBio Microfluidic Modulation Spectroscopy (MMS), and
  6. Dynamic Light Scattering (DLS) and SEC-MALS, including an in-line QELS detector to assess aggregation levels.
Figure 1: Representative single sample data for Intact Mass analysis of 96 batches of trastuzumab
Figure 1: Representative single sample data for Intact Mass analysis of 96 batches of trastuzumab

What This Combination of Techniques Delivers

A combination of these techniques provides an assessment of all the required structural attributes in a time efficient manner:

  • Intactness of the product.
  • Primary structure and an assessment of PTMs.
  • Oligosaccharide population.
  • Charge profile.
  • Secondary structure.
  • Aggregation state.

Case Study: A 96-Batch Trastuzumab Assessment

To achieve this, BioPharmaSpec conducted an internal assessment of 96 batches of Trastuzumab including both biosimilar and innovator products. This study was designed to rigorously test our methods, utilities and software packages built on 40+ years of in-depth understanding of product characterization across a broad range of molecule types.

High-Throughput Peptide Mapping

To perform the peptide mapping assessment on 96 samples, an efficient tryptic digest workflow was designed to minimize sample transfer and handling steps, using 96-well plates, multichannel pipettors, and rapid denaturation and digestion protocols.

The prepared samples were then assessed using tried and tested LC/MS platforms to handle and process targeted attributes, as well as to assess overall peptide mapping coverage. Processing tools were optimized for effective post-translational modification (PTM) assessment and to allow easy comparison of each sample batch alongside multiple control samples, assessing sample stability and instrument performance.

Figure 2: Peptide mapping (subset of samples overlay)
Figure 2: Peptide mapping (subset of samples overlay)

This single analysis identified several biosimilar candidate batches that fell outside the range of the reference standards analyzed alongside, based on Methionine oxidation levels and/or Heavy Chain C-terminal Lysine content.

Secondary Structure Assessment

Secondary structure was assessed using a high-throughput, 96-well plate format instrument via MMS (Microfluidic Modulation Spectroscopy). MMS sensitively measures protein structure in situ and reproducibly detects even very small differences between samples, that fall outside the capabilities of traditional technologies while providing rapid detection of structures including alpha helices, beta sheets and turns.

The data obtained from analysis of the 96 trial samples showed a similar secondary structure across the set, with no cause for concern.

Aggregation Analysis

Aggregation assessments were performed using SEC-MALS and DLS, with SEC-MALS optimized for shortened run times and higher flow rates to reliably detect monomer and dimer species.

Multiple biosimilar candidate batches were shown to contain elevated (two-fold) levels of dimer species relative to the reference material and could therefore be highlighted as poor candidates to continue in the production pipeline.

Figure 3: SEC-UV data obtained from analysis of 96 samples from an early-phase MAb product. Significant differences in the relative amount of dimer (peak around 2.4 mins) can be observed across the sample set.
Figure 3: SEC-UV data obtained from analysis of 96 samples from an early-phase MAb product. Significant differences in the relative amount of dimer (peak around 2.4 mins) can be observed across the sample set.

Key Findings

This study made clear that if titer alone had been used to assess the best cell line producing the largest amount of product, more than 90% of the Trastuzumab batches could have been selected for further development. However, characterizing each batch using the high-throughput tests outlined above told a different story: half of the biosimilar candidate batches could instead have been withdrawn, mitigating development risk at an earlier stage with a reduction of potential development costs further down the line.

What Our Client Say

"Early development of biopharmaceutical product candidates depends on the availability of as much product quality data as possible in order to take the best-informed decisions at every step in the development process. This applies to many different elements of product development, e.g. clone/cell line selection, fermentation process development, purification process development, formulation development, and stability evaluation. The more data points for important structural quality attributes that are available, the more qualified decisions can be made.

BioPharmaSpec's new High Throughput characterization service is designed to deliver just that and should for several reasons be attractive to many companies engaged in early development of biopharmaceuticals. BioPharmaSpec has a long-standing track record for delivering high-quality characterization data and their technical capabilities surpass most small- to mid-sized biopharmaceutical companies."

— Lead investigator from a long-term client

Would you like to learn how high-throughput characterization could support your biopharmaceutical development program?

Contact BioPharmaSpec to discuss your analytical requirements and discover how multi-attribute data can help reduce development risk at an earlier stage.

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